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Sample records for efficiency non-viral transfection

  1. Cholesterol improves the transfection efficiency of polyallylamine as a non-viral gene delivery vector

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    Reza Kazemi Oskuee

    2017-10-01

    Full Text Available ABSTRACT Cationic polymers such as polyallylamine (PAA having primary amino groups are poor transfection agents and possess a high cytotoxicity index when used without any chemical modification. In this study, PAA was modified with cholesterol in order to improve transfection efficiency and to reduce cytotoxicity. PAA polymers with molecular weights of 15 and 65 kDa were selected and grafted with cholesterol at percentages of 5, 10, 15, 30, and 50. After purification, the efficacy of the synthetic vectors was evaluated in terms of DNA condensation using the ethidium bromide test, buffering capacity, particle size, zeta potential, transfection efficiency, and cytotoxicity assay in Neuro2A cell lines. According to the ethidium bromide test, these vectors can condense DNA at moderate and high carrier to plasmid (C/P ratios. The buffering capacity of the prepared vector in both molecular weights was less than unmodified PAA. Particle size measurements demonstrated that modified PAAs were able to form nanoparticles ranging in size from 125 to 530 nm. The vectors based on PAA 15 kDa demonstrated a better transfection efficiency than the vectors made of PAA 65 kDa. Cytotoxicity studies showed that toxicity of all vectors was less than PAA. Some cholesterol modified polymers composed of PAA (15 kDa were suitable vectors for gene delivery with low cytotoxicity.

  2. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

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    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

  3. Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications.

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    Soto-Sánchez, Cristina; Martínez-Navarrete, Gema; Humphreys, Lawrence; Puras, Gustavo; Zarate, Jon; Pedraz, José Luis; Fernández, Eduardo

    2015-05-01

    This work demonstrates the successful long-term transfection in vivo of a DNA plasmid vector in rat visual cortex neurons using the magnetofection technique. The transfection rates reached values of up to 97% of the neurons after 30days, comparable to those achieved by viral vectors. Immunohistochemical treatment with anti-EGFP antibodies enhanced the detection of the EYFP-channelrhodopsin expression throughout the dendritic trees and cell bodies. These results show that magnetic nanoparticles offer highly efficient and enduring in vivo high-rate transfection in identified neurons of an adult mammalian brain and suggest that the magnetotechnique facilitates the introduction of large functional genetic material like channelrhodopsin with safe non-viral vectors using minimally invasive approaches. Gene therapy may be one of the treatment modalities for neurological diseases in the future. The use of viral transfection remains a concern due to restrictions to the size limit of the genetic material able to be packed, as well as safety issues. In this work, the authors evaluated magnetoplexes as an alternative vehicle. The results showed very promising data in that these nanoparticles could offer high transfection efficiency. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications

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    King, William J.; Kouris, Nicholas A.; Choi, Siyoung; Ogle, Brenda M.; Murphy, William L.

    2012-01-01

    Non-viral transfection is a promising technique which could be used to increase the therapeutic potential of stem cells. The purpose of this study was to explore practical culture parameters of relevance in potential human mesenchymal stem cell (hMSC) clinical and tissue engineering applications, including type of polycationic transfection reagent, N/P ratio and dose of polycation/pDNA polyplexes, cell passage number, cell density, and cell proliferation. The non-viral transfection efficiency was significantly influenced by N/P ratio, polyplex dose, cell density, and cell passage number. hMSC culture conditions that inhibited cell division also decreased transfection efficiency, suggesting that strategies to promote hMSC proliferation may be useful to enhance transfection efficiency in future tissue engineering studies. Non-viral transfection treatments influenced hMSC phenotype, including the expression level of the hMSC marker CD105, and the ability of hMSCs to differentiate down the osteogenic and adipogenic lineages. The parameters found here to promote hMSC transfection efficiency, minimize toxicity, and influence hMSC phenotype may be instructive in future non-viral transfection studies and tissue engineering applications. PMID:22277991

  5. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons.

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    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the "in vivo" transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs.

  6. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

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    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  7. Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

    NARCIS (Netherlands)

    Yang, X.; Walboomers, X.F.; Dolder, J. van den; Yang, F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2008-01-01

    Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and

  8. Nanomagnetic Gene Transfection for Non-Viral Gene Delivery in NIH 3T3 Mouse Embryonic Fibroblasts

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    Angeliki Fouriki

    2013-01-01

    Full Text Available The objective of this work was to examine the potential of oscillating nanomagnetic gene transfection systems (magnefect-nano™ for improving the transfection efficiency of NIH3T3 mouse embryonic fibroblasts (MEFs in comparison to other non-viral transfection techniques—static magnetofection™ and the cationic lipid agent, Lipofectamine 2000™. Magnetic nanoparticles (MNPs associated with the plasmid coding for green fluorescent protein (GFP were used to transfect NIH3T3 cells. The magnefect-nano system was evaluated for transfection efficiency, and any potential associated effects on cell viability were investigated. MNPs associated with the plasmid coding for GFP were efficiently delivered into NIH3T3 cells, and the magnefect-nano system significantly enhanced overall transfection efficiency in comparison to lipid-mediated gene delivery. MNP dosage used in this work was not found to affect the cell viability and/or morphology of the cells. Non-viral transfection using MNPs and the magnefect-nano system can be used to transfect NIH3T3 cells and direct reporter gene delivery, highlighting the wide potential of nanomagnetic gene transfection in gene therapy.

  9. Comparative Analysis of Non-viral Transfection Methods in Mouse Embryonic Fibroblast Cells

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    Lee, Migi; Chea, Kathleen; Pyda, Rajyalakshmi; Chua, Melissa; Dominguez, Isabel

    2017-01-01

    Mouse embryonic fibroblast (MEF) cells are an important in vitro model for developmental biology, disease, and reprogramming studies. However, as with other primary cells, they are challenging to transfect. Although viral gene-delivery methods achieve high gene-delivery efficiency, challenges with cell mutagenesis and safety among others have led to the use and improvement of non-viral gene-delivery methods in MEF cells. Despite the importance of gene delivery in MEF cells, there is limited comparison of method/reagent efficacy. In this study, we compared the effectiveness of different gene-delivery methods and several reagents currently available in MEF cells by introducing a plasmid containing enhanced green fluorescent protein (EGFP). We analyze transfection efficiency by EGFP fluorescence. Our results suggest that two gene-delivery methods—electroporation and magnetofection in combination with a lipid reagent, are the most efficient transfection methods in MEF cells. This study provides a foundation for the selection of transfection methods or reagents when using MEF cells. PMID:28507462

  10. [Expression patterns of non-viral transfection with GFP in the organ of Corti in vitro and in vivo. Gene therapy of the inner ear with non-viral vectors].

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    Praetorius, M; Pfannenstiel, S; Klingmann, C; Baumann, I; Plinkert, P K; Staecker, H

    2008-05-01

    Diseases of the inner ear such as presbycusis, tinnitus, sudden hearing loss, and vertigo affect many patients, but so far there are no specific therapy options. Gene therapy might become a potential modality of treatment. Viral vectors are standard in animal models to date. Future considerations, however, call for a further evaluation of non-viral transfection methods. The non-viral transfection agents Metafectene, Superfect, Effectene, and Mirus TransIT were incubated with a plasmid coding for GFP. In vivo, the plasmid-agent mix was injected via the membrane of the round window, and 48 h later the inner ear was perfused, harvested, decalcified, and histologically evaluated for GFP expression. Cationic lipids (Metafectene) and dendrimers (Superfect) were able to transfect cells in the area of the organ of Corti and lead to GFP expression. The polyamine (Mirus TransIT) did show expression of GFP in the area of Rosenthal's canal and in the area of the inner hair cell. The combination of a non-liposomal lipid with a DNA condensing component (Effectene) did not show transfection of the organ of Corti. In the area of the spiral ganglia cells, GFP expression was seen with all the transfection agents. Non-viral transfection agents are able to introduce a reporter gene in cells of the inner ear in vitro and in vivo. There are, however, differences in the efficiency of the transfection. They might be an alternative in gene therapy of the inner ear. Further investigations to elucidate their potential are needed.

  11. Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue

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    Gallego-Perez, Daniel; Pal, Durba; Ghatak, Subhadip; Malkoc, Veysi; Higuita-Castro, Natalia; Gnyawali, Surya; Chang, Lingqian; Liao, Wei-Ching; Shi, Junfeng; Sinha, Mithun; Singh, Kanhaiya; Steen, Erin; Sunyecz, Alec; Stewart, Richard; Moore, Jordan; Ziebro, Thomas; Northcutt, Robert G.; Homsy, Michael; Bertani, Paul; Lu, Wu; Roy, Sashwati; Khanna, Savita; Rink, Cameron; Sundaresan, Vishnu Baba; Otero, Jose J.; Lee, L. James; Sen, Chandan K.

    2017-10-01

    Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency). In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

  12. Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

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    Raisin, Sophie; Morille, Marie; Bony, Claire; Noël, Danièle; Devoisselle, Jean-Marie; Belamie, Emmanuel

    2017-08-22

    In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

  13. Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy.

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    Chang, Lingqian; Gallego-Perez, Daniel; Zhao, Xi; Bertani, Paul; Yang, Zhaogang; Chiang, Chi-Ling; Malkoc, Veysi; Shi, Junfeng; Sen, Chandan K; Odonnell, Lynn; Yu, Jianhua; Lu, Wu; Lee, L James

    2015-08-07

    Current transfection technologies lead to significant inter-clonal variations. Previously we introduced a unique electrotransfection technology, Nanochannel-Electroporation (NEP), which can precisely and benignly transfect small cell populations (~100-200 cells) with single-cell resolution. Here we report on the development of a novel 3D NEP system for large scale transfection. A properly-engineered array of nanochannels, capable of handling/transfecting ~60 000 cells cm(-2), was fabricated using cleanroom technologies. Positive dielectrophoresis was used to selectively position cells on the nanochannels, thus allowing highly efficient transfection. Single-cell dosage control was demonstrated using both small and large molecules, and different cell types. The potential clinical relevance of this system was tested with difficult-to-transfect natural killer cell suspensions, and plasmids encoding for the chimeric antigen receptor (CAR), a model of high relevance for adoptive immunotherapy. Our results show significantly higher CAR transfection efficiencies for the DEP-NEP system (>70% vs. <30%), as well as enhanced cell viabilities.

  14. Effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection

    NARCIS (Netherlands)

    Lin, C.; Engbersen, Johannes F.J.; Hennink, W.E.; Feijen, J.; Sam, A.P.

    2008-01-01

    The development of safe and efficient gene delivery vectors is an essential prerequisite for successful gene therapy. As viral vectors suffer from inherent disadvantages, cationic polymers as non-viral vectors have great potential in gene delivery, but their practical application so far is seriously

  15. Efficient transfection of MG-63 osteoblasts using magnetic nanoparticles and oscillating magnetic fields.

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    Fouriki, A; Clements, M A; Farrow, N; Dobson, J

    2014-03-01

    To examine the potential of magnetic nanoparticles (MNPs) in transfecting human osteosarcoma fibroblasts (MG-63) and investigate the effects of a novel non-viral oscillating nanomagnetic gene transfection system (magnefect-nano™) in enhancing transfection efficiency (TE). MG-63 cells were transfected using MNPs coupled with a GFP-carrying plasmid. The magnefect-nano system was evaluated for transfection efficiency and potential associated effects on cell viability. MG-63 cells were efficiently transfected using MNPs and the magnefect-nano system significantly enhanced overall transfection efficiency. MNPs were not found to affect cell viability and/or function of the cells. Non-viral transfection using MNPs and the magnefect-nano system can be used to transfect MG-63 cells and assist reporter gene delivery on a single cell basis, highlighting the wide potential of nanomagnetic gene transfection in gene therapy. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

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    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  17. Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach

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    Das, Debobrato

    Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF). With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

  18. Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-viral Gene Therapy for Leber's Congenital Amaurosis.

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    Sun, Da; Sahu, Bhubanananda; Gao, Songqi; Schur, Rebecca M; Vaidya, Amita M; Maeda, Akiko; Palczewski, Krzysztof; Lu, Zheng-Rong

    2017-06-16

    Development of a gene delivery system with high efficiency and a good safety profile is essential for successful gene therapy. Here we developed a targeted non-viral delivery system using a multifunctional lipid ECO for treating Leber's congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65(-/-) mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells.

    Science.gov (United States)

    Palchetti, Sara; Pozzi, Daniela; Marchini, Cristina; Amici, Augusto; Andreani, Cristina; Bartolacci, Caterina; Digiacomo, Luca; Gambini, Valentina; Cardarelli, Francesco; Di Rienzo, Carmine; Peruzzi, Giovanna; Amenitsch, Heinz; Palermo, Rocco; Screpanti, Isabella; Caracciolo, Giulio

    2017-02-01

    To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Preliminary study on non-viral transfection of F9 (factor IX gene by nucleofection in human adipose-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Susana Olmedillas López

    2016-04-01

    Full Text Available Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment. Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen. Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration. Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia.

  1. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons.

    Science.gov (United States)

    Vernon, Matthew M; Dean, David A; Dobson, Jon

    2015-08-17

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells.

  2. Biodegradable Tri-Block Copolymer Poly(lactic acid-poly(ethylene glycol-poly(L-lysine(PLA-PEG-PLL as a Non-Viral Vector to Enhance Gene Transfection

    Directory of Open Access Journals (Sweden)

    Na Zhang

    2011-02-01

    Full Text Available Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid-poly(ethylene glycol-poly(L-lysine (PLA-PEG-PLL copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z-NCA onto amine-terminated NH2-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ε-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells than PEI/DNA complexes (14.01% and 24.22%. These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.

  3. Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells.

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    Hornstein, Benjamin D; Roman, Dany; Arévalo-Soliz, Lirio M; Engevik, Melinda A; Zechiedrich, Lynn

    2016-01-01

    The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.

  4. Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

    Directory of Open Access Journals (Sweden)

    Claudia Merkl

    Full Text Available Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4 into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

  5. PEI-alginate nanocomposites: efficient non-viral vectors for nucleic acids.

    Science.gov (United States)

    Patnaik, Soma; Arif, Mohammed; Pathak, Atul; Singh, Naresh; Gupta, K C

    2010-01-29

    Branched polyethylenimine (PEI, 25 kDa) was ionically interacted with varying amount of alginic acid to block different proportion (2.6-5.7%) of amines in PEI to form a series of nanocomposites, PEI-Al. These nanocomposites, upon interaction with DNA, protected it against DNase I. Among various complexes evaluated, PEI-Al(4.8%)/DNA displayed the highest transfection efficiency in HEK293, COS-1 and HeLa cells that was approximately 2-8-folds higher than Superfect, Fugene, PEI (750 kDa)-Al(6.26%) and PEI alone. The projected nanocomposites were nearly non-toxic to cells in vitro. Furthermore, the concentration of PEI-Al(4.8%) needed to deliver GFP-specific siRNA in COS-1 cells was 20 times lower than PEI (750 kDa)-Al(6.26%). Intracellular trafficking of PEI-Al(4.8%) with or without complexed DNA in HeLa cells shows that both appear in the nucleus after 1 h. 2009 Elsevier B.V. All rights reserved.

  6. A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

    Science.gov (United States)

    Vankayala, Raviraj; Chiang, Chi-Shiun; Chao, Jui-I; Yuan, Chiun-Jye; Lin, Shyr-Yeu; Hwang, Kuo Chu

    2014-09-01

    Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Femtosecond optical transfection of cells:viability and efficiency

    National Research Council Canada - National Science Library

    D. Stevenson; B. Agate; X. Tsampoula; P. Fischer; C. T. A. Brown; W. Sibbett; A. Riches; F. Gunn-Moore; K. Dholakia

    2006-01-01

    .... However, there remains no study into the true efficiency of this procedure. Here, we present a detailed analysis of transfection efficiency and cell viability for femtosecond optical transfection using a titanium sapphire laser at 800 nm...

  8. Transfection efficiency influenced by aggregation of DNA/polyethylenimine max/magnetic nanoparticle complexes

    Energy Technology Data Exchange (ETDEWEB)

    Ota, Satoshi, E-mail: ota-satoshi-gw@ynu.ac.jp; Takahashi, Yoshiyuki [Yokohama National University, Faculty of Engineering (Japan); Tomitaka, Asahi [University of Washington, Department of Materials Science and Engineering (United States); Yamada, Tsutomu [Yokohama National University, Faculty of Engineering (Japan); Kami, Daisuke [Kyoto Prefectural University of Medicine, Department of Cardiac Supports (Japan); Watanabe, Masatoshi; Takemura, Yasushi [Yokohama National University, Faculty of Engineering (Japan)

    2013-05-15

    Gene delivery using magnetic nanoparticles (MNPs) is known as magnetofection and is an efficient non-viral gene delivery system. {gamma}-Fe{sub 2}O{sub 3} nanoparticles (primary diameter = 29 nm) and Fe{sub 3}O{sub 4} nanoparticles (primary diameter = 20-30 nm) coated with deacylated linear polyethylenimine (PEI max) were prepared and conjugated with DNA. The dependency of transfection efficiency on the weight of MNPs, viability of HeLa cells, and size of DNA/PEI max/MNP complexes was evaluated. Transfection efficiency initially increased with the weight of the complexes; however, it decreased with further increase in weight. In contrast, cell viability increased with further increase in weight. Cytotoxicity assay showed that the decline in transfection efficiency at higher weights was not attributable to cytotoxicity of DNA/PEI max/MNP complexes. The DNA/PEI max/MNP complexes aggregated because of DNA binding and pH interaction with the medium. Aggregation depending on the weight of MNPs was confirmed. The number of complexes was estimated from the size distribution. In addition, the dependency of the transfection efficiency on aggregation was assessed with respect to cellular endocytic pathways using the complexes. The complexes were internalized through clathrin-dependent endocytosis, which was a size-dependent pathway. This study reveals that decreased transfection efficiency was associated with the extent of aggregation, which was induced by high weight of MNPs.

  9. Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study

    Directory of Open Access Journals (Sweden)

    Ana V Oliveira

    2013-01-01

    Full Text Available Objective: Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently. Materials and Methods: Chitosan and thiolated chitosan nanoparticles (NPs were prepared in order to obtain a NH3 + :PO4− ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells. Results: In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency. Conclusion: The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery.

  10. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents.

    Science.gov (United States)

    Chettab, Kamel; Roux, Stéphanie; Mathé, Doriane; Cros-Perrial, Emeline; Lafond, Maxime; Lafon, Cyril; Dumontet, Charles; Mestas, Jean-Louis

    2015-01-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.

  11. Rigid aromatic linking moiety in cationic lipids for enhanced gene transfection efficiency.

    Science.gov (United States)

    Wang, Bing; Zhao, Rui-Mo; Zhang, Ji; Liu, Yan-Hong; Huang, Zheng; Yu, Qing-Ying; Yu, Xiao-Qi

    2017-08-18

    Although numerous cationic lipids have been developed as non-viral gene vectors, the structure-activity relationship (SAR) of these materials remains unclear and needs further investigation. In this work, a series of lysine-derived cationic lipids containing linkages with different rigidity were designed and synthesized. SAR studies showed that lipids with rigid aromatic linkage could promote the formation of tight liposomes and enhance DNA condensation, which is essential for the gene delivery process. These lipids could give much higher transfection efficiency than those containing more flexible aliphatic linkage in various cell lines. Moreover, the rigid aromatic linkage also affords the material higher serum tolerance ability. Flow cytometry assay revealed that the target lipids have good cellular uptake, while confocal microscopy observation showed weaker endosome escape than Lipofectamine 2000. To solve such problem and further increase the transfection efficiency, some lysosomotropic reagents were used to improve the endosome escape of lipoplex. As expected, higher transfection efficiency than Lipofectamine 2000 could be obtained via this strategy. Cytotoxicity assay showed that these lipids have lower toxicity in various cell lines than Lipofectamine 2000, suggesting their potential for further application. This work demonstrates that a rigid aromatic linkage might distinctly improve the gene transfection abilities of cationic lipids and affords information to construct safe and efficient gene vector towards practical application. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Elevation of Transfection Efficiency by Conjugation of Poly(amindoamine)-diethylenetriamine (PAM-DET) with Dexamethasone

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Yunseong; Park, Jihye; Jin, Geunwoo; Park, Jongsang [Seoul National Univ., Seoul (Korea, Republic of)

    2012-11-15

    We successfully conjugated hydrophobic group, dexamethasone onto the surface of PAM-DET to synthesize PAM-DET-DX to form polyplexes with enhanced stability against ionic strength. We evaluated its stability by measuring the size of its polyplexes; the conjugated PAM-DET polyplex showed decreased growth compared to the PAM-DET polyplex in an environment with increased ionic strength, which implies that the conjugated PAM-DET has enhanced stability against increased ionic strength. Furthermore, conjugation of hydrophobic group caused a slight increase in the transfection efficiency without inducing toxicity. Of course, it isn't a neglectable factor that nuclear localization effect of DX can drive the advanced transfection efficiency of PAM-DET-DX polyplex. It means that the hydrophobic moieties which have some other positive properties in transfection are good candidates that can be introduced to non-viral polymeric gene delivery carrier. This strongly indicates that the introduction of hydrophobic moiety on PAM-DET is a good method to enhance polyplex stability against ionic strength without diminishing its advantageous properties, such as high transfection efficiency and low cytotoxicity.

  13. Gene recombinant bone marrow mesenchymal stem cells as a tumor-targeted suicide gene delivery vehicle in pulmonary metastasis therapy using non-viral transfection.

    Science.gov (United States)

    Zhang, Tian-Yuan; Huang, Bing; Yuan, Zhong-Yue; Hu, Yu-Lan; Tabata, Yasuhiko; Gao, Jian-Qing

    2014-01-01

    One of the main limitations of anti-tumor gene therapy is the lack of an effective way to deliver therapeutic genes to tumor sites. Bone marrow mesenchymal stem cells (BMSCs) have been proposed as cellular delivery vehicles to tumor sites in tumor-targeted cancer gene therapy. Here, we investigated the therapeutic effects of cytomegalovirus-thymidine kinase expressing BMSCs (TK-BMSCs) on pulmonary melanoma metastasis combined with prodrug ganciclovir. BMSCs were successfully engineered through a non-viral gene vector. The gene recombinant BMSCs migrated to the pulmonary area and were found to have the tendency to target tumor nodules after systemic delivery. In vitro results demonstrate that the engineered BMSCs have significant suicide effects in the presence of ganciclovir in a dose-dependent manner and can exert a sufficient bystander effect on B16F10 tumor cells in co-culture experiments. In vivo studies confirmed the therapeutic effects of TK-BMSCs/ganciclovir on the metastasis tumor model. This study investigates the possibility of gene transfer via bone marrow mesenchymal stem cells in anti-cancer gene therapy using a metastatic melanoma model and cytomegalovirus-thymidine kinase expressing stem cells, demonstrating clear therapeutic effects. © 2013.

  14. Cationic Liposomes Modified with Polyallylamine as a Gene Carrier: Preparation, Characterization and Transfection Efficiency Evaluation

    Directory of Open Access Journals (Sweden)

    Reza Kazemi Oskuee

    2016-12-01

    Full Text Available Purpose: Cationic polymers and cationic liposomes have shown to be effective non-viral gene delivery vectors. In this study, we tried to improve the transfection efficiency by employing the advantages of both. Methods: For this purpose, modified polyallylamines (PAAs were synthesized. These modifications were done through the reaction of PAA (15 KDa with acrylate and 6-bromoalkanoic acid derivatives. Liposomes comprising of these cationic polymers and cationic lipid were prepared and extruded through polycarbonate filters to obtain desired size. Liposome-DNA nanocomplexes were prepared in three carrier to plasmid (C/P ratios. Size, zeta potential and DNA condensation ability of each complex were characterized separately and finally transfection efficiency and cytotoxicity of prepared vectors were evaluated in Neuro2A cell line. Results: The results showed that mean particle size of all these nanocomplexes was lower than 266 nm with surface charge of 22.0 to 33.9 mV. Almost the same condensation pattern was observed in all vectors and complete condensation was occurred at C/P ratio of 1.5. The lipoplexes containing modified PAA 15 kDa with 10% hexyl acrylate showed the highest transfection efficacy and lowest cytotoxicity in C/P ratio of 0.5. Conclusion: In some cases nanocomplexes consisting of cationic liposome and modified PAA showed better transfection activity and lower cytotoxicity compared to PAA.

  15. Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles.

    Science.gov (United States)

    Fan, Z; Chen, D; Deng, C X

    2013-09-28

    Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. In this study, we conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome, and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmids coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9%±2.2% (n=9), comparable with lipofection (7.5%±0.8%, n=9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Transfection efficiency along the regenerating soleus muscle of the rat.

    Science.gov (United States)

    Kósa, Magdolna; Zádor, Ernő

    2013-06-01

    We investigated the efficiency of a single plasmid transfection along the longitudinal axis of the regenerating soleus of young rats. This also reflected transfection efficiency along the fibers because the soleus is a nearly fusiform muscle in young animals. The complete regeneration was induced by notexin and the transfection was made by intramuscular injection of enhanced green fluorescent protein- or Discosoma red-coding plasmids after 4 days. One week after transfection the number of transfected fibers was higher at the place of injection (i.e., in the muscle belly) and lower or absent at the ends of the muscle. The inspection of longitudinal sections and neuromuscular endplates indicated that one of the reasons of uneven transfection might be the shortness of transfected myotubes and the other reason might be the limit of diffusion of transgenic proteins from the expressing nuclei. As a result, the efficiency of transfection in the whole regenerating muscle was much lower than it could be estimated from the most successfully transfected part.

  17. Optomizing Transfection Efficiency of Cervical Cancer Cells Transfected by Cationic Liposomes LipofectamineTM2000.

    Science.gov (United States)

    Huang, Fei; Zhao, Feng; Liang, Li-Ping; Zhou, Mei; Qu, Zhi-Ling; Cao, Yan-Zhen; Lin, Chen

    2015-01-01

    Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Lipofectamine(TM)2000 as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA , miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. The seeding density of Hela cell line and Siha are 1.5 x 10(4) (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (Ptransfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.

  18. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    Directory of Open Access Journals (Sweden)

    Helena Sork

    2016-01-01

    Full Text Available The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

  19. The Effect of Linear PEI on Characteristics and Transfection Efficiency of PEI-Based Cationic Nanoliposomes

    Directory of Open Access Journals (Sweden)

    Mohammad Ramezani

    2011-01-01

    Full Text Available Objective(sThe development of efficient and safe carrier system to transfer DNA into cells is essential in non-viral gene therapy. The aim of the present study was to evaluate the effect of linear polyetheneimine (lPEI (2500 Da on the physicochemical and biological properties of lipopolyplexes constructed from liposomes and lPEI. Materials and MethodsDifferent lipopolymers were synthesized from lPEI and acrylate derivatives. Nanocarriers were composed of the lipids (DOPE, DPPE and DOTAP and the synthesized lipopolymers. After characterization of the prepared vectors by determination of size and zeta potential, transfection activity was tested in Neuro2A cells. Ethidium bromide and MTT test were used to evaluate the DNA condensation ability and cytotoxicity of vectors, respectively. Results Vector’s size ranged from 95 to 337 nm and they had positive charge. The differences in DNA binding properties of lipopolyplexes were not significant. Among lipids, DOTAP showed better impact on transfection efficiency. The highest transfection activity was achieved by liposomal formulation consist of DOTAP and lipopolymer composed of lPEI and hexyl acrylate. The lipopolyplexes showed minimum cytotoxicity to the cultured cells in vitro. Conclusion The results of study confirmed that it is possible to improve gene expression using lipopolyplexes.

  20. Highly efficient transfection of human THP-1 macrophages by nucleofection.

    Science.gov (United States)

    Maeß, Marten B; Wittig, Berith; Lorkowski, Stefan

    2014-09-02

    Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

  1. Efficient non-viral reprogramming of myoblasts to stemness with a single small molecule to generate cardiac progenitor cells.

    Directory of Open Access Journals (Sweden)

    Zeeshan Pasha

    Full Text Available The current protocols for generation of induced pluripotent stem (iPS cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs using small molecules.SMs from young male Oct3/4-GFP(+ transgenic mouse were treated with DNA methyltransferase (DNMT inhibitor, RG108. Two weeks later, GFP(+ colonies of SM derived iPS cells (SiPS expressing GFP and with morphological similarity of mouse embryonic stem (ESCs were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs formation yielded spontaneously contracting EBs having morphological, molecular, and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group, extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group. A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed.Reprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function.

  2. Graphene substrates enhance optical transfection efficiency in pluripotent stem cells

    CSIR Research Space (South Africa)

    Khanyile, T

    2013-09-01

    Full Text Available on graphene coated glass resulted in an improvement in optical transfection efficiencies. Future perspectives involve the testing of cytokines, growth factors and integrins to investigate the stimulation of extracellular matrix through graphene...

  3. Biocompatible polymer enhances the in vitro and in vivo transfection efficiency of HVJ envelope vector.

    Science.gov (United States)

    Mima, Hidetoshi; Tomoshige, Ryuji; Kanamori, Toshihide; Tabata, Yasuhiko; Yamamoto, Seiji; Ito, Susumu; Tamai, Katsuto; Kaneda, Yasufumi

    2005-07-01

    Vector development is critical for the advancement of human gene therapy. However, the use of viral vectors raises many safety concerns and most non-viral methods are less efficient for gene transfer. One of the breakthroughs in vector technology is the combination of the vector with various polymers. HVJ (hemagglutinating virus of Japan) envelope vector (HVJ-E) has been developed as a versatile gene transfer vector. In this study, we combined HVJ-E with cationized gelatin to make it a more powerful tool and assessed its transfection efficiency in vitro and in vivo. In addition, we investigated the mechanism of the gene transfer by means of the inhibition of fusion or endocytosis. The combination of both protamine sulfate and cationized gelatin with HVJ-E, referred to as PS-CG-HVJ-E, further enhanced the in vitro transfection efficiency. In CT26 cells, the luciferase gene expression of PS-CG-HVJ-E was approximately 10 times higher than that of the combination of protamine sulfate with HVJ-E or the combination of cationized gelatin with HVJ-E, referred to as PS-HVJ-E or CG-HVJ-E, respectively. Furthermore, the luciferase gene expression in liver mediated by intravenous administration of CG-HVJ-E was much higher than the luciferase gene expression mediated by PS-HVJ-E or PS-CG-HVJ-E and approximately 100 times higher than that mediated by HVJ-E alone. Cationized gelatin-conjugated HVJ-E enhanced gene transfection efficiency both in vitro and in vivo. These results suggest that low molecular weight cationized gelatin may be appropriate for complex formation with various envelope viruses, such as retrovirus, herpes virus and HIV. Copyright 2005 John Wiley & Sons, Ltd.

  4. Enhanced Nanomagnetic Gene Transfection of Human Prenatal Cardiac Progenitor Cells and Adult Cardiomyocytes

    Science.gov (United States)

    Subramanian, Mahendran; Lim, Jenson; Dobson, Jon

    2013-01-01

    Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells. PMID:23936108

  5. Factors influencing the transfection efficiency of ultra low molecular weight chitosan/hyaluronic acid nanoparticles.

    Science.gov (United States)

    Duceppe, Nicolas; Tabrizian, Maryam

    2009-05-01

    The present work describes nanoparticles made of ultra low molecular weight chitosan (ULMWCh)/hyaluronic acid (HA) as novel potential carriers for gene delivery. Small and monodispersed nanoparticles with high in vitro transfection capabilities have been obtained by the complexation of these two polyelectrolytes. ULMWCh (over the higher molecular weight chitosan for clinical applications, namely increased solubility at physiological pH and improved DNA release. The ULMWCh:HA ratio and the HA molecular weights were varied with the aim of obtaining particles in the 100 nm range. Using chitosan (Ch) with a molecular weight of 5 kDa, HA with a molecular weight of 64 kDa, and a weight ratio of 4:1, nanoparticles with a Z-average size of 146+/-1 nm and narrow size distribution (polydispersity index: 0.073+/-0.030) were obtained. Nanoparticle images taken in dry conditions by SEM and AFM showed spherical particles. The optimal pH for transfection ranged from 6.4 to 6.8 for 0.25 microg of EGFP plasmid per well, with an incubation time of 4 h. Using these optimized parameters, DNA/ULMWCh:HA nanoparticles successfully transfected 25+/-1% of the 293T cells with pEGFP, compared to 0.7% obtained for DNA/ULMWCh under the same conditions. This high transfection efficiency of our non-viral gene delivery system could be attributed to the synergic effect of ULMWCh and low charge density of the HA chain for easy release of DNA which makes the system suitable for targeted gene delivery.

  6. Polyurethane dispersion containing quaternized ammonium groups: An efficient nanosize gene delivery carrier for A549 cancer cell line transfection.

    Science.gov (United States)

    Yousefpour Marzbali, Mahsa; Yari Khosroushahi, Ahmad; Movassaghpour, AliAkbar; Yeganeh, Hamid

    2016-01-25

    A novel polyurethane containing cationic ammonium groups (QPU) was synthesized and used as vector for gene therapy and cancer gene targeting. The synthesized QPU was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy methods. An agarose gel retardation electrophoresis assay was conducted to verify the complete complex formation between QPU and pDNA. The particles size and zeta potential of neat polymers, plasmid DNA, polymers/DNA polyplexes were determined by the dynamic light scattering technique. The polyplexes cytotoxicity was determined using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and its transfection efficiency was examined qualitatively by fluorescent microscopy and quantitatively by flow cytometery methods. The gel retardation assay, particle size and zeta potential measurements were confirmed that the synthesized cationic polymer could condense DNA efficiently in the physiologic condition. QPU polyplexes showed a significantly lower cytotoxicity compared to Polyfect polyplexes in the examined human cancerous (A549) or normal cells (KDR). Based on our findings, the transfection efficiency by QPU was 2.2 fold higher than Polyfect in the A549 cells whereas in the KDR cells, the cell transfection by Polyfect was 18.1 fold higher than QPU. Due to low cytotoxicity for normal cells and high transfection efficiency in cancer cells, the potential applicability of designed QPU as a non-viral gene carrier for targeting of cancer gene therapy was confirmed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. Influence of lipid components on gene delivery by polycation liposomes: Transfection efficiency, intracellular kinetics and in vivo tumor inhibition.

    Science.gov (United States)

    Chen, Jinliang; Sun, Xiaoyi; Yu, Zhenwei; Gao, Jianqing; Liang, Wenquan

    2012-01-17

    Transfection efficiency of non-viral gene vectors is influenced by many factors, including chemical makeup, cellular uptake pathway and intracellular delivery. To investigate the effect of lipid saturation on transfection efficiency of polycation liposomes (PCLs), a soybean phospholipids (SPL), egg phospholipids (EPL) and hydrogenated soybean phosphatidylcholine (HSPC) series was used to prepare PCLs. Testing these PCLs in a luciferase assay indicated that with increasing saturation (SPLexpression decreased. The effect of protamine combined with these PCLs was also studied in different cell lines. Improved transfection because of protamine incorporation was dependent on lipid saturation and on the cell line tested. The kinetics of cellular uptake and intracellular distribution was studied using flow cytometry and laser scanning confocal microscope, which showed that naked oligonucleotide (ODN) and PCLs/ODN complexes became equilibrium after 4h incubation. PCLs containing SPL (PCLs-S) and 1,2-dieleoyl-sn-glycero-3-phosphoethanolamine (PCLs-D) increased uptake rates by 2.20- and 5.45-fold, respectively. Furthermore, pCMV-IL-12 transfection mediated by PCLs-D showed excellent tumor inhibition efficiency compared with control and naked pCMV-IL-12 treatments in vivo. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Controlling DNA compaction with cationic amphiphiles for efficient delivery systems A step forward towards non-viral Gene Therapy

    Science.gov (United States)

    Savarala, Sushma

    The synthesis of pyridinium cationic lipids, their counter-ion exchange, and the transfection of lipoplexes consisting of these lipids with firefly luciferase plasmid DNA (6.7 KDa), into lung, prostate and breast cancer cell lines was investigated. The transfection ability of these newly synthesized compounds was found to be twice as high as DOTAP/cholesterol and Lipofectamine TM (two commercially available successful transfection agents). The compaction of the DNA onto silica (SiO2) nanoparticles was also investigated. For this purpose, it was necessary to study the stability and fusion studies of colloidal systems composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), a zwitterionic lipid, and mixtures of DMPC with cationic DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane).

  9. Hyperbranched-dendrimer architectural copolymer gene delivery using hyperbranched PEI conjugated to poly(propyleneimine) dendrimers: synthesis, characterization, and evaluation of transfection efficiency

    Science.gov (United States)

    Alavi, Seyyed Jamal; Gholami, Leila; Askarian, Saeedeh; Darroudi, Majid; Massoudi, Abdolhossein; Rezaee, Mehdi; Kazemi Oskuee, Reza

    2017-02-01

    The applications of dendrimer-based vectors seem to be promising in non-viral gene delivery because of their potential for addressing the problems with viral vectors. In this study, generation 3 poly(propyleneimine) (G3-PPI) dendrimers with 1, 4-diaminobutane as a core initiator was synthesized using a divergent growth approach. To increase the hydrophobicity and reduce toxicity, 10% of primary amines of G3-PPI dendrimers were replaced with bromoalkylcarboxylates with different chain lengths (6-bromohexanoic and 10-bromodecanoic). Then, to retain the overall buffering capacity and enhance transfection, the alkylcarboxylate-PPIs were conjugated to 10 kDa branched polyethylenimine (PEI). The results showed that the modified PPI was able to form complexes with the diameter of less than 60 nm with net-positive surface charge around 20 mV. No significant toxicity was observed in modified PPIs; however, the hexanoate conjugated PPI-PEI (PPI-HEX-10% PEI) and the decanoate conjugated PPI-PEI (PPI-DEC-10%-PEI) showed the best transfection efficiency in murine neuroblastoma (Neuro-2a) cell line, even PPI-HEX-10%-PEI showed transfection efficiency equal to standard PEI 25 kDa with reduced toxicity. This study suggested a new series of hyperbranched (PEI)-dendrimer (PPI) architectural copolymers as non-viral gene delivery vectors with high transfection efficiency and low toxicity.

  10. Hyperbranched–dendrimer architectural copolymer gene delivery using hyperbranched PEI conjugated to poly(propyleneimine) dendrimers: synthesis, characterization, and evaluation of transfection efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Alavi, Seyyed Jamal [Ferdowsi University of Mashhad, Department of Chemistry, Faculty of Science (Iran, Islamic Republic of); Gholami, Leila [Mashhad University of Medical Sciences, Department of Modern Sciences and Technologies, School of Medicine (Iran, Islamic Republic of); Askarian, Saeedeh [Mashhad University of Medical Sciences, Department of Medical Biotechnology, School of Medicine (Iran, Islamic Republic of); Darroudi, Majid [Mashhad University of Medical Sciences, Nuclear Medicine Research Center (Iran, Islamic Republic of); Massoudi, Abdolhossein [University of Payam noor, Department of Chemistry (Iran, Islamic Republic of); Rezaee, Mehdi; Kazemi Oskuee, Reza, E-mail: Oskueekr@mums.ac.ir [Mashhad University of Medical Sciences, Department of Medical Biotechnology, School of Medicine (Iran, Islamic Republic of)

    2017-02-15

    The applications of dendrimer-based vectors seem to be promising in non-viral gene delivery because of their potential for addressing the problems with viral vectors. In this study, generation 3 poly(propyleneimine) (G3-PPI) dendrimers with 1, 4-diaminobutane as a core initiator was synthesized using a divergent growth approach. To increase the hydrophobicity and reduce toxicity, 10% of primary amines of G3-PPI dendrimers were replaced with bromoalkylcarboxylates with different chain lengths (6-bromohexanoic and 10-bromodecanoic). Then, to retain the overall buffering capacity and enhance transfection, the alkylcarboxylate–PPIs were conjugated to 10 kDa branched polyethylenimine (PEI). The results showed that the modified PPI was able to form complexes with the diameter of less than 60 nm with net-positive surface charge around 20 mV. No significant toxicity was observed in modified PPIs; however, the hexanoate conjugated PPI–PEI (PPI-HEX-10% PEI) and the decanoate conjugated PPI–PEI (PPI-DEC-10%-PEI) showed the best transfection efficiency in murine neuroblastoma (Neuro-2a) cell line, even PPI-HEX-10%-PEI showed transfection efficiency equal to standard PEI 25 kDa with reduced toxicity. This study suggested a new series of hyperbranched (PEI)–dendrimer (PPI) architectural copolymers as non-viral gene delivery vectors with high transfection efficiency and low toxicity.

  11. D-Glucosamine Promotes Transfection Efficiency during Electroporation

    Directory of Open Access Journals (Sweden)

    Kazunari Igawa

    2014-01-01

    Full Text Available D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP in the cultured cells (osteoblasts; NOS-1 cells. The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

  12. [Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells].

    Science.gov (United States)

    Li, Ji; Liu, Zuojin

    2015-12-01

    To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0×10(8) TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells. Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (Ptransfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.

  13. A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines.

    Science.gov (United States)

    Figueroa, Elizabeth; Bugga, Pallavi; Asthana, Vishwaratn; Chen, Allen L; Stephen Yan, J; Evans, Emily Reiser; Drezek, Rebekah A

    2017-05-02

    Gold-polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. However, we have observed that certain cell types are more amenable to Au-PAMAM transfection than others. Here we utilized two representative cell lines-a "difficult to transfect" CT26 cell line and an "easy to transfect" SK-BR3 cell line-and attempted to determine the underlying mechanism for differential transfection in both cell types. Using a commonly established poly-cationic polymer similar to PAMAM (polyethyleneimine, or PEI), we additionally sought to quantify the relative transfection efficiencies of each vector in CT26 and SK-BR3 cells, in the hopes of elucidating any mechanistic differences that may exist between the two transfection vectors. A comparative time course analysis of green fluorescent protein reporter-gene expression and DNA uptake was conducted to quantitatively compare PEI- and AuPAMAM-mediated transfection in CT26 and SK-BR3, while flow cytometry and confocal microscopy were used to determine the contribution of cellular uptake, endosomal escape, and cytoplasmic transport to the overall gene delivery process. Results from the time course analysis and flow cytometry studies revealed that initial complex uptake and cytoplasmic trafficking to the nucleus are likely the two main factors limiting CT26 transfectability. The cell type-dependent uptake and intracellular transport mechanisms impacting gene therapy remain largely unexplored and present a major hurdle in the application-specific design and efficiency of gene delivery vectors. This systematic investigation offers insights into the intracellular mechanistic processes that may account for cell-to-cell differences, as well as vector-to-vector differences, in gene transfectability.

  14. Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

    Directory of Open Access Journals (Sweden)

    Escriou Virginie

    2003-09-01

    Full Text Available Abstract Background Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. Results We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl-6-maleimidyl hexanamide (TFPAM-6. When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. Conclusions The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

  15. Disulfide-bond-containing agamatine-cystaminebisacrylamide polymer demonstrates better transfection efficiency and lower cytotoxicity than polyethylenimine in NIH/3T3 cells.

    Science.gov (United States)

    Yang, Zhen; Sun, Yanping; Xian, Lei; Xun, Zhe; Yu, Jiankun; Yang, Tianzhi; Zhao, Xiaoyun; Cai, Cuifang; Wang, Dongkai; Ding, Pingtian

    2017-08-10

    Previously, we synthesized a non-viral vector containing disulfide bond by polymerization of agamatine (AGM) and N,N'-cystaminebisacrylamide (CBA). In this study, we investigated the transfection efficiency of disulfide bond (SS) containing AGM-CBA polymer in gene delivery into NIH/3T3 cells, and examined the factors affecting its transfection efficiency by comparing with polyethylenimine (PEI). In addition, experiments were carried out to determine the mechanisms of cell entry pathways and intracellular behavior of AGM-CBA/pDNA polyplexes. The transfection efficiency of AGM-CBA/pDNA with different weight ratios and different amounts of pDNA was measured and the pathways mediated transfection processes were studied by using various endocytosis inhibitors. To determine the intracellular behavior of AGM-CBA/pDNA polyplexes, the transfection efficiencies of AGM-CBA/pDNA and PEI/pDNA polyplexes with different combination structures were determined by using reporter gene and fake plasmid DNA. The transfection efficiency of AGM-CBA/pDNA polyplexes was correlated with its weight ratio of AGM-CBA and pDNA, and the amount of pDNA. Both AGM-CBA/pDNA and PEI/pDNA polyplexes enter into cell by clathrin- and caveolae-mediated endocytic pathways. However, AGM-CBA/pDNA showed different intracellular behavior in NIH/3T3 cells compared to PEI/pDNA polyplexes. It was hypothesized that disulfide bond in AGM-CBA could be an important factor contributing to its intracellular behavior and better transfection efficiency. Overall, AGM-CBA demonstrated better transfection efficiency and lower cytotoxicity than PEI in NIH/3T3 cells as a gene delivery vector. © 2017 Wiley Periodicals, Inc.

  16. A Biodegradable Polyethylenimine-Based Vector Modified by Trifunctional Peptide R18 for Enhancing Gene Transfection Efficiency In Vivo.

    Directory of Open Access Journals (Sweden)

    Jing Hu

    Full Text Available Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI derivate was synthesized by crosslinking low-molecular-weight (LMW PEI with N-octyl-N-quaternary chitosan (OTMCS, and then adopting a designed trifunctional peptide (RGDC-TAT-NLS with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100-300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo.

  17. A Biodegradable Polyethylenimine-Based Vector Modified by Trifunctional Peptide R18 for Enhancing Gene Transfection Efficiency In Vivo.

    Science.gov (United States)

    Hu, Jing; Zhu, Manman; Liu, Kehai; Fan, Hua; Zhao, Wenfang; Mao, Yuan; Zhang, Yaguang

    2016-01-01

    Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI) derivate was synthesized by crosslinking low-molecular-weight (LMW) PEI with N-octyl-N-quaternary chitosan (OTMCS), and then adopting a designed trifunctional peptide (RGDC-TAT-NLS) with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100-300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo.

  18. Efficient gene transfer from innervated muscle into rat peripheral and central nervous systems using a non-viral haemagglutinating virus of Japan (HVJ)-liposome method.

    Science.gov (United States)

    Kato, Naoki; Nakanishi, Kuniaki; Nemoto, Koichi; Morishita, Ryuichi; Kaneda, Yasufumi; Uenoyama, Maki; Ikeda, Tomosumi; Fujikawa, Kyosuke

    2003-05-01

    We evaluated the feasibility of gene delivery into the peripheral and central nervous systems via retrograde axonal transport following injection of a haemagglutinating virus of Japan (HVJ)-liposome-DNA complex vector into an innervated muscle. Transfection efficiency was assessed by measuring luciferase activity, and was compared statistically with that achieved using a liposome-DNA control vector. High luciferase activity was observed in the injected muscle, the ipsilateral sciatic nerve, and the ipsilateral dorsal root ganglia on day 1 after gene transfer. The spinal cord also showed luciferase activity, although this was lower than in the other tissues. However, no activity was observed in the contralateral sciatic nerve or the contralateral dorsal root ganglia. In addition, we performed gene transfer twice, with a 1-week interval, to evaluate the feasibility of repeated therapeutic gene delivery. Again, a high transfection efficiency was observed immediately, even after the second gene transfer, and transfection efficiency was significantly higher at each defined time-point using the HVJ-liposome complex vector than using a control vector. These results indicate that this method could be used for repeated therapeutic gene delivery into muscle, nerve, dorsal root ganglia, and possibly spinal cord, without the need for a surgical approach, making it well suited to clinical applications.

  19. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    NARCIS (Netherlands)

    Sork, Helena; Nordin, Joel Z; Turunen, Janne J; Wiklander, Oscar Pb; Bestas, Burcu; Zaghloul, Eman M; Margus, Helerin; Padari, Kärt; Duru, Adil D; Corso, Giulia; Bost, Jeremy; Vader, Pieter; Pooga, Margus; Smith, Ci Edvard; Wood, Matthew Ja; Schiffelers, Raymond M; Hällbrink, Mattias; Andaloussi, Samir El

    2016-01-01

    The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of

  20. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    Science.gov (United States)

    2011-01-01

    Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet

  1. High-Efficient Transfection of Human Embryonic Stem Cells by Single-Cell Plating and Starvation.

    Science.gov (United States)

    Liu, Hui; Ren, Caiping; Zhu, Bin; Wang, Lei; Liu, Weidong; Shi, Jia; Lin, Jianxing; Xia, Xiaomeng; Zeng, Fei; Chen, Jiawen; Jiang, Xingjun

    2016-03-15

    Nowadays, the low efficiency of small interfering RNA (siRNA) or plasmid DNA (pDNA) transfection is a critical issue in genetic manipulation of human embryonic stem (hES) cells. Development of an efficient transfection method for delivery of siRNAs and plasmids into hES cells becomes more and more imperative. In this study, we tried to modify the traditional transfection protocol by introducing two crucial processes, single-cell plating and starvation, to increase the transfection efficiency in hES cells. Furthermore, we comparatively examined the transfection efficiency of some commercially available siRNA or pDNA transfection reagents in hES cells. Our results showed that the new developed method markedly enhanced the transfection efficiency without influencing the proliferation and pluripotency of hES cells. Lipofectamine RNAiMAX exhibited much higher siRNA transfection efficiency than the other reagents, and FuGENE HD was identified as the best suitable reagent for efficient pDNA transfection of hES cells among the tested reagents.

  2. The Effect of Environmental pH on Polymeric Transfection Efficiency

    Science.gov (United States)

    Kang, Han Chang; Samsonova, Olga; Kang, Sun-Woong; Bae, You Han

    2011-01-01

    Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6~7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1~2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers. PMID:22130563

  3. Use of Primary and Secondary Polyvinylamines for Efficient Gene Transfection.

    Science.gov (United States)

    Dréan, Mathilde; Debuigne, Antoine; Goncalves, Cristine; Jérôme, Christine; Midoux, Patrick; Rieger, Jutta; Guégan, Philippe

    2017-02-13

    Gene transfection with polymeric carrier remains a challenge; particularly, high transfection levels combined with low toxicity are hard to achieve. We herein revisit polyvinylamines, an old and neglected family of cationic polymers. They can be readily obtained by controlled hydrolysis of polyvinylamides prepared through (controlled) radical polymerization. A series of tailor-made and well-defined polyvinylamines bearing primary amino groups, and poly(N-methylvinylamine) bearing secondary amines, were evaluated for the transfection of cells with pDNA as a function of their molar mass, molar mass distribution, and degree of deacetylation. Unexpected high transfection levels, in combination with low cytotoxicity were recorded for both series. Surprisingly, a great impact of the molar mass was observed for the primary amine polyvinylamine series, whereas the results were mostly independent of molar mass or dispersity for the polymer bearing secondary amine. It was further established that a certain percentage of acetamide groups increased the transfection level, while maintaining low cytotoxicity. These results highlight for the first time the real potential of polyvinylamines as gene carriers, and make these polymers very attractive for further development in gene therapy.

  4. Epizone: Interlaboratory Ring Trial to Compare Dna Transfection Efficiencies

    DEFF Research Database (Denmark)

    Dory, Daniel; Albina, Emmanuel; Kwiatek, Olivier

    Chemical-based transfection of DNA into cultured cells is routinely used to study for example viral or cellular gene functions involved in virus replication, to analyse cellular defence mechanisms or develop specific strategies to interfere with virus replication. Other applications include rescu...

  5. A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol-block-poly(disulfide histamine Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Kangkang An

    2014-05-01

    Full Text Available A novel 4-arm poly(ethylene glycol-b-poly(disulfide histamine copolymer was synthesized by Michael addition reaction of poly(ethylene glycol (PEG vinyl sulfone and amine-capped poly(disulfide histamine oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter with almost neutral surface charge (+(5–10 mV. Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment, but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment. The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment. In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0. Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.

  6. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

    Science.gov (United States)

    Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M.; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean

    2017-01-01

    Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. PMID:28863132

  7. Enhanced Non-Viral Gene Delivery to Human Embryonic Stem Cells via Small Molecule-Mediated Transient Alteration of Cell Structure.

    Science.gov (United States)

    Yen, Jonathan; Yin, Lichen; Cheng, Jianjun

    Non-viral gene delivery into human embryonic stem cells (hESCs)is an important tool for controlling cell fate. However, the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. We herein report a novel approach to enhance non-viral gene delivery to hESCs by transiently altering the cell and colony structure. (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632), a small molecule that inhibits the rho-associated protein kinase pathway, is utilized to induce transient colony spreading which leads to increased transfection efficiency by 1.5 to 2 folds in a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y-27632 post-transfection, cells can revert back to its normal state and do not show alteration of pluripotency. This approach provides a simple, effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation.

  8. Solid lipid nanoparticles mediate non-viral delivery of plasmid DNA to dendritic cells

    Science.gov (United States)

    Penumarthi, Alekhya; Parashar, Deepti; Abraham, Amanda N.; Dekiwadia, Chaitali; Macreadie, Ian; Shukla, Ravi; Smooker, Peter M.

    2017-06-01

    There is an increasing demand for novel DNA vaccine delivery systems, mainly for the non-viral type as they are considered relatively safe. Therefore, solid lipid nanoparticles (SLNs) were investigated for their suitability as a non-viral DNA vaccine delivery system. SLNs were synthesised by a modified solvent-emulsification method in order to study their potential to conjugate with plasmid DNA and deliver them in vitro to dendritic cells using eGFP as the reporter plasmid. The DNA-SLN complexes were characterised by electron microscopy, gel retardation assays and dynamic light scattering. The cytotoxicity assay data supported their biocompatibility and was used to estimate safe threshold concentration resulting in high transfection rate. The transfection efficiency of these complexes in a dendritic cell line was shown to increase significantly compared to plasmid alone, and was comparable to that mediated by lipofectamine. Transmission electron microscopy studies delineated the pathway of cellular uptake. Endosomal escape was observed supporting the mechanism of transfection.

  9. The mechanism of high transfection efficiency of human serum albumin conjugated polyethylenimine in A549 cells

    Directory of Open Access Journals (Sweden)

    Ssu-Wei Peng

    2015-01-01

    Full Text Available Background: In our previous study, HSA-PEI demonstrated high pDNA transfection efficiency and low cytotoxicity. Materials and Methods: In this study, the relationship between albumin receptors and the high pDNA transfection efficiency of HSA-PEI was investigated by fluorescent microscopy and flow-cytometer in A549 cells. Results: According to our results, the presence of an albumin receptor on A549 cells was confirmed via the application of a fluorescent tracer, FITC-HSA, and the dose-dependent competition of HSA. The transfection efficiency of HSA-PEI/pEGFP showed a dose-dependent inhibition when different amounts of HSA were added to the culture medium of the A549 cells. However, the inhibitory effect of HSA did not affect the transfection efficiency of some cationic transfection enhancement reagents, such as lipofectamine, jetPEI-RGD or PEI; their transfection was a result of contact between the positively charged reagents and the negatively charged cell surface. Conclusion: It was determined that, unlike other cationic reagents, the high transfection efficiency of HSA-PEI was not from the electronic effect but, instead, predominantly from its ligand effect.

  10. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  11. Non-viral gene therapy that targets motor neurons in vivo

    Directory of Open Access Journals (Sweden)

    Mary-Louise eRogers

    2014-10-01

    Full Text Available A major challenge in neurological gene therapy is safe delivery of transgenes to sufficient cell numbers from the circulation or periphery. This is particularly difficult for diseases involving spinal cord motor neurons such as amyotrophic lateral sclerosis (ALS. We have examined the feasibility of non-viral gene delivery to spinal motor neurons from intraperitoneal injections of plasmids carried by ‘immunogene’ nanoparticles targeted for axonal retrograde transport using antibodies. PEGylated polyethylenimine (PEI-PEG12 as DNA carrier was conjugated to an antibody (MLR2 to the neurotrophin receptor p75 (p75NTR. We used a plasmid (pVIVO2 designed for in vivo gene delivery that produces minimal immune responses, has improved nuclear entry into post mitotic cells and also expresses green fluorescent protein (GFP. MLR2-PEI-PEG12 carried pVIVO2 and was specific for mouse motor neurons in mixed cultures containing astrocytes. While only 8% of motor neurons expressed GFP 72 h post transfection in vitro, when the immunogene was given intraperitonealy to neonatal C57BL/6J mice GFP specific motor neuron expression was observed in 25.4% of lumbar, 18.3% of thoracic and 17.0 % of cervical motor neurons, 72 h post transfection. PEI-PEG12 carrying pVIVO2 by itself did not transfect motor neurons in vivo, demonstrating the need for specificity via the p75NTR antibody MLR2. This is the first time that specific transfection of spinal motor neurons has been achieved from peripheral delivery of plasmid DNA as part of a non-viral gene delivery agent. These results stress the specificity and feasibility of immunogene delivery targeted for p75NTR expressing motor neurons, but suggests that further improvements are required to increase the transfection efficiency of motor neurons in vivo.

  12. Megalin-targeted enhanced transfection efficiency in cultured human HK-2 renal tubular proximal cells using aminoglycoside-carboxyalkyl- polyethylenimine -containing nanoplexes.

    Science.gov (United States)

    Oroojalian, Fatemeh; Rezayan, Ali Hossein; Shier, Wayne Thomas; Abnous, Khalil; Ramezani, Mohammad

    2017-05-15

    Non-viral vectors are of interest as therapeutic gene delivery agents in gene therapy, because they are simple to prepare, easy to modify and have definable safety profiles compared to viral vectors. The potential of gene therapy in the treatment of renal diseases is limited by a lack of effective kidney-targeted gene delivery systems. Aminoglycoside antibiotics gentamicin and neomycin were connected by amide linkages to carboxyl groups on carboxyalkylated-PEI25 (25kDa PEI) or carboxyalkylated-PEI10 (10kDa PEI). Aminoglycoside-carboxyalkylated-PEI conjugates were characterized with respect to size, surface charge density, DNA condensation ability, and buffering capacity. Polyplexes prepared by electrostatic interaction between aminoglycoside-carboxyalkylated-PEIs and enhanced green fluorescent protein-expressing (EGFP) plasmid DNA had appropriate nano-scale size (143-173nm). Their targeting potential was investigated in cultured HK-2 immortalized human cortex/proximal tubule kidney epithelial cells, which expresses megalin, a scavenger receptor that mediates endocytosis of a diverse group of ligands, including aminoglycoside antibiotics. Aminoglycoside-carboxyalkylated-PEIs significantly increased EGFP gene transfection efficiency in HK-2 cells by ∼13-fold for aminoglycoside-carboxyalkylated-PEI25 and ∼7-fold increase for aminoglycoside-carboxyalkylated-PEI10 relative to the corresponding PEIs without aminoglycosides. The transfection efficiency of polyplexes was dependent on the weight ratio of aminoglycoside-containing ligand in the carrier. In the presence of a range of concentrations of human serum albumin, which competes for megalin binding, aminoglycoside-carboxyalkylated-PEI-mediated transfection was reduced to background levels. These results suggest that aminoglycoside-carboxyalkylated-PEI polyplexes can target megalin-expressing kidney-derived cells in vitro resulting in improved transfection efficiency with low cytotoxicity. Copyright © 2017

  13. Acidity-responsive gene delivery for "superfast" nuclear translocation and transfection with high efficiency.

    Science.gov (United States)

    Zhu, Jing-Yi; Zeng, Xuan; Qin, Si-Yong; Wan, Shuang-Shuang; Jia, Hui-Zhen; Zhuo, Ren-Xi; Feng, Jun; Zhang, Xian-Zheng

    2016-03-01

    In principle, not only efficient but rapid transfection is required since it can maximize the bioavailability of vector-carried gene prior to the cellular excretion. However, the "rapid" goal has been paid few attentions so far in the research field of vector-aided transfection. As a pioneering attempt, the present study designed a lysosome-targeting acidity-responsive nanoassembly as gene vectors, which proved the amazing potency to mediate the "Superfast" transnuclear gene transport and gene transfection with high efficiency in vitro and in vivo. The nanoassembly was constructed on the pH-reversible covalent boronic acid-diol coupling between 1,3-diol-rich oligoethylenimine (OEI-EHDO) and phenylboronic acid modified cholesterol (Chol-PBA). The rapid and efficient nuclei-tropic delivery and transfection was demonstrated to highly rely on the lysosome-acidity induced assembly destruction followed by the easy liberation of gene payloads inside cells. The nanoassembly-mediated transfection at 8 h can afford the outcome even comparable to that achieved at 48 h by the golden standard of PEI25k, and the transfection efficiency can still remain at a high level during 48 h. In contrast, time-dependent efficiency enhancement was identified for the transfections using PEI25k and OEI-EHDO as delivery vectors. Moreover, owing to the hydroxyl-rich surface, this delivery nanosystem presented strong tolerance to the serum-induced transfection inhibition that frequently occurred for the polycationic gene vectors such as PEI25k. The in vitro and in vivo results manifested the low toxicity of this bio-decomposable nanoassembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis.

    Directory of Open Access Journals (Sweden)

    Huading Lu

    Full Text Available The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS with a low molecular weight (Mw PEI (Mw = 1.8 kDa. Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w. The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.

  15. Effect of Eudragit on In Vitro Transfection Efficiency of PEI-DNA Complexes.

    Science.gov (United States)

    Kanthamneni, Naveen; Yung, Bryant; Lee, Robert J

    2016-01-01

    Eudragit® E 100 (EE100) was used to improve the transfection efficiency of polyethylenimine (PEI). Mobility of PEI-DNA complexes with and without EE100 were visualized by agarose gel electrophoresis and their transfection efficiencies were investigated in KB human oral carcinoma cells by flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of transfected cells. Gel electrophoresis illustrated formation of complete complexes at N/P ratios above 5. PEI had the highest transfection efficiency at an N/P ratio of 15, whereas in combination with EE100, the transfection efficiency was highest at an N/P ratio of 7.5. High concentrations of EE100 in combination with PEI were found to reduce cell viability. The results show a synergistic action of EE100 in transfection of DNA at low N/P ratios compared to PEI alone. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  16. Lipofectamine RNAiMAX: an efficient siRNA transfection reagent in human embryonic stem cells.

    Science.gov (United States)

    Zhao, Ming; Yang, Hong; Jiang, Xingjun; Zhou, Wen; Zhu, Bin; Zeng, Ying; Yao, Kaitai; Ren, Caiping

    2008-09-01

    RNA interference methodology suppresses specific gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. Lipofectamine RNAiMAX, a new transfection reagent, has been confirmed high efficiency in delivering small interfering RNA (siRNA) into mesenchymal stem cells and neural stem cells. In this study, we used three transfection reagents (Lipofectamine RNAiMAX, Oligofectamine and Lipofectamine 2000) to deliver siRNA into human embryonic stem (hES) cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and Oct4, a key regulator of pluripotency. As a result, siRNA can be delivered into hES cells more efficiently by Lipofectamine RNAiMAX compared with the other two transfection reagents and high efficient knockdown of target genes was obtained by Lipofectamine RNAiMAX even at a low concentration of siRNA. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells.

  17. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    to be valuable in studying the evolution of enzymes. Some of these newly discovered enzymes have been useful in numerous practical applications in medicine and biotechnology, and have contributed to our understanding of the structural basis of nucleoside and nucleoside analogue activation....... of great medical interest. However, during the last 20 years, research on dNKs has gone into non-mammalian organisms. In this review, we focus on non-viral dNKs, in particular their diversity and their practical applications. The diversity of this enzyme family in different organisms has proven...

  18. HVJ-based non-viral gene transfer method: successful gene therapy using HGF and VEGF genes in experimental ischemia.

    Science.gov (United States)

    Shimamura, Munehisa; Sato, Naoyuki; Yoshimura, Shinichi; Kaneda, Yasufumi; Morishita, Ryuichi

    2006-01-01

    VEGF and HGF are pleiotropic factors that regulate cell growth, cell motility, and morphogenesis of various types of cells. The receptors of these growth factors are expressed in neurons and endothelial cells, and are identified as neurotrophic, neuroprotective, and angiogenic factors. Indeed, gene therapy using viral vectors encoding the VEGF or HGF gene has been reported to be effective for preventing the expansion of ischemic injury. However, the safety issue of viral vectors is a major problem in clinical application. To overcome this problem, we have developed an HVJ-based non-viral vector, which achieves high-efficiency transfection rates of viral vectors with the safety of liposomes. This review discusses the feasibility of gene therapy using an HVJ-based non-viral vector containing the VEGF or HGF gene for cerebral ischemia.

  19. Gene therapy for haemophilia...yes, but...with non-viral vectors?

    Science.gov (United States)

    Liras, A; Olmedillas, S

    2009-05-01

    High-purity plasma-derived and recombinant factors are currently safe and efficient treatment for haemophilia. The mid-term future of haemophilia treatment will involve the use of modified recombinant factors to achieve advantages such as decreased immunogenicity in inhibitor formation and enhanced efficacy as a result of their longer half-life. In the long-term, gene therapy and cell therapy strategies will have to be considered. Achievements in cell therapy to date have been using embryonic stem cells and hepatic sinusoidal endothelial cells. Current gene therapy strategies for haemophilia are based on gene transfer using adeno-associated viruses and non-viral vectors. Gene therapy for haemophilia is justified because it is a chronic disease and because a very regular factor infusion is required that may involve fatal risks and because it is very expensive. Haemophilia is a very good candidate for use of gene therapy protocols because it is a monogenic disease, and even low expression is able to achieve reversion from a severe to a moderate phenotype. The current trends in haemophilia using adeno-associated viral vectors are safe but also involve immunogenicity problems. The other alternatives are non-viral vectors. There have been in recent years relevant advances in non-viral transfection that raise hope for considering this possibility. Several research groups are opting for this experimental alternative. An expression over 5%, representing a moderate phenotype, for a few months with a high safety, regarding vector, transfected cells, and implantation procedure, would already be a great success. This may represent an intermediate protocol in which the expression levels and times obtained are lower and shorter respectively as compared to viral vectors, but which provide a potential greater patient safety. This may more readily win acceptance among both patients and haematologists because fatal events in the past due to HIV/HCV infection may constrain the

  20. Faktor Risiko Non Viral Pada Karsinoma Nasofaring

    Directory of Open Access Journals (Sweden)

    Sukri Rahman

    2015-09-01

    Full Text Available Abstrak           Latar belakang: Karsinoma nasofaring adalah tumor ganas epitel nasofaring yang sampai saat ini penyebabnya belum diketahui, infeksi virus Epstein Barr dilaporkan sebagai faktor dominan terjadinya karsinoma nasofaring tetapi faktor non viral juga berperan untuk timbulnya keganasan nasofaring. Tujuan: Untuk mengetahui faktor non viral  yang dapat meningkatkan kejadian karsinoma nasofaring sehingga dapat mencegah dan menghindari faktor-faktor non viral tersebut. Tinjauan Pustaka: Karsinoma nasofaring merupakan tumor ganas epitel nasofaring yang penyebabnya berhubungan dengan faktor viral dan non viral diantaranya asap rokok, ikan asin, formaldehid, genetik, asap kayu bakar , debu kayu, infeksi kronik telinga hidung tenggorok, alkohol dan obat tradisional. Kesimpulan: Pembuktian secara klinis dan ilmiah terhadap faktor non viral sebagai penyebab timbulnya karsinoma nasofaring masih belum dapat dijelaskan secara pasti. Faktor non viral merupakan salah satu faktor risiko yang dapat meningkatkan angka kejadian timbulnya keganasan nasofaring Kata kunci: karsinoma nasofaring, faktor risiko, non viral AbstractBackground: Nasopharyngeal carcinoma is a malignant epithelial nasopharyngeal tumor that until now the cause still unknown, Epstein barr virus infection had reported as predominant occurance of nasopharyngeal carcinoma but non viral factors may also contribute to the onset of the incidence of nasopharyngeal malignancy. Purpose: To find non viral factors that may increase the incidence of nasopharyngel carcinoma in order to prevent and avoid non-viral factors Literature: Nasopharyngeal carcinoma is a malignant tumor that causes nasopharyngeal epithelium associated with viral and non-viral factors such as cigarette smoke, salt fish, formaldehyde, genetic, wood smoke ,wood dust, ear nose throat chronic infections, alcohol, and traditional medicine. Conclusion: Clinically and scientifically proving the non-viral factors as

  1. PEI-alginate nanocomposites as efficient in vitro gene transfection agents.

    Science.gov (United States)

    Patnaik, Soma; Aggarwal, Anita; Nimesh, Surendra; Goel, Anita; Ganguli, Munia; Saini, Neeru; Singh, Y; Gupta, K C

    2006-09-12

    The positive charge on PEI was partially shielded by forming ionic nanocomposites with a polysaccharide, alginic acid, in aqueous solution, bypassing tedious chemical synthesis. The content of alginic acid was varied systematically to obtain a series of nanocomposites. The nanocomposites were first characterized by assessing the surface charge (zeta potential), size (DLS) and morphology (AFM) followed by evaluation for their DNA interaction ability, cytotoxicity and transfection efficiency on various cell lines. The transfection efficiency of PEI-alginate (6.26%) nanocomposites improved dramatically (2-16-fold over native PEI) in all the cell lines studied. However, a decrease in transfection efficiency was observed on deviating from this optimal concentration of alginic acid in nanocomposites. Cytotoxicity of PEI-alginate/DNA complexes was nearly abolished on increasing the concentration of alginic acid in nanocomposites. PEI-alginate (6.26%) nanocomposites also delivered SiRNAs efficiently into mammalian cells, resulting in 80% suppression of GFP expression. The cellular uptake and endosomal escape of PEI-alginate nanocomposites and PEI were found to follow a similar route when transfection was carried out in presence of chloroquine, bafilomycin A1, cytochalasin B and methyl-beta-cyclodextrin. The results demonstrate a versatile vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids.

  2. Enhanced transfection efficiency and reduced cytotoxicity of novel lipid-polymer hybrid nanoplexes

    DEFF Research Database (Denmark)

    Jain, Sanyog; Kumar, Sandeep; Agrawal, Ashish Kumar

    2013-01-01

    The present study reports the development, characterization, and evaluation of novel polyelectrolytes stabilized lipoplexes as a nonviral vector for gene delivery. In order to achieve the advantage of both DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and PEI (high transfection efficiency...

  3. A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses.

    Science.gov (United States)

    Rosa, Filipa; Sales, Kevin C; Cunha, Bernardo R; Couto, Andreia; Lopes, Marta B; Calado, Cecília R C

    2015-10-01

    Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R (2) ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R (2) = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.

  4. Sendai F/HN viroplexes for efficient transfection of leukemic T cells.

    Science.gov (United States)

    Kim, Jung Seok; Lee, Yeon Kyung; Jeong, Hwa Yeon; Kang, Seong Jae; Kim, Min Woo; Ryu, Seung Hyun; Kim, Hong Sung; Kim, Keun Sik; Kim, Dong-Eun; Park, Yong Serk

    2013-09-01

    Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells. The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a ζ-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR. The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA. This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines.

  5. Efficiency of two different transfection reagents for use with human NTERA2 cells.

    Science.gov (United States)

    Aluigi, M G; Hofreiter, S; Falugi, C; Pestarino, M; Candiani, S

    2007-01-01

    The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany) and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA). Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity.

  6. Efficiency of two different transfection reagents for use with human NTERA2 cells

    Directory of Open Access Journals (Sweden)

    MG Aluigi

    2009-08-01

    Full Text Available The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA. Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity.

  7. High-efficient nonviral transfection of the rat thyroid cell line FRTL-5.

    Science.gov (United States)

    Klagge, A; Krause, K; Müller, K; Haag, J; Fuhrer, D

    2010-11-01

    FRTL-5 cells are used in many laboratories as an in vitro system of thyroid follicular cells since they share many properties of human thyrocytes. However, the use of FRTL-5 cells for experimental modifications is limited by low transfection efficiencies of lipid-based transfections and the need for cumbersome viral transduction protocols. A new technology - nucleofection - has become available for cell lines that are difficult to transfect. Here, we report the application and optimization of this method in FRTL-5 cells. Using the green fluorescent protein (GFP) as a reporter gene, FRTL-5 cells were easily transfectable with efficiencies over 60%. In addition, the simultaneous transfer of siRNA against GFP was feasible and allowed suppression of GFP over at least 4 days. Furthermore nucleofection was successful for establishing stable FRTL-5 cell clones. In conclusion, this optimized fast and efficient nucleofection protocol offers new properties for the experimental use of FRTL-5 cells. © Georg Thieme Verlag KG Stuttgart · New York.

  8. Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging.

    Science.gov (United States)

    Horibe, Tomohisa; Torisawa, Aya; Akiyoshi, Ryutaro; Hatta-Ohashi, Yoko; Suzuki, Hirobumi; Kawakami, Koji

    2014-02-01

    The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Efficient Transfection of siRNA by Peptide Dendrimer-Lipid Conjugates.

    Science.gov (United States)

    Kwok, Albert; Eggimann, Gabriela A; Heitz, Marc; Reymond, Jean-Louis; Hollfelder, Florian; Darbre, Tamis

    2016-12-02

    Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target-gene-specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure-activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure-activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer-based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off-target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine-tune transfection and cell specificity in the future. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A simple and efficient method to transfect small interference RNA into bovine SCNT embryos.

    Science.gov (United States)

    Zhang, Hui; Wang, LiJun; Li, WenZhe; Mao, QingFu; Wang, YongSheng; Li, Qian; Hua, Song; Zhang, Yong

    2015-10-01

    RNA interference is an important tool to study the gene function. Microinjection and electroporation are usually used to transfer DNA, small interference RNA (siRNA), morpholinos, and protein into oocytes or embryos. This study used a simple and effective method to transfect siRNA into bovine somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of platinum microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted DNA methyltransferase-1 (DNMT1) was chosen to verify the siRNA transfection efficiency of this approach. First, a suitable concentration of siRNA was mixed with Zimmermann's fusion medium. Reconstructed embryos were then added into the microdrops of the mixed fusion medium to simultaneously transfect the siRNA and electrofuse the SCNT embryos. Our results showed that transfecting DNMT1 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of DNMT1 messenger RNA, DNMT1 protein, and global DNA methylation levels in the SCNT embryos. Meanwhile, the survival rate after electrofusion (90.4% vs. 89.4% vs. 89.1%, P > 0.05) and developmental rates of the SCNT embryos (72.8% vs. 74.9% vs. 72.4%, P > 0.05; 29.7% vs. 31.7% vs. 29.7%, P > 0.05) were not significantly affected. In summary, siRNAs were effectively transfected into the SCNT embryos via the proposed method and exert their functions, and the normal development of preimplantation SCNT embryos was not affected by the method used. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. DOTAP/DOPE ratio and cell type determine transfection efficiency with DOTAP-liposomes.

    Science.gov (United States)

    Kim, Bieong-Kil; Hwang, Guen-Bae; Seu, Young-Bae; Choi, Jong-Soo; Jin, Kyeong Sik; Doh, Kyung-Oh

    2015-10-01

    The effects of lipid compositions on their physicochemical properties and transfection efficiencies were investigated. Four liposome formulations with different 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) to dioleoylphosphatidylethanolamine (DOPE) weight ratios were investigated, that is, weight ratios 1:0 (T1P0), 3:1 (T3P1), 1:1 (T1P1), and 1:3 (T1P3). Mean sizes of liposomes were influenced by their lipid composition and the preparation concentration at the time of sonication. Zeta potentials of liposomes were inversely correlated with their liposome sizes. However, neither liposome sizes nor zeta potentials were correlated with transfection efficiency. The optimum composition of liposomes was cell-line dependent (T1P0 and T3P1 for Huh7 and AGS, T3P1 and T1P1 for COS7, and T1P1 and T1P3 for A549). The shape of lipoplexes was changed from lamellar to inverted hexagonal structure according to the increased ratio of DOPE, but there was no definite advantage of specific structure in transfection efficiency throughout all used cell lines. However, cellular internalization was consistently faster in T1P0, T3P1, T1P1 compared to T1P3 in all cell lines, suggesting the importance of endosomal escape. Our findings show that the transfection efficiency of DOTAP liposomes is mainly influenced by lipid composition and cell type, and not by size or zeta potential. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Effect of N-pyridinium positions of quaternized chitosan on transfection efficiency in gene delivery system.

    Science.gov (United States)

    Sajomsang, Warayuth; Gonil, Pattarapond; Ruktanonchai, Uracha Rungsardthong; Petchsangsai, Maleenart; Opanasopit, Praneet; Puttipipatkhachorn, Satit

    2014-04-15

    Methylated N-pyridylmethyl chitosan chlorides (M-PyMeChCs) with a similar total degree of quaternization (DQT) and molecular weight but different N-pyridinium positions were synthesized by reductive amination and methylation, respectively. The effect of N-pyridinium positions on transfection efficiency and cytotoxicity was investigated in human hepatoma (Huh7) cell lines. The results revealed that M-PyMeChCs are able to form a complete complex formation with DNA since there is an N/P ratio of 5. The particle sizes of M-PyMeChCs/DNA nanopolyplexes were approximately 300 nm and indicated a positive charge. The morphology of these nanopolyplexes was found to be in a spherical shape which was investigated by using the transmission electron microscopy (TEM). The M4-PyMeChC/DNA nanopolyplexes showed highest in vitro transfection efficiency in Huh7 cells at N/P ratio of 20 compared to M2-PyMeChC/DNA and M3-PyMeChC/DNA nanopolyplexes. In comparison to M3-PyMeChC/DNA nanopolyplexes, M2-PyMeChC/DNA and M4-PyMeChC/DNA nanopolyplexes showed lower cytotoxicity in Huh7 cells. Our result demonstrated that N-pyridinium positions of M-PyMeChCs are related to transfection efficiency and cytotoxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Enhanced transfection efficiency and reduced cytotoxicity of novel lipid-polymer hybrid nanoplexes.

    Science.gov (United States)

    Jain, Sanyog; Kumar, Sandeep; Agrawal, Ashish Kumar; Thanki, Kaushik; Banerjee, Uttam Chand

    2013-06-03

    The present study reports the development, characterization, and evaluation of novel polyelectrolytes stabilized lipoplexes as a nonviral vector for gene delivery. In order to achieve the advantage of both DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and PEI (high transfection efficiency) a system was hypothesized in which DOTAP/phosphatidyl choline (PC) lipoplexes were electrostatically coated with anionic poly(acrylic acid) (PAA) and cationic polyethylenimine (PEI) alternatively to finally shape a robust structure PEI-PAA-DOTAP/PC-lipoplexes (nanoplexes). The nanoplexes were found to have size of 242.6 ± 9.4 nm and zeta potential of +23.1 ± 1.5 mV. Following development nanoplexes were evaluated for cellular uptake, nuclear colocalization, transfection efficiency, and cellular toxicity in MCF-7, HeLa, and HEK-293 cell lines. In support of our hypothesis nanoplexes exhibited higher uptake and nuclear colocalization in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, and PEI polyplexes. Nanoplexes also exhibited 50-80, 11-12, 6-7, and 5-6 fold higher transfection efficiency in comparison with DOTAP/PC-lipoplexes, DOTAP/DOPE-lipoplexes, PEI-polyplexes, and lipofectamine, respectively, and significantly lower toxicity in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, PEI polyplexes, and commercial lipofectamine.

  14. Gene delivery to carcinoma cells via novel non-viral vectors: nanoparticle tracking analysis and suicide gene therapy.

    Science.gov (United States)

    Gebremedhin, Senait; Singh, Aruna; Koons, Stephen; Bernt, William; Konopka, Krystyna; Duzgunes, Nejat

    2014-08-18

    Suicide gene therapy of oral squamous cell carcinoma (OSCC) may be a viable approach to the treatment of this cancer. However, human OSCC cells are relatively resistant to efficient transfection by non-viral vectors. To identify an optimal vector for gene delivery, we compared the transfection activities and efficiencies of Glycofect, Metafectene, Metafectene Pro, Metafectene Easy and FuGENE HD, using the OSCC cell line, HSC-3, and the cervical carcinoma cell line, HeLa. The size distribution and ζ-potential of the complexes of these vectors with plasmid DNA were assessed by nanoparticle tracking analysis and electrophoretic mobility measurements, respectively. Metafectene Easy and FuGENE HD mediated the highest transfection activity (measured as luciferase expression) and efficiency (measured as the percentage of cells transfected with ß-galactosidase). These vectors were used to deliver a plasmid encoding herpes simplex virus thymidine kinase, followed by ganciclovir treatment. By day 9, HeLa cell viability was 22±3% of controls with FuGENE HD and 26±3% with Metafectene Easy. The viability of HSC-3 cells was 42±25% with FuGENE HD, and 58±28% with Metafectene Easy. The reduction in viability was statistically significant in both cases (p⩽0.005; average of 3 independent experiments), although there was considerable variability between experiments with the HSC-3 cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Impact of Polyethylenimine Conjugation Mode on the Cell Transfection Efficiency of Silica Nanovectors.

    Science.gov (United States)

    Wang, Xiaolin; Masse, Sylvie; Laurent, Guillaume; Hélary, Christophe; Coradin, Thibaud

    2015-10-13

    The conjugation of polyethylenimine (PEI) to silica nanoparticles has emerged as a useful strategy in gene delivery. Here we investigate the influence of the PEI conjugation mode on the transfection ability of plain silica nanoparticles. Surface functionalization with sulfonate- and chloride-bearing silanes modulates the amount and conformation of PEI and therefore the particles' affinity for the plasmid, without impacting on cytotoxicity. However, transfection efficiency in both immortalized and primary cells is more directly correlated to the nature and strength of the particle-PEI interactions. It suggests that PEI detachment from the particle surface at the stage of endosomal escape is a key event in the plasmid delivery process. These data should provide fruitful guidelines for the fine tuning of colloidal surfaces intended for intracellular delivery of bioactive molecules.

  16. Junk DNA enhances pEI-based non-viral gene delivery

    NARCIS (Netherlands)

    Gaal, E.V.B. van; Oosting, R.S.; Hennink, W.E.; Crommelin, D.J.A.; Mastrobattista, E.

    Gene therapy aims at delivering exogenous DNA into the nuclei of target cells to establish expression of a therapeutic protein. Non-viral gene delivery is examined as a safer alternative to viral approaches, but is presently characterized by a low efficiency. In the past years several non-viral

  17. The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection.

    Science.gov (United States)

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2010-10-15

    An unsaturated hydrocarbon chain in phospholipid was reported to affect a phase transition and a fusogenic activity after mixing membranes, and consequently to achieve a high DNA transfection efficiency. We previously showed that a biosurfactant mannosylerythritol lipid-A (MEL-A) enhances the gene transfection efficiency of cationic liposomes. Here, we have studied the effects of unsaturated fatty acid ratio of MEL-A on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid ratios (9.1%, 21.5%, and 46.3%). The gene transfer efficiency of cationic liposomes containing MEL-A (21.5%) was much higher than that of those containing MEL-A (9.1%) and MEL-A (46.3%). MEL-A (21.5%)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release. Imaging analysis revealed that MEL-A (21.5%)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3%)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1%)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. Thus, it is understandable that the unsaturated fatty acid ratio of MEL-A strongly influences the gene transfection efficiency of cationic liposomes. Copyright 2010 Elsevier B.V. All rights reserved.

  18. [Aspirin-PEI-beta-CyD as a novel non-viral vector for gene transfer].

    Science.gov (United States)

    Wang, Zhong-Ren; Chen, Dan; Zhou, Jun; Tang, Gu-Ping

    2009-01-01

    To develop a novel non-viral gene delivery vector based on PEI-beta-CyD as backbone modified with aspirin, and to identify its physicochemical characters. 1, 1-carbonyldiimidazole (CDI) was used to bind aspirin onto PEI-beta-CyD to form PEI-beta-CyD-ASP. (1)H-NMR, FT-IR, UV and XRD were used to confirm the polymer structure. The ability of condensation was demonstrated by gel retardation assay. MTT assay was used to test the cell viability in B16, Hela and A293 cell lines. Transfection efficiency of the polymer was tested in B16 cells. The structure of PEI-beta-CyD-ASP was confirmed by (1)H-NMR, FT-IR, UV and XRD, which efficiently condensed plasmid DNA at the N/P ratio of 4. The copolymer showed low cytotoxicity and high transfection efficiency in B16 cells. The synthesized aspirin-PEI-beta-CyD might be a potential gene delivery vector.

  19. The density of GM1-enriched lipid rafts correlates inversely with the efficiency of transfection mediated by cationic liposomes.

    Science.gov (United States)

    Kovács, Tamás; Kárász, Andrea; Szöllosi, János; Nagy, Peter

    2009-08-01

    Although cationic liposome-mediated transfection has become a standard procedure, the mechanistic details of the process are unknown. It has been suggested that endocytic uptake of lipoplexes is efficient, and transfectability is largely determined by later steps. In this article, we stained GM1-enriched membrane microdomains, a subclass of lipid rafts, with subunit B of cholera toxin and correlated transfection efficiency with their density by quantitatively evaluating microscopic images. We found a strong anticorrelation between the density of GM1-enriched membrane microdomains and the efficacy of transfection monitored by measuring the expression level of GFP in different cell lines transfected by lipofection using two different transfection agents. These findings imply that GM1-enriched membrane microdomains interfere with the process of lipofection. The blocked step must be endocytosis since the accumulation of fluorescently labeled plasmids was lower in cells with high content of GM1-enriched membrane microdomains. Such a correlation was not observed in cells transfected by electroporation. By comparing the efficiency of lipofection in several cell lines we found that those with a high density of GM1-enriched membrane microdomains were the most resistant to transfection. We conclude that the inhibition of lipofection by GM1-enriched membrane microdomains is a general rule, and that endocytosis of lipoplexes can be rate limiting in cells with high density of GM1-enriched membrane rafts. Copyright 2009 International Society for Advancement of Cytometry.

  20. Polyethyleneimine-poly(ethylene glycol)-star-copolymers as efficient and biodegradable vectors for mammalian cell transfection.

    Science.gov (United States)

    Ladewig, Katharina; Xu, Zhi Ping; Gray, Peter; Max Lu, G Q

    2014-07-01

    High molecular weight (MW) polyethyleneimine (PEI) has been successfully used for the transfection of a broad variety of cell lines. In contrast to low MW PEI, which exhibits low transfection efficiencies but also low cytotoxicity, high MW PEI-mediated transfection achieves much higher efficiencies but at the cost of cell viability; therefore its use in commercial scale transfection and clinical application is limited. In this work we address this problem by constructing biodegradable high MW PEI mimics built from low MW PEI building blocks. The end-groups of small 5-arm star polyethylene glycol (PEG) prepolymers were decorated with linear oligo-ethyleneimine (OEI)/PEI arms of various MW via azomethine linkages. The resultant PEI-PEG-star-copolymers were investigated for their ability to complex plasmid DNA. Polymer/DNA complexes were characterized using techniques such as dynamic light scattering and transmission electron microscopy. Having established their cytotoxicity limits, they were tested as gene delivery vehicles for the transfection of suspension adapted Chinese hamster ovary (CHO-S) cells under serum-free conditions and adherent human embryonic kidney cells (HEK293T) in serum containing medium. Our PEI-PEG-star-copolymers showed a reduced cytotoxicity compared to high MW PEI while maintaining the ability to complex plasmid DNA and transfect mammalian cells, with significant transfection efficiencies. The effects of the optimum parameters on the transfection of mammalian cells using such novel polymers are discussed. © 2013 Wiley Periodicals, Inc.

  1. Bioreducible poly(amido amine)s for non-viral gene delivery

    NARCIS (Netherlands)

    Lin, C.

    2008-01-01

    This thesis describes the design and development of bioreducible poly(amido amine)s as non-viral vectors for gene delivery in vitro and in vivo. The structural influences of these polymers on their physico-chemical properties and gene delivery properties, transfection capability and cytotoxicity in

  2. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    Science.gov (United States)

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

    2014-03-01

    Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

  3. An Electrostatically Self-Assembled Ternary Nanocomplex as a Non-Viral Vector for the Delivery of Plasmid DNA into Human Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Sun-Hee Cho

    2016-04-01

    Full Text Available In this study, we developed electrostatically self-assembled ternary nanocomplexes as a safe and effective non-viral vector for the delivery of plasmid DNA (pDNA into human adipose-derived stem cells (hASCs. Although polyethylenimine (PEI polymers initially showed excellent performance as gene delivery carriers, their broad use has been limited by cytotoxicity resulting from their strong positive charge. To reduce the cytotoxicity, we utilized anionic hyaluronic acid (HA as a corona layer material for pDNA/PEI binary nanocomplexes. HA was also introduced to increase the targeting efficiency of pDNA/PEI nanocomplexes because HA has can bind CD44 that is highly expressed on the surface of hASCs. We confirmed that the addition of HA changed the surface charge of pDNA/PEI nanocomplexes from positive to negative. The pDNA/PEI/HA ternary nanocomplexes showed high transfection efficiency and low cytotoxicity compared with commercially available products. When hASCs were pretreated with HA to passivate CD44, the transfection efficiency of pDNA/PEI/HA nanocomplexes was significantly reduced. These results suggest that HA that can act as a targeting ligand to CD44 contributed to the improved transfection of pDNA into hASCs. Our novel pDNA/PEI/HA nanocomplexes may be used as an effective non-viral pDNA delivery system for hASCs.

  4. Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery.

    Science.gov (United States)

    Zhou, Yangbo; Tang, Zhaomin; Shi, Chunli; Shi, Shuai; Qian, Zhiyong; Zhou, Shaobing

    2012-11-01

    Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (-M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.

  5. Efficiency of two different transfection reagents for use with human NTERA2 cells

    OpenAIRE

    MG Aluigi; S Hofreiter; Falugi, C; M Pestarino; S Candiani

    2009-01-01

    The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO...

  6. Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

    Directory of Open Access Journals (Sweden)

    Mat Yunus Abdul Masani

    Full Text Available Genetic engineering remains a major challenge in oil palm (Elaeis guineensis because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

  7. Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

    Science.gov (United States)

    Masani, Mat Yunus Abdul; Noll, Gundula A; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

    2014-01-01

    Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

  8. Efficient Gene Transfection into Mammalian Cells Mediated by Cross-linked Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Zichun Hua

    2007-02-01

    Full Text Available 25 kDa branched polyethylenimine (PEI has successfully been used for in vitroand in vivo gene delivery approaches, but it is cytotoxic. Smaller PEIs are usually non-cytotoxic but less efficient. In order to enhance the gene delivery efficiency and minimizecytotoxicity of PEI, we explored to synthesize cross-linked PEIs with degradable bonds byreacting amines of small branched 2000 Da PEI with small diacrylate (1,4-butanedioldiacrylate or ethyleneglycol dimethacrylate for 2-6 hours. The efficiency of the cross-linkedPEIs during in vitro delivering plasmid containing enhanced green fluorescent protein(EGFP gene reporter and their cytotoxicity were assessed in melanoma B16F10 cell andother cell lines. In vivo gene delivery efficiency was evaluated by direct injection delivery ofthe EGFP plasmid/ cross-linked PEI complexes into mice and by estimating the EGFPexpression in animal muscles. Compared to commercially available 25-kDa branched PEI,the cross-linked PEIs reported here could mediate more efficient expression of reporter genethan the 25-kDa PEI control, 19-fold more efficiently in B16F10 cells, 17-fold in 293T cells, 2.3-fold in 3T3 cells, and they exhibited essentially nontoxic at their optimized condition for gene delivery. Furthermore the transfection activity of polyplexs was preserved in the presence of serum proteins. The muscle transfected with the cross-linked PEI prepared here exhibited normal morphology and excellent gene expression. The cross-linked PEIs reported here were evidently more efficient than the commercial 25-kD PEI control and had less cytotoxicity in gene delivery in vitro and in vivo.

  9. Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency.

    Science.gov (United States)

    Brgles, Marija; Šantak, Maja; Halassy, Beata; Forcic, Dubravko; Tomašić, Jelka

    2012-01-01

    Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one. Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/-) in the range of 1-50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering. Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity. It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.

  10. Correlation between cationic lipid-based transfection and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael, E-mail: michael@elbaum.ac.il

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. - Highlights: • Cationic lipid-based transfection supports protein expression without cell division. • Protein expression is unrelated to cell cycle status at the time of transfection. • Time-lapse imaging provides direct evaluation without statistical averaging. • Lipoplex dissociation is a likely target for improvement of transfection efficiency.

  11. Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

    Science.gov (United States)

    Chernousova, S; Epple, M

    2017-05-01

    The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

  12. Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

    Science.gov (United States)

    Vernon, Matthew Martin

    Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

  13. Influence of histidine incorporation on buffer capacity and gene transfection efficiency of HPMA-co-oligolysine brush polymers.

    Science.gov (United States)

    Shi, Julie; Schellinger, Joan G; Johnson, Russell N; Choi, Jennifer L; Chou, Brian; Anghel, Ersilia L; Pun, Suzie H

    2013-06-10

    One of the major intracellular barriers to nonviral gene delivery is efficient endosomal escape. The incorporation of histidine residues into polymeric constructs has been found to increase endosomal escape via the proton sponge effect. Statistical and diblock copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), oligolysine, and oligohistidine were synthesized via reversible-addition fragmentation chain transfer (RAFT) polymerization and tested for in vitro transfection efficiency, buffering ability, and polyplex uptake mechanism via the use of chemical endocytic inhibitors. Interestingly, histidine-containing statistical and diblock polymers exhibited increased buffer capacity in different endosomal pH ranges. Statistical copolymers transfected better than block copolymers that contained similar amounts of histidine. In addition, only the polymer containing the highest incorporation of oligohistidine residues led to increases in transfection efficiency over the HPMA-oligolysine base polymer. Thus, for these polymer architectures, high histidine incorporation may be required for efficient endosomal escape. Furthermore, inhibitor studies indicate that nonacidified caveolae-mediated endocytosis may be the primary route of transfection for these copolymers, suggesting that alternative approaches for increasing endosomal escape may be beneficial for enhancing transfection efficiency with these HPMA-oligolysine copolymers.

  14. Dexamethasone conjugation to polyamidoamine dendrimers G1 and G2 for enhanced transfection efficiency with an anti-inflammatory effect.

    Science.gov (United States)

    Kim, Jin Young; Ryu, Jae Hwan; Hyun, Hyesun; Kim, Hyun Ah; Choi, Joon Sig; Yun Lee, Dong; Rhim, Taiyoun; Park, Jeong Hyun; Lee, Minhyung

    2012-09-01

    Polyamidoamine (PAM) dendrimers with low generation such as PAM generation 1 (PAMG1) and PAM generation 2 (PAMG2) have been widely used as a gene carrier due to low toxicity, albeit their low transfection efficiency. In this study, dexamethasone was conjugated to PAMG1 and PAMG2 in order to increase the transfection efficiency. In a gel retardation assay, the dexamethasone conjugated PAMG1 and PAMG2 (PAMG1-Dexa and PAMG2-Dexa) retarded plasmid DNA (pDNA) completely at 5:1 and 3:1 weight ratios (polymer:pDNA), respectively. In transfection assays, PAMG1-Dexa and PAMG2-Dexa had the highest transfection efficiency at 20:1 and 10:1 weight ratios, respectively. In addition, PAMG1-Dexa and PAMG2-Dexa had higher transfection efficiencies than PAMG1, PAMG2, PEI25k, and lipofectamine. In a MTT assay, PAMG1-Dexa and PAMG2-Dexa were less cytotoxic than lipofectamine. In addition, PAMG1-Dexa and PAMG2-Dexa decreased the TNF-α level more efficiently than dexamethasone only in the lipopolysaccharide (LPS)-induced Raw264.7 cells. Therefore, PAMG1-Dexa and PAMG2-Dexa may prove to be useful as gene delivery carriers with an anti-inflammatory effect.

  15. Acceleration of gene transfection efficiency in neuroblastoma cells through polyethyleneimine/poly(methyl methacrylate) core-shell magnetic nanoparticles

    Science.gov (United States)

    Tencomnao, Tewin; Klangthong, Kewalin; Pimpha, Nuttaporn; Chaleawlert-umpon, Saowaluk; Saesoo, Somsak; Woramongkolchai, Noppawan; Saengkrit, Nattika

    2012-01-01

    Background The purpose of this study was to demonstrate the potential of magnetic poly(methyl methacrylate) (PMMA) core/polyethyleneimine (PEI) shell (mag-PEI) nanoparticles, which possess high saturation magnetization for gene delivery. By using mag-PEI nanoparticles as a gene carrier, this study focused on evaluation of transfection efficiency under magnetic induction. The potential role of this newly synthesized nanosphere for therapeutic delivery of the tryptophan hydroxylase-2 (TPH-2) gene was also investigated in cultured neuronal LAN-5 cells. Methods The mag-PEI nanoparticles were prepared by one-step emulsifier-free emulsion polymerization, generating highly loaded and monodispersed magnetic polymeric nanoparticles bearing an amine group. The physicochemical properties of the mag-PEI nanoparticles and DNA-bound mag-PEI nanoparticles were investigated using the gel retardation assay, atomic force microscopy, and zeta size measurements. The gene transfection efficiencies of mag-PEI nanoparticles were evaluated at different transfection times. Confocal laser scanning microscopy confirmed intracellular uptake of the magnetoplex. The optimal conditions for transfection of TPH-2 were selected for therapeutic gene transfection. We isolated the TPH-2 gene from the total RNA of the human medulla oblongata and cloned it into an expression vector. The plasmid containing TPH-2 was subsequently bound onto the surfaces of the mag-PEI nanoparticles via electrostatic interaction. Finally, the mag-PEI nanoparticle magnetoplex was delivered into LAN-5 cells. Reverse-transcriptase polymerase chain reaction was performed to evaluate TPH-2 expression in a quantitative manner. Results The study demonstrated the role of newly synthesized high-magnetization mag-PEI nanoparticles for gene transfection in vitro. The expression signals of a model gene, luciferase, and a therapeutic gene, TPH-2, were enhanced under magnetic-assisted transfection. An in vitro study in neuronal cells

  16. Genetic engineering of mesenchymal stem cells by non-viral gene delivery.

    Science.gov (United States)

    Wang, Weiwei; Xu, Xun; Li, Zhengdong; Lendlein, Andreas; Ma, Nan

    2014-01-01

    Mesenchymal stem cells (MSCs) are an ideal cell source for tissue engineering and regenerative medicine as they possess self-renewal properties and multilineage differentiation potential. They can be isolated from various tissues and expanded easily through normal cell culture techniques. Genetic modifications of MSCs to further improve their therapeutic efficacy have been widely studied and extensively researched. Compared to viral gene delivery methods, non-viral methods generate less toxicity and immunogenicity and thus represent a promising and effective tool for the genetic engineering of MSCs. In the last decades, various non-viral gene delivery strategies have been developed and some of them have been applied for MSC transfection. This paper gives an overview of the techniques, influencing factors and potential applications of non-viral methods used for the genetic engineering of MSCs.

  17. Comparison of Transfection Agents in Forming Complexes with Ferumoxides, Cell Labeling Efficiency, and Cellular Viability

    Directory of Open Access Journals (Sweden)

    Ali Syed Arbab

    2004-01-01

    Full Text Available By complexing ferumoxides or superparamagnetic iron oxide (SPIO to transfection agents (TAs, it is possible to magnetically label mammalian cells. There has been no systematic study comparing TAs complexed to SPIO as far as cell labeling efficiency and viability. This study investigates the toxicity and labeling efficiency at various doses of FEs complexed to different TAs in mammalian cells. Different classes of TAs were used, such as polycationic amines, dendrimers, and lipid-based agents. Cellular toxicity was measured using doses of TAs from 1 to 50 μg/mL in incubation media. Iron incorporation efficiency was measured by combining various amounts of FEs and different doses of TAs. Lipofectamine2000 showed toxicity at lowest dose (1 μg/mL, whereas FuGENE6 and low molecular weight poly-L-lysine (PLI. showed the least toxicity. SPIO labeling efficiency was similar with high-molecular-weight PIX (388.1 kDa and superfect, whereas FuGENE6 and low-molecular-weight PLL were inefficient in labeling cells. Concentrations of 25 to 50 μg/mL of FEs complexed to TAs in media resulted in sufficient endocytosis of the SPIO into endosomes to detect cells on cellular magnetic resonance imaging.

  18. Evaluation of the magnetic field requirements for nanomagnetic gene transfection

    Directory of Open Access Journals (Sweden)

    A. Fouriki

    2010-07-01

    Full Text Available The objective of this work was to examine the effects of magnet distance (and by proxy, field strength on nanomagnetic transfection efficiency. Methods: non-viral magnetic nanoparticle-based transfection was evaluated using both static and oscillating magnet arrays. Results: Fluorescence intensity (firefly luciferase of transfected H292 cells showed no increase using a 96-well NdFeB magnet array when the magnets were 5 mm from the cell culture plate or nearer. At 6 mm and higher, fluorescence intensity decreased systematically. Conclusion: In all cases, fluorescence intensity was higher when using an oscillating array compared to a static array. For distances closer than 5 mm, the oscillating system also outperformed Lipofectamine 2000™.

  19. Biophysics and protein corona analysis of Janus cyclodextrin-DNA nanocomplexes. Efficient cellular transfection on cancer cells.

    Science.gov (United States)

    Martínez-Negro, M; Caracciolo, G; Palchetti, S; Pozzi, D; Capriotti, A L; Cavaliere, C; Laganà, A; Ortiz Mellet, C; Benito, J M; García Fernández, J M; Aicart, E; Junquera, E

    2017-07-01

    The self-assembling processes underlining the capabilities of facially differentiated ("Janus") polycationic amphiphilic cyclodextrins (paCDs) as non-viral gene nanocarriers have been investigated by a pluridisciplinary approach. Three representative Janus paCDs bearing a common tetradecahexanoyl multitail domain at the secondary face and differing in the topology of the cluster of amino groups at the primary side were selected for this study. All of them compact pEGFP-C3 plasmid DNA and promote transfection in HeLa and MCF-7 cells, both in absence and in presence of human serum. The electrochemical and structural characteristics of the paCD-pDNA complexes (CDplexes) have been studied by using zeta potential, DLS, SAXS, and cryo-TEM. paCDs and pDNA, when assembled in CDplexes, render effective charges that are lower than the nominal ones. The CDplexes show a self-assembling pattern corresponding to multilamellar lyotropic liquid crystal phases, characterized by a lamellar stacking of bilayers of the CD-based vectors with anionic pDNA sandwiched among them. When exposed to human serum, either in the absence or in the presence of pDNA, the surface of the cationic CD-based vector becomes coated by a protein corona (PC) whose composition has been analyzed by nanoLC-MS/MS. Some of the CDplexes herein studied showed moderate-to-high transfection levels in HeLa and MCF-7 cancer cells combined with moderate-to-high cell viabilities, as determined by FACS and MTT reduction assays. The ensemble of data provides a detail picture of the paCD-pDNA-PC association processes and a rational base to exploit the protein corona for targeted gene delivery on future in vivo applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Multifunctional cationic polyurethanes designed for non-viral cancer gene therapy.

    Science.gov (United States)

    Cheng, Jian; Tang, Xin; Zhao, Jie; Shi, Ting; Zhao, Peng; Lin, Chao

    2016-01-01

    Nano-polyplexes from bioreducible cationic polymers have a massive promise for cancer gene therapy. However, the feasibility of cationic polyurethanes for non-viral gene therapy is so far not well studied. In this work, a linear cationic polyurethane containing disulfide bonds, urethane linkages and protonable tertiary amino groups was successfully generated by stepwise polycondensation reaction between 2,2'-dithiodiethanol bis(p-nitrophenyl carbonate) and 1,4-bis(3-aminopropyl)piperazine (BAP). We confirmed that the cationic polyurethane (denoted as PUBAP) displayed superior gene delivery properties to its cationic polyamide analogue, thus causing higher in vitro transfection efficiency in MCF-7 and SKOV-3 cells. Besides, further folate-PEGylation and hydrophobic deoxycholic acid (DCA) conjugation to amino-containing PUBAP can be conducted to afford multifunctional polyurethane gene delivery system. After optimization, folate-decorated nano-polyplexes from the PUBAP conjugated with 8 folate-PEG chains and 12 DCA residues exhibited superb colloidal stability under physiological conditions, and performed rapid uptake via folate receptor-mediated endocytosis, efficient intracellular gene release and nucleus translocation into SKOV-3 cells in vitro and in vivo. Importantly, PUBAP based polyplexes possess low cytotoxicity as a result of PUBAP biodegradability. Therefore, marked growth inhibition of SKOV-3 tumor xenografted in Balb/c nude mice was achieved with negligible side effects on the mouse health after intravenous administration of PUBAP based polyplexes with a therapeutic plasmid encoding for TNF-related apoptosis-inducing ligand. This work provides a new insight into biomedical application of bio-responsive polyurethanes for cancer therapy. In this study, we have confirmed that disulfide-based cationic polyurethane presents a new non-viral vector for gene transfer and cancer gene therapy. The significance of this work includes: (1) design and synthesis of a

  1. Temperature variable and the efficiency of sperm mediated transfection of HPV16 DNA into cells.

    Science.gov (United States)

    Kadze, Ruslana; Chan, Philip J; Jacobson, John D; Corselli, Johannah U; King, Alan

    2002-09-01

    To pretreat sperm at various temperatures before exposure to human papillomavirus (HPV) 16 DNA fragments and to assess the efficiency of HPV carrier sperm to transfect cumulus cells. Cumulus cells from follicular aspirates were obtained, pooled and divided into culture dishes containing Sybr Gold-stained HPV DNA carrying sperm that were either pretreated at 4 degree C, 37 degree C or 40 degree C (n = 5). The cells were incubated in 5% CO(2) in air mixture at 37 degree C for 24 hours. The efficiency of sperm to take up fluorescent HPV DNA was determined at hour 0. After incubation, cumulus cell viability was assessed using the eosin method and the percentages of fluorescent cumulus cells determined. Over half of all the cumulus cells became fluorescent with the highest percentage in the 37 degree C group. Sperm pretreated at 4 degree C had the greatest amount of HPV DNA fragments. Total sperm motility was similar for the 3 pretreatment groups. There were no differences in cumulus viability among the groups. Sperm pretreated at 37 degree C transferred the greatest amount of fluorescent HPV DNA fragments to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data suggested the possibility of sperm as a vector for the transmission of HPV DNA to the cumulus cells surrounding ovulated oocytes, which might lead to early implantation failures.

  2. Efficiency of gene transfection reagents in NG108-15, SH-SY5Y and CHO-K1 cell lines.

    Science.gov (United States)

    Martín-Montañez, E; López-Téllez, J F; Acevedo, M J; Pavía, J; Khan, Z U

    2010-06-01

    Several gene delivery reagents were analyzed for their transfection efficiency. Genes studied belonged to the class of mammalian proteins termed regulators of G-protein signaling (RGS), ranged in size up to 2.2 Kb long and were transfected into the NG108-15, SH-SY5Y and CHO-K1 cell lines. Prior to transfection, genes were cloned into a nonviral vector pcDNA 6.2/EmGFP, so as to express a green fluorescent protein tag at the 3' end. Flow cytometry was used to analyze cell fluorescent activity and thereby transfection efficiency. Gene delivery reagents Lipofectamine 2000 and ExGen 500 produced more effective transfection in NG108-15 cells whereas Lipofectamine 2000, ExGen 500 and TurboFectin 8.0 were more effective in CHO-K1 cells. In both these cell lines, transfection efficiency reached 60-80%. In SH-SY5Y cells, TurboFectin 8.0 produced the best transfection result; however efficiency level was only 5%. Gene size had no effect on transfection efficiency. Unlike Lipofectamine 2000, cells transfected using ExGen 500 showed morphological deformation. Our results suggest that Lipofectamine 2000 is the most suitable transfection medium for gene delivery to NG108-15 and CHO-K1 cells. Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

  3. Triazine dendrimers as non-viral vectors for in vitro and in vivo RNAi: The effects of peripheral groups and core structure on biological activity

    Science.gov (United States)

    Merkel, Olivia M.; Mintzer, Meredith A.; Librizzi, Damiano; Samsonova, Olga; Dicke, Tanja; Sproat, Brian; Garn, Holger; Barth, Peter J.; Simanek, Eric E.; Kissel, Thomas

    2010-01-01

    A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv.) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this non-viral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents. PMID:20524664

  4. A Low Protein Binding Cationic Poly(2-oxazoline) as Non-Viral Vector

    KAUST Repository

    He, Zhijian

    2015-04-02

    © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Developing safe and efficient non-viral gene delivery systems remains a major challenge. We present a new cationic poly(2-oxazoline) (CPOx) block copolymer for gene therapy that was synthesized by sequential polymerization of non-ionic 2-methyl-2-oxazoline and a new 2-oxazoline monomer, 2-(N-methyl, N-Boc-amino)-methyl-2-oxazoline, followed by deprotection of the pendant secondary amine groups. Upon mixing with plasmid DNA (pDNA), CPOx forms small (diameter ≈80 nm) and narrowly dispersed polyplexes (PDI <0.2), which are stable upon dilution in saline and against thermal challenge. These polyplexes exhibited low plasma protein binding and very low cytotoxicity in vitro compared to the polyplexes of pDNA and poly(ethylene glycol)-b-poly(L-lysine) (PEG-b-PLL). CPOx/pDNA polyplexes at N/P = 5 bound considerably less plasma protein compared to polyplexes of PEG-b-PLL at the same N/P ratio. This is a unique aspect of the developed polyplexes emphasizing their potential for systemic delivery in vivo. The transfection efficiency of the polyplexes in B16 murine melanoma cells was low after 4 h, but increased significantly for 10 h exposure time, indicative of slow internalization of polyplexes. Addition of Pluronic P85 boosted the transfection using CPOx/pDNA polyplexes considerably. The low protein binding of CPOx/pDNA polyplexes is particularly interesting for the future development of targeted gene delivery.

  5. Bioreducible Poly-L-Lysine-Poly[HPMA] Block Copolymers Obtained by RAFT-Polymerization as Efficient Polyplex-Transfection Reagents.

    Science.gov (United States)

    Tappertzhofen, Kristof; Beck, Simone; Montermann, Evelyn; Huesmann, David; Barz, Matthias; Koynov, Kaloian; Bros, Matthias; Zentel, Rudolf

    2016-01-01

    Polylysine-b-p[HPMA] block copolymers containing a redox-responsive disulfide bond between both blocks are synthesized by RAFT polymerization of pentafluorphenyl-methacrylate with a macro-CTA from Nϵ-benzyloxycarbonyl (Cbz) protected polylysine (synthesized by NCA polymerization). This polylysine-b-p[PFMA] precursor block copolymer is converted to polylysine(Cbz)-b-p[HPMA] by postpolymerization modification with 2-hydroxypropylamine. After removal of the Cbz protecting group, cationic polylysine-b-p[HPMA] copolymers with a biosplittable disulfide moiety became available, which can be used as polymeric transfection vectors. These disulfide linked polylysine-S-S-b-p[HPMA] block copolymers show low cytotoxicity and increased transfection efficiencies (HEK-293T cells) compared to analogous blockcopolymers without disulfide group making them interesting for the transfection of sensitive immune cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains

    Science.gov (United States)

    Wu, Shi-Hao; Liao, Zhi-Xing; Rizak, Joshua D.; Zheng, Na; Zhang, Lin-Heng; Tang, Hen; He, Xiao-Bin; Wu, Yang; He, Xia-Ping; Yang, Mei-Feng; Li, Zheng-Hui; Qin, Dong-Dong; Hu, Xin-Tian

    2017-01-01

    Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non-human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin Ⅰ, or Ca2+/calmodulin-dependent protein kinase Ⅱ promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs). These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates. PMID:28409504

  7. High‐throughput screening of clinically approved drugs that prime polyethylenimine transfection reveals modulation of mitochondria dysfunction response improves gene transfer efficiencies

    Science.gov (United States)

    Nguyen, Albert; Beyersdorf, Jared; Riethoven, Jean‐Jack

    2016-01-01

    Abstract Nonviral gene delivery methods are advantageous over viral vectors in terms of safety, cost, and flexibility in design and application, but suffer from lower gene transfer efficiency. In addition to modifications to nucleic acid design and nonviral carriers, new tools are sought to enhance transfection. Priming is the pharmacological modulation of transfection efficiency and transgene expression, and has demonstrated transfection increase in several compounds, for example, chloroquine and glucocorticoids. To develop a library of transfection priming compounds, a high‐throughput screen was performed of the NIH Clinical Collection (NCC) to identify clinical compounds that prime polyethylenimine (PEI) transfection. HEK293T cells were treated with priming compounds, then transfected with enhanced green fluorescent protein (EGFP)‐encoding plasmid by PEI. After 48‐hr culture, primed and transfected cells were assayed for transfection, cell proliferation, and cell viability by fluorescence measurement of EGFP reporter, Hoechst 33342 nuclei stain, and resazurin metabolic assay. From the microscope image analysis and microplate measurements, transfection fold‐changes were determined, and compounds resulting in statistically significant transfection fold‐change were identified. NCC compounds were clustered using PubChem fingerprint similarity by Tanimoto coefficients in ChemmineTools. Fold‐changes for each compound were linked to drug clusters, from which drug classes that prime transfection were identified. Among the identified drugs classes that primed transfection increases were antioxidants, GABAA receptor modulators, and glucocorticoids. Resveratrol and piceid, stilbenoid antioxidants found in grapes, and zolpidem, a GABAA modulator, increased transfection nearly three‐fold. Literature indicate interaction of the identified transfection priming drug clusters with mitochondria, which may modulate mitochondrial dysfunction known to be associated

  8. Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination.

    Science.gov (United States)

    Tan, Peng H; Beutelspacher, Sven C; Wang, Yao-He; McClure, Myra O; Ritter, Mary A; Lombardi, Giovanna; George, Andrew J T

    2005-05-01

    Genetic manipulation of dendritic cells (DCs) is important in the context of using either mature DCs to immunize patients or immature DCs to induce tolerance. Here, we describe a novel method of transfecting monocyte-derived human DCs using immunolipoplexes containing anti-CD71 or anti-CD205 monoclonal Abs. This results in up to 20% transfection, which can be increased to 20-30% if the immunolipoplexes are used to transfect CD14+ monocytes prior to differentiation into DCs. Transfected DCs can be substantially enriched using a drug-selection protocol during differentiation. Unlike adenoviral transduction, this nonviral transfection does not alter the expression of costimulatory molecules or the production of proinflammatory cytokines by DCs. In addition, DC function is unaltered, as assessed by mixed lymphocyte reactions. To test the feasibility of the immunolipoplexes and selection protocol for therapeutic intervention, we transfected DCs with the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Allogeneic T cells exposed to IDO-expressing DCs did not proliferate, secreted more IL-10 and less Th1 and Th2 cytokines, and had a higher amount of apoptosis than T cells incubated with control DCs. Furthermore the remaining T cells were rendered anergic to further stimulation by allogeneic DC. These immunolipoplexes, which can be easily and rapidly assembled, have potential for clinical immunization, in particular for tolerance induction protocols.

  9. AP-4F, antennapedia peptide linked to an amphipathic alpha helical peptide, increases the efficiency of Lipofectamine-mediated gene transfection in endothelial cells.

    Science.gov (United States)

    Ou, Jingsong; Geiger, Tracy; Ou, Zhijun; Ackerman, Allan W; Oldham, Keith T; Pritchard, Kirkwood A

    2003-06-06

    Typically, endothelial cells are difficult to transfect. In this study, we report that antennapedia peptide (AP) linked to L-4F, a water-soluble, amphipathic alpha helical peptide that avidly binds lipids (AP-4F) increases Lipofectamine 2000-mediated transfection of bovine coronary endothelial cell cultures. Transfection efficiency was monitored by flow cytometry and fluorescent microscopy. Lipofectamine 2000 transfection of endothelial cell cultures with green fluorescence protein (GFP)-DNA typically yields transfection efficiencies of 35.4+/-3.3% with low levels of cell death (8.1+/-1.0%). Pre-treatment of the Lipofectamine 2000-GFP-DNA complexes with AP-4F for 5 min increased transfection to 58.2+/-2.8% without increasing cell death. AP-4F increases Lipofectamine 2000-mediated transfection in a time-dependent fashion (within 10-20 min). Systematic studies reveal that the individual components of AP-4F, i.e., AP and L-4F alone, are ineffective in increasing Lipofectamine 2000-mediated transfection and that AP-4F must be directly associated with DNA liposomes prior to transfection for optimal uptake by endothelial cells. These observations demonstrate that AP-4F may be useful for increasing the transfection efficiency of endothelial cell cultures with standard commercially available reagents.

  10. PEGylated chitosan complexes DNA while improving polyplex colloidal stability and gene transfection efficiency.

    Science.gov (United States)

    Maurstad, Gjertrud; Stokke, Bjørn T; Vårum, Kjell M; Strand, Sabina P

    2013-04-15

    Chitosan is widely explored as a gene delivery vehicle due to its ability to condense DNA, facilitate transport, and subsequent release allowing gene expression, as well as protecting the DNA. Here, we investigate the enhancement of chitosan-DNA dispersion stability while maintaining transfection efficacy by PEGylation of chitosan. Molecular properties of fully deacetylated chitosans and degree of PEGylation were investigated with respect to compaction of DNA, stability and transfection efficacy. Each of the three chitosan samples with varying chain lengths was PEGylated at three different degrees. The chitosans with degree of PEGylation from 0.6 to 1.9% made polyplexes with DNA. PBS induced colloidal aggregation of polyplexes with initial radius of about 100 nm observed for nonPEGylated chitosans was suppressed for 1.9% PEGylated chitosans. The observed increase in transfection efficacy coinciding with increased polyplex colloidal stability suggests that aggregation of gene-delivery packages may reduce the transfection efficacy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Application of HVJ-liposome mediated gene transfer in lung transplantation-distribution and transfection efficiency in the lung.

    Science.gov (United States)

    Ohmori, Kenichi; Takeda, Shin-ichi; Miyoshi, Shinichiro; Minami, Masato; Nakane, Shigeru; Ohta, Mitsunori; Sawa, Yoshiki; Matsuda, Hikaru

    2005-05-01

    A novel hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer system has been shown to have benefits of a high efficiency of transfection and low immunogenicity. The aims of this study were to determine the effect of re-transfection of the HVJ-liposome system via the airway, and to quantify the distribution of gene expression between transtracheal and transplantation approaches. Beta-galactosidase (beta-gal) plasmid DNA was introduced into lung tissues using the HVJ-liposome method. Two groups of Sprague-Dawley (SD) rats received intratracheal instillation of 10 microg of the beta-gal gene, once on Day 0 in 1 group (Group Tb-1, n=4) and 3 times on Days 0-2 in another (Group Tb-3, n=4). In a third group of SD rats (Group Tx, n=5), an orthotopic left lung transplantation was performed after the donor lung was flushed with an HVJ-liposome complex solution and preserved for 1h. Gene expression and distribution in lung tissue was then quantified by counting the X-gal stained cells. Both the transtracheal and transplantation approaches resulted in low levels of transfection in the vascular endothelial cells (0.2+/-0.1 and 4.0+/-1.8%), respectively, but a moderate degree of transfection to the airway (11.0+/-7.1 and 28.0+/-20.7%) and alveolar cells (3.0+/-1.8 and 6.0+/-3.6%). Three repetitive injections via the airway increased gene expression in airway epithelial cells of 41.0+/-12.0% compared with the single administration of 11.0+/-4.3%. Our results suggest that the repeated transtracheal gene transfection using HVJ-liposome may have benefits for treatment of problems after lung transplantation. In addition, gene transfer using a flushing solution during harvest may provide an opportunity for gene manipulation in the setting of lung transplantation.

  12. Click modification of helical amylose by poly(L-lysine) dendrons for non-viral gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jia-Dong [PCFM Lab and GDHPPC Lab, Institute of Polymer Science, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen (Zhongshan) University, Guangzhou 510275 (China); Zhuang, Bao-Xiong [Second Affiliated Hospital, Sun Yat-sen (Zhongshan) University, Guangzhou 510102 (China); Mai, Kaijin [PCFM Lab and GDHPPC Lab, Institute of Polymer Science, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen (Zhongshan) University, Guangzhou 510275 (China); Chen, Ru-Fu [Second Affiliated Hospital, Sun Yat-sen (Zhongshan) University, Guangzhou 510102 (China); Wang, Jie, E-mail: sumsjw@163.com [Second Affiliated Hospital, Sun Yat-sen (Zhongshan) University, Guangzhou 510102 (China); Zhang, Li-Ming, E-mail: ceszhlm@mail.sysu.edu.cn [PCFM Lab and GDHPPC Lab, Institute of Polymer Science, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen (Zhongshan) University, Guangzhou 510275 (China)

    2015-04-01

    Although amylose as a naturally-occurring helical polysaccharide has been widely used for biomedical applications, few studies have dealt with its chemical modification for non-viral gene delivery. In this work, the click modification of amylose by poly(L-lysine) dendrons was carried out and then characterized by Fourier transform infrared spectroscopy, wide-angle X-ray diffraction and elemental analyses. Such a modified polysaccharide exhibited excellent ability to condense plasmid pMSCV-GFP-PARK2 into compact and spherical nanoparticles. Moreover, it displayed much lower cytotoxicity when compared to branched polyethylenimine (bPEI, 25 kDa), a commercially available gene vector. Similar to bPEI, it had a dose-dependent gene transfection activity in human embryonic kidney 293T cells, as observed by confocal laser scanning microscopy and flow cytometry. At each optimized N/P ratio, the percentage of transfected cells by this modified polysaccharide was found to be comparable to that by bPEI. Western blot and cell apoptosis analyses confirmed its effectiveness for the delivery of plasmid pMSCV-GFP-PARK2 to 293T cells. - Highlights: • The click modification of amylose by poly(L-lysine) dendrons was carried out. • This modified amylose could condense plasmid pMSCV-GFP-PARK2 into nanocomplexes. • This modified amylose exhibited much lower cytotoxicity than commercial polyethylenimine. • This modified amylose could delivery efficiently plasmid pMSCV-GFP-PARK2 to 293T cells.

  13. Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

    Directory of Open Access Journals (Sweden)

    Markus eSmolny

    2014-10-01

    Full Text Available Microglial activation is a central event in neurodegeneration. Novel technologies are sought for that specifically manipulate microglial function in order to delineate their role in onset and progression of neuropathologies. We investigated for the first time whether non-viral gene delivery based on polyethyleneglycol (PEG-polyethyleneimine (PEI conjugated to the monoclonal anti-CD11b antibody OX42 (‘OX42-immunogene’ could be used to specifically target microglia. We identified the microglial integrin receptor CD11b as a potential target for receptor-mediated gene transfer based on its cellular specificity in mixed glia culture and in vivo and found that the OX42 antibody is rapidly internalized and trafficked to acidic organelles in absence of activation of the respiratory burst. We then showed that the OX42-immunogene although internalised was degraded intracellularly and did not cause substantial gene expression in microglia. Investigation of specific barriers to microglial gene transfer revealed that aggregated OX42-immunogene polyplexes stimulated the respiratory burst that likely involved Fcγ-receptors. Transfections in the presence of the endosomolytic agent chloroquine improved transfection efficiency indicating that endosomal escape may be limited. This study identifies CD11b as an entry point for antibody-mediated gene transfer into microglia and takes important steps towards the further development of OX42-immunogenes.

  14. Establishment of a cell line with high transfection efficiency from zebrafish Danio rerio embryos and its susceptibility to fish viruses.

    Science.gov (United States)

    Jin, Y L; Chen, L M; Le, Y; Li, Y L; Hong, Y H; Jia, K T; Yi, M S

    2017-10-01

    A cell line ZBE3 isolated from a continuous cell culture derived from zebrafish Danio rerio blastomeres by clonal growth was characterized. ZBE3 cells had been subcultured for >120 passages since the initial primary culture of the blastomeres. The ZBE3 cells grow stably at temperature from 20 to 32° C with an optimum temperature of 28° C in ESM2 or ESM4 medium with 15% foetal bovine serum (FBS). The optimum FBS concentration for ZBE3 cell growth ranged from 15 to 20%. Cytogenetical analysis indicated that the modal chromosome number of ZBE3 cells was 50, the same as the diploid chromosome number of D. rerio. Significant cytopathic effect was observed in ZBE3 cells after infection with redspotted grouper nervous necrosis virus, Singapore grouper iridovirus and grass carp reovirus, and the viral replication in the cells was confirmed by real-time quantitative PCR and transmission electron microscopy, indicating the susceptibility of ZBE3 cells to the three fish viruses. After transfected with pEGFP-N3 plasmid, ZBE3 cells showed a transfection efficiency of about 40% which was indicated by the percentage of cells expressing green fluorescence protein. The stable growth, susceptibility to fish viruses as well as high transfection efficiency make ZBE3 cells be a useful tool in transgenic manipulation, fish virus-host cell interaction and immune response in fish. © 2017 The Fisheries Society of the British Isles.

  15. Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent

    OpenAIRE

    Chernousova, S; Epple, M.

    2017-01-01

    The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP ...

  16. [Transfection Efficiency of Ad5F11p-GFP on CIK and NK-92 Cells and Its Influence on Biological Characteristics].

    Science.gov (United States)

    Xu, Zan-Mei; Lu, Ying; Zhao, Lan-Jun; Liu, Jin; Hu, Xian-Wen; Wu, Chu-Tse; Duan, Hai-Feng

    2016-06-01

    To study transfection efficiency of Ad5F11p-GFP and its influence on biological characteristics of CIK and NK-92 cells in order to predict the application of Ad5F11p vector in immunotherapy. Two kinds of immune cells, cytokine-induced killer (CIK) cells and natural-killer (NK) cell line NK-92 cells, were transfected by Ad5F11p-GFP at different multiplicity of transfection (MOI), and untransfected immune cells were used as negative control. GFP expression was determined by flow cytometry, the cell morphology was observed with microscope, the cell proliferation was analyzed by trypan blue staining, specific cytotoxicity of NK-92 cells was determined by LDH assay. About 90% of transfection efficiency for NK-92 cells could be achieved at a MOI of 25, while the transfection efficiency for CIK was less than 40% at a MOI of 200. In addition, the transfection efficiency basically unchanged at the same MOI for 48 h and 96 h, and the immune cells transfected with the virus trended to form agglomeration, displaying slower proliferation, increase of IFN-γ release and enhancement of tumor killing activity. Ad5F11p- modified NK-92 shows a good prospect for adoptive immunotherapy.

  17. A simple, highly efficient method for heterologous expression in mammalian primary neurons using cationic lipid-mediated mRNA transfection

    Directory of Open Access Journals (Sweden)

    Damian J Williams

    2010-11-01

    Full Text Available Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The postmitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro-transcribed mRNA and the cationic lipid transfection reagent Lipofectamine 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R, a G protein inwardly-rectifying K+ channel (GIRK4 and a dominant-negative G protein α-subunit mutant (GoA G203T indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

  18. Transfection of nerve cells.

    Science.gov (United States)

    Salozhin, S V; Bol'shakov, A P

    2010-03-01

    Transfection is a method of transforming cells based on the introduction into living cells of plasmids encoding a particular protein or RNA. This review describes the main methods of transfection and considers their advantages and disadvantages. Most attention is paid to lentivirus transduction as one of the most efficient methods for transforming nerve cells. The development of current transfection systems based on lentivirus vectors is described and a brief review of studies performed using in vivo and in vitro lentivirus transfection of nerve cells is presented.

  19. Cholesterol Domains Enhance Transfection

    Science.gov (United States)

    Betker, Jamie L.; Kullberg, Max; Gomez, Joe; Anchordoquy, Thomas J.

    2014-01-01

    The formation of cholesterol domains in lipoplexes has been associated with enhanced serum stability and transfection rates both in cell culture and in vivo. This study utilizes the ability of saturated phosphatidylcholines to promote the formation of cholesterol domains at much lower cholesterol contents than have been utilized in previous work. The results show that lipoplexes with identical cholesterol and cationic lipid contents exhibit significantly improved transfection efficiencies when a domain is present, consistent with previous work. In addition, studies assessing transfection rates in the absence of serum demonstrate that the ability of domains to enhance transfection is not dependent on interactions with serum proteins. Consistent with this hypothesis, characterization of the adsorbed proteins composing the corona of these lipoplex formulations did not reveal a correlation between transfection and the adsorption of a specific protein. Finally, we show that the interaction with serum proteins can promote domain formation in some formulations, and thereby result in enhanced transfection only after serum exposure. PMID:23557286

  20. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells.

    Science.gov (United States)

    Yang, Shaozhe; Shi, Haijun; Chu, Xinran; Zhou, Xiaoling; Sun, Pingnan

    2016-09-01

    To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

  1. Synthesis, and Characterization, and Evaluation of Cellular Effects of the FOL-PEG-g-PEI-GAL Nanoparticles as a Potential Non-Viral Vector for Gene Delivery

    Directory of Open Access Journals (Sweden)

    S. Ghiamkazemi

    2010-01-01

    Full Text Available In this manuscript, we synthesized the potential non viral vector for gene delivery with proper transfection efficiency and low cytotoxicity. Polyethylenimine (PEI is a well-known cationic polymer which has high positive surface charge for condensing plasmid DNA. However; it is highly cytotoxic in many cell lines because of the high surface charge, non-biodegradability and non-biocompatibility. To enhance PEI biodegradability, the graft copolymer “PEG-g-PEI” was synthesized. To target cancer liver cells, two targeting ligands folic acid and galactose (lactobionic acid which are over expressed on human hepatocyte carcinoma were attached to graft copolymer and “FOL-PEG-g-PEI-GAL” copolymer was synthesized. Composition of this grafted copolymer was characterized using 1H-NMR and FTIR spectra. The molecular weight and zeta potential of this copolymer was compared to PEI. The particle size and zeta potential of FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratio were measured using dynamic light scattering (DLS. Cytotoxicity of the copolymer was also studied in cultured HepG2 human hepatoblastoma cell line. The FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratios exhibited no cytotoxicity in HepG2 cell line compared to PEI 25K as a control. The novel copolymer showed enhanced biodegradability in physiological conditions in compared with PEI and targeted cultured HepG2 cells. More importantly, significant transfection efficiency was exhibited in cancer liver cells. Together, our results showed that “FOL-PEG-g-PEI-GAL” nanoparticals could be considered as a useful non-viral vector for targeted gene delivery.

  2. Integration of antimicrobial peptides with gold nanoparticles as unique non-viral vectors for gene delivery to mesenchymal stem cells with antibacterial activity.

    Science.gov (United States)

    Peng, Li-Hua; Huang, Yan-Fen; Zhang, Chen-Zhen; Niu, Jie; Chen, Ying; Chu, Yang; Jiang, Zhi-Hong; Gao, Jian-Qing; Mao, Zheng-Wei

    2016-10-01

    Gold nanoparticles (AuNPs) have emerged as attractive non-viral gene vectors. However their application in regenerative medicine is still limited partially due to a lack of an intrinsic capacity to transfect difficult-to-transfect cells such as primary cells or stem cells. In current study, we report the synthesis of antimicrobial peptide conjugated cationic AuNPs (AuNPs@PEP) as highly efficient carriers for gene delivery to stem cells with antibacterial ability. The AuNPs@PEP integrate the advantages of cationic AuNPs and antibacterial peptides: the presence of cationic AuNPs can effectively condense DNA and the antimicrobial peptides are essential for the cellular & nucleus entry enhancement to achieve high transfection efficiency and antibacterial ability. As a result, antimicrobial peptides conjugated AuNPs significantly promoted the gene transfection efficiency in rat mesenchymal stem cells than pristine AuNPs, with a similar extent to those expressed by TAT (a well-known cell-penetrating peptide) modified AuNPs. More interestingly, the combinational system has better antibacterial ability than free antimicrobial peptides in vitro and in vivo, possibly due to the high density of peptides on the surface of AuNPs. Finally we present the concept-proving results that AuPs@PEP can be used as a carrier for in vivo gene activation in tissue regeneration, suggesting its potential as a multifunctional system with both gene delivery and antibacterial abilities in clinic. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Synthesis of Mannosylated Polyethylenimine and Its Potential Application as Cell-Targeting Non-Viral Vector for Gene Therapy

    Directory of Open Access Journals (Sweden)

    Ying Hu

    2014-10-01

    Full Text Available Mannose polyethylenimine with a molecular weight of 25 k (Man-PEI25k was synthesized via a phenylisothiocyanate bridge using mannopyranosylphenyl isothiocyanate as a coupling reagent, and characterized by 1H NMR (nuclear magnetic resonance and FT-IR (Fourier transform infrared spectroscopy analysis. Spherical nanoparticles were formed with diameters of 80–250 nm when the copolymer was mixed with DNA at various charge ratios of copolymer/DNA (N/P. Gel electrophoresis demonstrated that the DNA had been condensed and retained by the PEI derivates at low N/P ratios. The Man-PEI25k/DNA complexes were less cytotoxic than the PEI complexes with a molecular weight of 25 k (PEI25k at the same N/P ratio. Laser scan confocal microscopy and flow cytometry confirmed that the Man-PEI25k/DNA complexes gave higher cell uptake efficiency in (Dendritic cells DC2.4 cells than HeLa cells. The transfection efficiency of Man-PEI25k was higher than that of PEI25k towards DC2.4 cells. These results indicated that Man-PEI25k could be used as a potential DC-targeting non-viral vector for gene therapy.

  4. Ultrasound treatment increases transfection efficiency of low molecular weight chitosan in fibroblasts but not in KB cells.

    Directory of Open Access Journals (Sweden)

    Ureporn Kedjarune-Leggat

    Full Text Available The aim of this study was to optimize transfection efficiency (TE of the depolymerized low molecular weight (LW chitosan with molecular weight (Mw at 16 kDa and 54% degree of deacetylation (DDA on three primary cells of fibroblast (F, dental pulp (P, and periodontal ligament (PDL. The effect of low frequency ultrasound treatment on the chitosan-DNA complexes prior transfection on TE was also evaluated. This LW chitosan required high N/P ratio (>34 to bind DNA completely. An N/P ratio above 56 tended to improve TE in most primary cells nearly at the level of Lipofectamine. Ultrasonication can reduce the aggregation and sizes of the chitosan-DNA microparticles. It increased TE of F cells at an N/P ratio above 34, which was higher than Lipofectamine. However, this ultrasound treatment caused loss of TE in KB cells. MTT assay of these chitosan-DNA complexes revealed no significant cytotoxicity to both KB and F cells. This LW chitosan has potential for further development into a safer alternative to gene delivery systems in various cells of interest; however the optimal conditions have to be adjusted, depending on each cell source.

  5. Advances in Non-Viral DNA Vectors for Gene Therapy

    Directory of Open Access Journals (Sweden)

    Cinnamon L. Hardee

    2017-02-01

    Full Text Available Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic

  6. Unsatisfactory gene transfer into bone-resorbing osteoclasts with liposomal transfection systems

    Directory of Open Access Journals (Sweden)

    Laitala-Leinonen Tiina

    2005-08-01

    Full Text Available Abstract Background Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Many of the details of osteoclast formation, activation and motility remain unsolved. Therefore, there is an interest among bone biologists to transfect the terminally differentiated osteoclasts and follow their responses to the transgenes in vitro. Severe difficulties in transfecting the large, adherent osteoclasts have been encountered, however, making the use of modern cell biology tools in osteoclast research challenging. Transfection of mature osteoclasts by non-viral gene transfer systems has not been reported. Results We have systematically screened the usefulness of several commercial DNA transfection systems in human osteoclasts and their mononuclear precursor cell cultures, and compared transfection efficacy to adenoviral DNA transfection. None of the liposome-based or endosome disruption-inducing systems could induce EGFP-actin expression in terminally differentiated osteoclasts. Instead, a massive cell death by apoptosis was found with all concentrations and liposome/DNA-ratios tested. Best transfection efficiencies were obtained by adenoviral gene delivery. Marginal DNA transfection was obtained by just adding the DNA to the cell culture medium. When bone marrow-derived CD34-positive precursor cells were transfected, some GFP-expression was found at the latest 24 h after transfection. Large numbers of apoptotic cells were found and those cells that remained alive, failed to form osteoclasts when cultured in the presence of RANKL and M-CSF, key regulators of osteoclast formation. In comparison, adenoviral gene delivery resulted in the transfection of CD34-positive cells that remained GFP-positive for up to 5 days and allowed osteoclast formation. Conclusion Osteoclasts and their precursors are sensitive to liposomal transfection systems, which induce osteoclast apoptosis. Gene transfer to mononuclear

  7. Uptake and transfection efficiency of PEGylated cationic liposome-DNA complexes with and without RGD-tagging.

    Science.gov (United States)

    Majzoub, Ramsey N; Chan, Chia-Ling; Ewert, Kai K; Silva, Bruno F B; Liang, Keng S; Jacovetty, Erica L; Carragher, Bridget; Potter, Clinton S; Safinya, Cyrus R

    2014-06-01

    Steric stabilization of cationic liposome-DNA (CL-DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL-DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL-DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL-DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL-DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL-DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Uptake and transfection efficiency of PEGylated cationic liposome–DNA complexes with and without RGD-tagging

    Science.gov (United States)

    Majzoub, Ramsey N.; Chan, Chia-Ling; Ewert, Kai K.; Silva, Bruno F. B.; Liang, Keng S.; Jacovetty, Erica L.; Carragher, Bridget; Potter, Clinton S.; Safinya, Cyrus R.

    2014-01-01

    Steric stabilization of cationic liposome–DNA (CL–DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL–DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL–DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL–DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL-DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and subsequent transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL–DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface. PMID:24661552

  9. Spatio-temporally controlled transfection by quantitative injection into a single cell.

    Science.gov (United States)

    Kwon, Hyosung; Park, Hang-soo; Yu, Jewon; Hong, Sunghoi; Choi, Yeonho

    2015-10-01

    Transfection-based cellular control has been widely used in biology; however, conventional transfection methods cannot control spatio-temporal differences in gene expression or the quantity of delivered materials such as external DNA or RNA. Here, we present a non-viral and spatio-temporally controlled transfection technique of a quantitative injection into a single cell. DNA was quantitatively injected into a single cell at a desired location and time, and the optimal gene delivery and expression conditions were determined based on the amount of the delivered DNA and the transfection efficacy. Interestingly, an injection of 1500 DNAs produced an about average 30% gene expression efficiency, which was the optimal condition, and gene expression was sustained for more than 14 days. In a single cell, fluorescent intensity and polymerase chain reaction (PCR) results were compared for the quantity of gene expression. The high coincidence of both results suggests that the fluorescence intensity can reveal gene expression level which was investigated by PCR. In addition, 3 multiple DNA genes were successfully expressed in a single cell with different ratio. Overall, these results demonstrate that spatio-temporally controlled transfection by quantitative transfection is a useful technique for regulating gene expression in a single cell, which suggests that this technique may be used for stem cell research, including the creation of induced pluripotent stem (iPS) cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Poly[N-(2-aminoethyl)ethyleneimine] as a New Non-Viral Gene Delivery Carrier : The Effect of Two Protonatable Nitrogens in the Monomer Unit on Gene Delivery Efficiency

    NARCIS (Netherlands)

    Khazaie, Yahya; Novo, Luis; van Gaal, Ethlinn; Fassihi, Afshin; Mirahmadi-Zareh, Seyedeh Zohreh; Esfahani, Mohammad Hossein Nasr; van Nostrum, Cornelus F.; Hennink, Wim E.; Dorkoosh, Farid

    2014-01-01

    Purpose. The aim of this study was to investigate the in vitro gene delivery efficiency of poly[N-(2-aminoethyl)ethylene-imine](PAEEI), a polymer with a linear Polyethyleneimine (LPEI) backbone and with aminoethyl side groups that has two protonatable nitrogen atoms per monomer unit instead of one

  11. Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

    OpenAIRE

    Hunt, Michelle A.; Currie, Margaret J.; Robinson, Bridget A.; Dachs, Gabi U.

    2010-01-01

    Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for trans...

  12. In vitro labeling of neural stem cells with poly-L-lysine coated super paramagnetic nanoparticles for green fluorescent protein transfection.

    Science.gov (United States)

    Albukhaty, Salim; Naderi-Manesh, Hossein; Tiraihi, Taki

    2013-04-01

    The magnetic nanoparticle-based transfection method is a relatively new technique for delivery of functional genes to target tissues. We aimed to evaluate the transfection efficiency of rat neural stem cell (NSC) using poly-L-lysine hydrobromide (PLL)-coated super paramagnetic iron oxide nanoparticles (SPION). The SPION was prepared and coated with PLL as transfection agent and the transfection efficiency was evaluated in rat NSC using enhanced green fluorescent protein-N1 plasmid containing GFP as a reporter gene. NSC was incubated for 24 h in cell culture media containing 25 µg/ml SPION and in different concentrations of PLL (0.25, 0.50, 0.75, 1 and 2 µg/ml). Cell viability was determined before and after transfection for every concentration using Trypan blue assay. Characterization of prepared uncoated (SPION) and coated (SPION-PLL) complexes were evaluated by a transmission electron microscope and the zeta potential. PLL at 0.75 μg/ml showed optimal results with 25 μg/ml SPION concentration compared with other PLL concentrations (0.25, 0.50, 1 and 2 μg/ml). The 18% efficiency of the transfected cells showed green fluorescence. Transfection with SPION is an efficient, non-viral gene transfere method.

  13. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.

    Science.gov (United States)

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-05-11

    Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.

  14. Development of a confocal ultrasound device using an inertial cavitation control for transfection in-vitro

    Science.gov (United States)

    Mestas, J. L.; Chettab, K.; Roux, S.; Prieur, F.; Lafond, M.; Dumontet, C.; Lafon, C.

    2015-12-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. We developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (peGFP- C1) in adherent and non-adherent cell lines. The frequency spectrum of the signal receive by a hydrophone is used to compute a cavitation index (CI) representative of the inertial cavitation activity. The influence of the CI on transfection efficiency, as well as reproducibility were determined. A real-time feedback loop control on CI was integrated in the process to regulate the cavitation level during sonoporation. In both adherent and non-adherent cell lines, the sonoporation device produced a highly efficient transfection of peGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. Moreover, the sonoporation of non-adherent cell lines Jurkat and K562 was found to be equivalent to nucleofection in terms of efficiency and toxicity while these two cell lines were resistant to transfection with lipofection.

  15. Discovery of Cationic Polymers for Non-viral Gene Delivery using Combinatorial Approaches

    Science.gov (United States)

    Barua, Sutapa; Ramos, James; Potta, Thrimoorthy; Taylor, David; Huang, Huang-Chiao; Montanez, Gabriela; Rege, Kaushal

    2015-01-01

    Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems. PMID:21843141

  16. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    Science.gov (United States)

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Diagnosis and management of common non-viral oral ulcerations ...

    African Journals Online (AJOL)

    The aetiology, diagnosis and management of the most common non-viral ulcerative disorders of the oral mucosa are discussed. These include traumatic ulcers, recurrent aphthous stomatitis, malignancy as well as oral ulceration associated with cutaneous pathology. South African Family Practice Vol. 49 (8) 2007: pp. 20-26 ...

  18. High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts

    DEFF Research Database (Denmark)

    Adler, Andrew F; Speidel, Alessondra T; Christoforou, Nicolas

    2011-01-01

    Optimization of nonviral gene delivery typically focuses on the design of particulate carriers that are endowed with desirable membrane targeting, internalization, and endosomal escape properties. Topographical control of cell transfectability, however, remains a largely unexplored parameter. Eme...

  19. Adhesion Receptors Mediate Efficient Non-viral Gene Delivery

    NARCIS (Netherlands)

    Zuhorn, Inge S.; Kalicharan, Dharamdajal; Robillard, George T.; Hoekstra, Dick

    2007-01-01

    For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors,

  20. Low Molecular Weight pDMAEMA-block-pHEMA Block-Copolymers Synthesized via RAFT-Polymerization: Potential Non-Viral Gene Delivery Agents?

    Directory of Open Access Journals (Sweden)

    Thomas Kissel

    2011-03-01

    Full Text Available The aim of this study was to investigate non-viral pDNA carriers based on diblock-copolymers consisting of poly(2-(dimethyl aminoethyl methacrylate (pDMAEMA and poly(2-hydroxyethyl methacrylate (pHEMA. Specifically the block-lengths and molecular weights were varied to determine the minimal requirements for transfection. Such vectors should allow better transfection at acceptable toxicity levels and the entire diblock-copolymer should be suitable for renal clearance. For this purpose, a library of linear poly(2-(dimethyl aminoethyl methacrylate-block-poly(2-hydroxyl methacrylate (pDMAEMA-block-pHEMA copolymers was synthesized via RAFT (reversible addition-fragmentation chain transfer polymerization in a molecular weight (Mw range of 17–35.7 kDa and analyzed using 1H and 13C NMR (nuclear magnetic resonance, ATR (attenuated total reflectance, GPC (gel permeation chromatography and DSC (differential scanning calorimetry. Copolymers possessing short pDMAEMA-polycation chains were 1.4–9.7 times less toxic in vitro than polyethylenimine (PEI 25 kDa, and complexed DNA into polyplexes of 100–170 nm, favorable for cellular uptake. The DNA-binding affinity and polyplex stability against competing polyanions was comparable with PEI 25 kDa. The zeta-potential of polyplexes of pDMAEMA-grafted copolymers remained positive (+15–30 mV. In comparison with earlier reported low molecular weight homo pDMAEMA vectors, these diblock-copolymers showed enhanced transfection efficacy under in vitro conditions due to their lower cytotoxicity, efficient cellular uptake and DNA packaging. The homo pDMAEMA115 (18.3 kDa self-assembled with DNA into small positively charged polyplexes, but was not able to transfect cells. The grafting of 6 and 57 repeating units of pHEMA (0.8 and 7.4 kDa to pDMAEMA115 increased the transfection efficacy significantly, implying a crucial impact of pHEMA on vector-cell interactions. The intracellular trafficking, in vivo transfection

  1. Biodegradable Nanoparticles of mPEG-PLGA-PLL Triblock Copolymers as Novel Non-Viral Vectors for Improving siRNA Delivery and Gene Silencing

    Directory of Open Access Journals (Sweden)

    Qiu-Sheng Shi

    2012-01-01

    Full Text Available Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs made from monomethoxypoly(ethylene glycol-poly(lactic-co-glycolic acid-poly-l-lysine (mPEG-PLGA-PLL triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

  2. Biodegradable Nanoparticles of mPEG-PLGA-PLL Triblock Copolymers as Novel Non-Viral Vectors for Improving siRNA Delivery and Gene Silencing

    Science.gov (United States)

    Du, Jing; Sun, Ying; Shi, Qiu-Sheng; Liu, Pei-Feng; Zhu, Ming-Jie; Wang, Chun-Hui; Du, Lian-Fang; Duan, You-Rong

    2012-01-01

    Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing. PMID:22312268

  3. Normalization of transfection efficiency using the beta-lactamase gene of the pGL3 luciferase vector in primary anterior pituitary cells.

    Science.gov (United States)

    Kang, Seong W; You, Seungkwon; Wong, Eric A; El Halawani, Mohamed E

    2002-08-01

    The beta-galactosidase reporter gene is commonly used as a control for transfection efficiency in the promoter reporter assay system. While investigating vasoactive intestinal peptide response elements in the promoter of the prolactin gene, we found that primary pituitary cells from turkey hens highly expressed endogenous beta-galactosidase. Therefore, we developed a new protocol for determining transfection efficiency using the beta-lactamase gene, which is present on many expression vectors. Transcript levels of beta-lactamase were measured by RT-PCR after transfection of different amounts of the pGL3-basic and pGL3-control vectors. A high correlation was observed between the amount of plasmid transfected and beta-lactamase mRNA levels. Although no eukaryotic promoter was present, there was apparently leaky expression of the beta-lactamase gene. Expression of beta-lactamase was independent of expression from the simian virus 40 or turkey prolactin promoters cloned upstream of the luciferase gene.

  4. Non-viral delivery of genome-editing nucleases for gene therapy.

    Science.gov (United States)

    Wang, M; Glass, Z A; Xu, Q

    2017-03-01

    Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

  5. Microencapsulation of recombinant adenovirus within poly-DL-lactide-poly(ethylene glycol) microspheres for enhanced gene transfection efficiency and inhibitory effects on hepatocellular carcinoma cells in vitro.

    Science.gov (United States)

    Xia, Dong; Feng, Li-Bo; Wu, Xiao-Long; Xia, Guo-Dong; Xu, Liang

    2015-08-01

    When gene therapy is performed for the treatment of malignant tumors, gene transfer efficiency and selectivity are highly important. Polymer vehicle microspheres are a novel type of therapy, which have been developed rapidly in recent years and are able to control drug release, prolong the biological half-life of drugs, decrease side effects and achieve targeted delivery. The present study was designed to construct a polymer microsphere-encapsulated recombinant adenovirus with human tissue inhibitors of the matrix metalloproteinase-1 (TIMP-1) gene, and to discuss its characterization for the purpose of liver cancer gene therapy. The microsphere was prepared from biodegradable poly-DL-lactide-poly(ethylene glycol) (PELA) encapsulating rAdTIMP-1, the recombinant adenovirus carrying TIMP-1, by a modified double-emulsion method. The particle morphology, diameter, virus encapsulation, loading rate and release kinetics of the rAd-microspheres were determined in vitro. Hepatocellular carcinoma (HCC) HepG2 cells were transfected with the rAd-microsphere and the efficiency of transfection was assessed by fluorescent microscopy. The production and expression of TIMP-1 was identified by gelatin zymography and western blot analysis, and the invasiveness was detected by a matrigel matrix invasion assay. The microsphere encapsulating rAdTIMP-1 was successfully constructed with a diameter of 1.965 μm, encapsulation efficiency of 60.0%, a viral load of 10.5 x 10(8)/mg, a virus release of ~60% within 120 h and a total release time of >240 h. The resultant rAd-microspheres were able to efficiently transfect HepG2 cells with the transfection efficiency enhanced by ~90%. As a result, the transfected HepG2 cells had significantly increased TIMP-1 enzyme activity and the expression of TIMP-1 was detected by western blot analysis. In addition, the proliferation and invasion ability of the HCC cells was markedly inhibited by the rAd-microspheres. The resultant rAd-microspheres, PELA

  6. Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.

    Science.gov (United States)

    Teo, Pei Yun; Yang, Chuan; Hedrick, James L; Engler, Amanda C; Coady, Daniel J; Ghaem-Maghami, Sadaf; George, Andrew J T; Yang, Yi Yan

    2013-10-01

    ~100% substitution of primary amine groups leads to significantly lower gene transfection efficiency. These findings provide insights to modification of PEI for development of effective and non-cytotoxic non-viral vectors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. The shape and size effects of polycation functionalized silica nanoparticles on gene transfection.

    Science.gov (United States)

    Lin, Xinyi; Zhao, Nana; Yan, Peng; Hu, Hao; Xu, Fu-Jian

    2015-01-01

    Silica nanoparticles are attractive candidates for the development of safe and efficient non-viral gene carriers, owing to their controlled morphologies, potential of facile surface modification and excellent biocompatibility as well as in vivo biodegradability. Conversely, the size and shape of nanoparticles are considered to have an intense influence on their interaction with cells and biological systems, but the effects of particle size and shape on gene transfection are poorly understood. In this work, a series of novel gene carriers were designed employing polycation modified silica nanoparticles with five different morphologies, while keeping uniform zeta potential and surface functionality. Then the effects of particle size and shape of these five different carriers on gene transfection were investigated. The morphology of silica nanoparticles is demonstrated to play an important role in gene transfection, especially when the amount of polycation is low. Chiral nanorods with larger aspect ratio were found to fabricate the most efficient gene carriers with compromised cytotoxicity. It was also noted that hollow nanosphere-based carriers exhibited better gene transfection performance than did solid counterparts. These results may provide new strategies to develop promising gene carriers and useful information for the application of nanoparticles in biomedical areas. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  8. Peptide Dendrimer/Lipid Hybrid Systems Are Efficient DNA Transfection Reagents: Structure–Activity Relationships Highlight the Role of Charge Distribution Across Dendrimer Generations

    Science.gov (United States)

    2013-01-01

    Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure–activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6–10-fold over commercial reagents under their respective optimal conditions. Emerging structure–activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity. PMID:23682947

  9. Graphene and carbon nanotube nanocomposite for gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Hollanda, L.M. [Laboratory of Biotechnology, Department of Biochemistry, Institute of Biology at UNICAMP, Rua Monteiro Lobato 255, Campinas, SP CEP 13083-862 (Brazil); Lobo, A.O. [Laboratory of Biomedical Nanotechnology, Institute of Research and Development at the UNIVAP, Av. Shishima Hifumi, 2911, CEP: 12244-000 Sao Jose dos Campos, SP (Brazil); Lancellotti, M. [Laboratory of Biotechnology, Department of Biochemistry, Institute of Biology at UNICAMP, Rua Monteiro Lobato 255, Campinas, SP CEP 13083-862 (Brazil); Berni, E. [Biological Chemistry Laboratory, Department of Physical Chemistry, Institute of Chemistry at UNICAMP, R. José de Castro, Campinas, SP CEP 13083-970 (Brazil); Corat, E.J. [Associated Laboratory of Sensors and Materials of the INPE, Av. dos Astronautas 1758, Sao Jose dos Campos CEP: 12227-010 SP (Brazil); Zanin, H., E-mail: hudsonzanin@gmail.com [Associated Laboratory of Sensors and Materials of the INPE, Av. dos Astronautas 1758, Sao Jose dos Campos CEP: 12227-010 SP (Brazil)

    2014-06-01

    Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5 m{sup 2}/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency. - Highlights: • Facile water dispersible GCN was prepared exfoliating MWCNTs by oxygen plasma etching. • Oxygen content groups were attached on the GCN surfaces changing its hydrophobicity. • Low concentrations of GCN showed low cytotoxicity for NIH-3T3 and NG97 line cells. • The GCN and pIRES plasmid conjugated with the GFP gene. • Gene transfection was studied using this new material.

  10. Protein expression following non-viral delivery of plasmid DNA coding for basic FGF and BMP-2 in a rat ectopic model.

    Science.gov (United States)

    Rose, Laura C; Kucharski, Cezary; Uludağ, Hasan

    2012-04-01

    Non-viral delivery of genes involved in stimulation of bone formation has been pursued for clinical bone repair, but no effort has been made to assess protein expression levels after in vivo delivery. This is critical to better understand gene delivery efficiencies and to compare different modes of non-viral delivery. This study investigated expression levels of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) after delivering expression vectors (plasmid DNA) with polymeric carriers in a rat subcutaneous implant model. The polymers used were a 2 kDa molecular weight polyethylenimine modified with linoleic acid (PEI-LA) and the 25 kDa PEI (PEI25) used for non-viral gene delivery in animal models. The PEI-LA mediated delivery of the plasmid DNAs in 293T cells led to ∼3.5 and ∼13 ng/10(6) cells/day secretion of bFGF and BMP-2 in vitro, respectively. Using the reporter protein, Green Fluorescence Protein (GFP), transfection in implants was readily detected by the presence of GFP-positive cells and a polymeric carrier was needed for this GFP expression. No bFGF and BMP-2 were detected in the scaffolds due to high background in detection assays and/or rapid diffusion of the secreted proteins from the implant site. However, using an ex vivo culture system, significant levels of BMP-2, but not bFGF, secretion were observed from the scaffolds. The BMP-2 secretion from PEI-LA delivered expression vector was equivalent and/or superior to PEI25 depending on the plasmid DNA implant dose. Gelatin scaffolds were able to sustain ∼0.3 ng/sponge/day BMP-2 secretion as compared to collagen scaffolds (∼0.1 ng/sponge/day). These values were equivalent to secretion rates reported with some viral delivery systems from independent studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Ultrasound mediated gene transfection

    Science.gov (United States)

    Williamson, Rene G.; Apfel, Robert E.; Brandsma, Janet L.

    2002-05-01

    Gene therapy is a promising modality for the treatment of a variety of human diseases both inherited and acquired, such as cystic fibrosis and cancer. The lack of an effective, safe method for the delivery of foreign genes into the cells, a process known as transfection, limits this effort. Ultrasound mediated gene transfection is an attractive method for gene delivery since it is a noninvasive technique, does not introduce any viral particles into the host and can offer very good temporal and spatial control. Previous investigators have shown that sonication increases transfection efficiency with and without ultrasound contrast agents. The mechanism is believed to be via a cavitation process where collapsing bubble nuclei permeabilize the cell membrane leading to increased DNA transfer. The research is focused on the use of pulsed wave high frequency focused ultrasound to transfect DNA into mammalian cells in vitro and in vivo. A better understanding of the mechanism behind the transfection process is also sought. A summary of some in vitro results to date will be presented, which includes the design of a sonication chamber that allows us to model the in vivo case more accurately.

  12. In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by gene-gun biolistics

    NARCIS (Netherlands)

    K. Eizema (Karin); H.A. van Heugten (Han); K. Bezstarosti (Karel); M.C. van Setten (Marga); J.M.J. Lamers (Jos)

    1999-01-01

    textabstractThe transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the

  13. A family of cationic polyamides for in vitro and in vivo gene transfection.

    Science.gov (United States)

    Zhang, Chengnan; Jin, Rong; Zhao, Peng; Lin, Chao

    2015-08-01

    The purpose of this study is to develop biodegradable cationic polyamides for non-viral gene delivery and elucidate their structural effects on gene transfection activity. To this end, a group of novel cationic polyamides were synthesized by polycondensation reaction between different di-p-nitrophenyl esters and tertiary amine-containing primary diamines. These linear polyamides have flexible alkylene group (ethylene or propylene), protonable amino group and bioreducible disulfide linkage in the polyamide main chain. The alkylene group and disulfide linkage in these polyamides have a distinct effect on their gene delivery properties including buffering capacity, gene binding ability and intracellular gene release profile. Those cationic polyamides containing disulfide linkage and 1,4-bis(3-aminopropyl)piperazine (BAP) residue exhibited high buffering capacity (endosomal escape ability), high gene binding ability, and intracellular gene release ability, thus inducing fast gene nucleus translocation and robust gene transfection in vitro against different cell lines and rat bone marrow mesenchymal stem cells. Moreover, the transfection efficiencies in vitro were comparable or higher than those of 25 kDa branched polyethylenimine and Lipofectamine 2000 transfection agent as positive controls. These cationic polyamides and their polyplexes were of low cytotoxicity when an optimal transfection efficacy was achieved. In vivo transfection tests showed that bioreducible BAP-based polyamides were applicable for intravenous gene delivery in a mouse model, leading to higher level of transgene expression in the liver as compared to 22 kDa linear polyethylenimine as a positive control. These cationic polyamides provide a useful platform to elucidate the relationship between chemical functionalities and gene transfection activity. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  14. Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes.

    Science.gov (United States)

    Penacho, Nuno; Simões, Sérgio; de Lima, Maria C Pedroso

    2009-05-01

    Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.

  15. Tunable pDNA/DODAB:MO lipoplexes: the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency.

    Science.gov (United States)

    Silva, João P Neves; Oliveira, Ana C N; Lúcio, Marlene; Gomes, Andreia C; Coutinho, Paulo J G; Oliveira, M Elisabete C D Real

    2014-09-01

    Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293T mammalian cell line with pSV-β-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODAB's tendency to form bilayer structures and MO's propensity to form inverted non-lamellar structures. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic light scattering (DLS), zeta (ζ) potential, cryo-transmission electron microscopy (cryo-TEM) and ethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50°C. Experimental results indicate that pDNA:DODAB:MO's structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50°C. Increase in temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation, causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but also its transfection efficiency. Altogether, these results

  16. Correlation between cationic lipid-based transfection and cell division.

    Science.gov (United States)

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. Copyright © 2016. Published by Elsevier Inc.

  17. An Evaluation on Transfection Efficiency of pHRE-Egr1-EGFP in Hepatocellular Carcinoma Cells Bel-7402 Mediated by PEI-MZF-NPs

    Directory of Open Access Journals (Sweden)

    Mei Lin

    2011-01-01

    Full Text Available To improve transfection and expression efficiency of target gene, especially under cancer anoxic microenvironment, we have developed pHRE-Egr1-EGFP/PEI-MZF-NPs nanosystem, in which pHRE-Egr1-EGFP, eukaryotic gene expression plasmid, is constructed by combining radiation promoter Egr1 with anoxia induction components (HRE, forming anoxic radiation double sensitive HRE/Egr1 promoter to activate reporter gene EGFP expression. MZF-NPs (Mn0.5 Zn0.5 Fe2O4 magnetic nanoparticles, obtained by coprecipitation method, are coated with cation poly(ethylenimine (PEI. We transferred pHRE-Egr1-EGFP into hepatocellular carcinoma Bel-7402 cells, using PEI-MZF-NPs as the carrier and tested some relevant efficacy. The results show that PEI-MZF-NPs have good DNA-binding ability, protection ability, release ability, little toxicity, and high transfection efficiency, obviously superior to those of the liposome method and electricity perforation method. Moreover, the expression level of EGFP gene induced by anoxia and radiation was significantly higher than that of single radiation activation. It is therefore concluded that HRE/Egr1 can induce and improve target gene expression efficiency in cancer anoxic microenvironment, and that PEI-MZF-NPs can be used as a novel nonviral gene vector which offers a viable approach to the mediated radiation gene therapy of cancer.

  18. A novel Poly(ε-caprolactone-Pluronic-Poly(ε-caprolactone grafted Polyethyleneimine(PCFC-g-PEI, Part 1, synthesis, cytotoxicity, and in vitro transfection study

    Directory of Open Access Journals (Sweden)

    Zhao Xia

    2009-07-01

    Full Text Available Abstract Background Polyethyleneimine (PEI, a cationic polymer, is one of the successful and widely used vectors for non-viral gene transfection in vitro. However, its in vivo application was greatly limited due to its high cytotoxicity and short duration of gene expression. To improve its biocompatibility and transfection efficiency, PEI has been modified with PEG, folic acid, and chloroquine in order to improve biocompatibility and enhance targeting. Results Poly(ε-caprolactone-Pluronic-Poly(ε-caprolactone (PCFC was synthesized by ring-opening polymerization, and PCFC-g-PEI was obtained by Michael addition reaction with GMA-PCFC-GMA and polyethyleneimine (PEI, 25 kD. The prepared PCFC-g-PEI was characterized by 1H-NMR, SEC-MALLS. Meanwhile, DNA condensation, DNase I protection, the particle size and zeta potential of PCFC-g-PEI/DNA complexes were also determined. According to the results of flow cytometry and MTT assay, the synthesized PCFC-g-PEI, with considerable transfection efficiency, had obviously lower cytotoxicity against 293 T and A549 cell lines compared with that of PEI 25 kD. Conclusion The cytotoxicity and in vitro transfection study indicated that PCFC-g-PEI copolymer prepared in this paper was a novel gene delivery system with lower cytotoxicity and considerable transfection efficiency compared with commercial PEI (25 kD.

  19. Zn(II)-dipicolylamine-based metallo-lipids as novel non-viral gene vectors.

    Science.gov (United States)

    Su, Rong-Chuan; Liu, Qiang; Yi, Wen-Jing; Zhao, Zhi-Gang

    2017-08-01

    In this study, a series of Zn(II)-dipicolylamine (Zn-DPA) based cationic lipids bearing different hydrophobic tails (long chains, α-tocopherol, cholesterol or diosgenin) were synthesized. Structure-activity relationship (SAR) of these lipids was studied in detail by investigating the effects of several structural aspects including the type of hydrophobic tails, the chain length and saturation degree. In addition, several assays were used to study their interactions with plasmid DNA, and results reveal that these lipids could condense DNA into nanosized particles with appropriate size and zeta-potentials. MTT-based cell viability assays showed that lipoplexes 5 had low cytotoxicity. The in vitro gene transfection studies showed the hydrophobic tails clearly affected the TE, and hexadecanol-containing lipid 5b gives the best TE, which was 2.2 times higher than bPEI 25k in the presence of 10% serum. The results not only demonstrate that these lipids might be promising non-viral gene vectors, but also afford us clues for further optimization of lipidic gene delivery materials.

  20. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

    Science.gov (United States)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

    2017-07-01

    Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

  1. Bioactive Core-Shell Nanofiber Hybrid Scaffold for Efficient Suicide Gene Transfection and Subsequent Time Resolved Delivery of Prodrug for Anticancer Therapy.

    Science.gov (United States)

    Sukumar, Uday Kumar; Packirisamy, Gopinath

    2015-08-26

    Nanofiber scaffold's ability to foster seemingly nonexistent interface with the cells enables them to effectively deliver various bioactive molecules to cells in the vicinity. Among such bioactive molecules, therapeutically active nucleic acid has been the most common candidate. In spite of such magnanimous efforts in this field, it remains a paradox that suicide gene delivery by nanofibers has never been sought for anticancer application. To investigate such a possibility, in the present work, a composite core-shell nanofiberous scaffold has been realized which could efficiently transfect suicide gene into cancer cells and simultaneously deliver prodrug, 5-Fluorocytosine (5-FC) in a controlled and sustained manner. The scaffold's ability to instigate apoptosis by suicide gene therapy in nonsmall lung cancer cells (A549) was ascertained at both phenotypic and genotypic levels. A cascade of events starting from suicide gene polyplex release from nanofibers, transfection, and expression of cytosine deaminase-uracil phosphoribosyltransferase (CD::UPRT) suicide gene by A549; subsequent prodrug release; and its metabolic conversion into toxic intermediates which finally culminates in host cells apoptosis has been monitored in a time-dependent manner. This work opens up new application avenues for nanofiber-based scaffolds which can effectively manage cancer prognosis.

  2. Fundamental studies on the synthesis, characterization, stabilization, 3-D scaffolds, and trafficking mechanisms of nano-structured calcium phosphates (NanoCaPs) for non-viral gene delivery

    Science.gov (United States)

    Olton, Dana

    Non-viral transfer of therapeutic genes into mammalian cells represents a potentially viable approach to (1) treat and cure acute and chronic genetically transferred congenital disorders and to (2) aid in tissue regeneration. Non-viral vectors have been praised for their potential to circumvent some of the limitations associated with viral vectors including immunogenicity, cytotoxicity and insertional mutagenesis. Among the various types of non-viral gene delivery vectors, nano-structured ceramic particles, particularly, particles of calcium phosphate (CaP) remain an attractive option because of their safety, biocompatibility, biodegradability, ease of handling as well as their adsorptive capacity for DNA. CaP-DNA complexes have been used in vitro since the 1970s and have recently been tested in vivo. However, despite CaPs' extensive use, concerns still remain regarding the synthesis and colloidal instability of this vector. Also, towards the development of a more efficient gene delivery agent, there is a need to understand the mechanisms involved in both the cellular uptake as well as in the subsequent intracellular processing of CaP-DNA complexes. Moreover, although significant advances have been made in the synthesis and design of tissue engineered constructs, the development of a safe, effective scaffold has yet to be realized. As such, the focus of this thesis has been to address these four concerns. In this work, we begin by presenting a novel aqueous-based approach to synthesize nano-particles of CaP (NanoCaPs). Our results show that this approach generates nano-crystalline hydroxyapatite particles. When tested in vitro, transfection of these complexes resulted in higher, more consistent levels of gene expression when compared to particles synthesized via manual mixing. The optimized forms of these particles both effectively bound (90% efficient) and condensed (70% efficient) plasmid DNA (pDNA) and possessed negative zeta potentials of approximately -20m

  3. Oral sex and transmission of non-viral STIs

    Science.gov (United States)

    Edwards, S.; Carne, C.

    1998-01-01

    OBJECTIVES: To review the literature on the role of oral sex in the transmission of non-viral sexually transmitted infections (STIs). METHOD: A Medline search was performed using the keywords oro-genital sex, and those specific to each infection. Further references were then taken from each article read. CONCLUSIONS: Oral sex is a common sexual practice between both heterosexual and homosexual couples. Oro-genital sex is implicated as a route of transmission for gonorrhoea, syphilis, Chlamydia trachomatis, chancroid, and Neisseria meningitidis. Other respiratory organisms such as streptococci, Haemophilus influenzae, and Mycoplasma pneumoniae could also be transmitted by this route. Fellatio confers risk for acquisition of infection by the oral partner. Cunnilingus appears to predispose to recurrent vaginal candidiasis although the mechanism for this is unclear, while a link between oro-genital sex and bacterial vaginosis is currently being studied. Oro-anal sex is implicated in the transmission of various enteric infections. In view of the increased practice of oral sex this has become a more important potential route of transmission for oral, respiratory, and genital pathogens. 


 PMID:9634339

  4. The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine.

    Science.gov (United States)

    Mochizuki, Shinichi; Kanegae, Naho; Nishina, Koichi; Kamikawa, Yumi; Koiwai, Kazunori; Masunaga, Hiroyasu; Sakurai, Kazuo

    2013-02-01

    Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE=1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

    Science.gov (United States)

    Ng, Quinn Kwan Tai

    Gene transfer or gene delivery is described as the process in which foreign DNA is introduced into cells. Over the years, gene delivery has gained the attention of many researchers and has been developed as powerful tools for use in biotechnology and medicine. With the completion of the Human Genome Project, such advances in technology allowed for the identification of diseases ranging from hereditary disorders to acquired ones (cancer) which were thought to be incurable. Gene therapy provides the means necessary to treat or eliminate genetic diseases from its origin, unlike traditional medicine which only treat symptoms. With ongoing clinical trials for gene therapy increasing, the greatest difficulty still lies in developing safe systems which can target cells of interest to provide efficient delivery. Nature, over millions of years of evolution, has provided an example of one of the most efficient delivery systems: viruses. Although the use of viruses for gene delivery has been well studied, the safety issues involving immunogenicity, insertional mutagenesis, high cost, and poor reproducibility has provided problems for their clinical application. From understanding viruses, we gain insight to designing new systems for non-viral gene delivery. One of these techniques utilized by adenoviruses is the clustering of ligands on its surface through the use of a protein called a penton base. Through the use of nanotechnology we can mimic this basic concept in non-viral gene delivery systems. This dissertation research is focused on developing and applying a novel system for displaying the integrin binding ligand (RGD) in a constrained manner to form a clustered integrin ligand binding platform to be used to enhance the targeting and efficiency of non-viral gene delivery vectors. Peptide mixed monolayer protected gold nanoparticles provides a suitable surface for ligand clustering. A relationship between the peptide ratios in the reaction solution used to form these

  6. Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

    Directory of Open Access Journals (Sweden)

    Busch Melanie

    2009-12-01

    Full Text Available Abstract Background Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC on complete Freund's adjuvant-induced inflammatory pain in rats. Results POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin. Conclusion Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.

  7. Droplet-based Microfluidic Systems for Production and Transfection In Vitro of Non-Viral Vectors for Gene Delivery

    OpenAIRE

    Hala Hamed; Amani Miskey; Razan Alkurd; Zena Ghazal; Razan Sami; Nageed Abduljaleel; Moayad Shahwan

    2015-01-01

    Sleeping is a natural repetitive state of rest for the mind and body which is essential to life. Sufficient sleep is extremely important for one’s mental and physical health. But sleep loss is a remarkable problem in modern society. The objective of this study was designed to encourage students to seek healthier sleep habits, by using academic success as indicator. It was a cross sectional study conducted among different studying levels of AUST undergraduate medical students during February 2...

  8. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    Science.gov (United States)

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

  9. Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

    OpenAIRE

    Perkins, J.B.; Dean, D. H.

    1983-01-01

    DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.

  10. In vitro non-viral lipofectamine delivery of the gene for glial cell line-derived neurotrophic factor to human umbilical cord blood CD34+ cells.

    Science.gov (United States)

    Yu, Guolong; Borlongan, Cesar V; Ou, Yali; Stahl, Christine E; Yu, SeongJin; Bae, EungKyung; Kaneko, Yuji; Yang, Tianlun; Yuan, Chunjun; Fang, Li

    2010-04-14

    Using a lipofection technique, we explored a non-viral delivery of plasmid DNA encoding a rat pGDNF (glial cell line-derived neurotrophic factor) to CD34+ cells derived from human umbilical cord blood (HUCB) cells in order to obtain cells stably expressing the GDNF gene. The target gene GDNF was amplified from cortex cells of newborn Sprague-Dawley rats by reverse transcriptase polymerase chain reaction (RT-PCR) and inserted into vector pEGFP-N1 to construct the eukaryotic expression vector pEGFP/GDNF. The positive clones were identified by sequencing and endonuclease digestion. The expression of pEGFP/GDNF-transfected HUCB cells CD34+ was examined by ELISA. Single fragment of 640 bp was obtained after the rat GDNF cDNA was amplified by RT-PCR. Two fragments of about 4.3 kb and 640 pb were obtained after digestion of recombinant plasmid pEGFP/GDNF with XhoI/KpnI. The nucleic acid fragment of 640 bp was confirmed to agree well with the sequence of GDNF gene published by GenBank. The expression of GDNF mRNA and the level of GDNF from pEGFP/GDNF-transfected CD34+ cells were increased substantially, compared with pEGFP control plasmid transfected CD34+ cells (P<0.05). Moreover, co-culture of primary rat cells with the pEGFP/GDNF-transfected CD34+ cells promoted enhanced neuroprotection against oxygen-glucose deprivation induced cell dysfunctions. The present results support the use of the non-viral plasmid liposome for therapeutic gene expression for stem cell therapy. Copyright 2010 Elsevier B.V. All rights reserved.

  11. Non-virally engineered human adipose mesenchymal stem cells produce BMP4, target brain tumors, and extend survival.

    Science.gov (United States)

    Mangraviti, Antonella; Tzeng, Stephany Y; Gullotti, David; Kozielski, Kristen L; Kim, Jennifer E; Seng, Michael; Abbadi, Sara; Schiapparelli, Paula; Sarabia-Estrada, Rachel; Vescovi, Angelo; Brem, Henry; Olivi, Alessandro; Tyler, Betty; Green, Jordan J; Quinones-Hinojosa, Alfredo

    2016-09-01

    There is a need for enabling non-viral nanobiotechnology to allow safe and effective gene therapy and cell therapy, which can be utilized to treat devastating diseases such as brain cancer. Human adipose-derived mesenchymal stem cells (hAMSCs) display high anti-glioma tropism and represent a promising delivery vehicle for targeted brain tumor therapy. In this study, we demonstrate that non-viral, biodegradable polymeric nanoparticles (NPs) can be used to engineer hAMSCs with higher efficacy (75% of cells) than leading commercially available reagents and high cell viability. To accomplish this, we engineered a poly(beta-amino ester) (PBAE) polymer structure to transfect hAMSCs with significantly higher efficacy than Lipofectamine™ 2000. We then assessed the ability of NP-engineered hAMSCs to deliver bone morphogenetic protein 4 (BMP4), which has been shown to have a novel therapeutic effect by targeting human brain tumor initiating cells (BTIC), a source of cancer recurrence, in a human primary malignant glioma model. We demonstrated that hAMSCs genetically engineered with polymeric nanoparticles containing BMP4 plasmid DNA (BMP4/NP-hAMSCs) secrete BMP4 growth factor while maintaining their multipotency and preserving their migration and invasion capacities. We also showed that this approach can overcome a central challenge for brain therapeutics, overcoming the blood brain barrier, by demonstrating that NP-engineered hAMSCs can migrate to the brain and penetrate the brain tumor after both intranasal and systemic intravenous administration. Critically, athymic rats bearing human primary BTIC-derived tumors and treated intranasally with BMP4/NP-hAMSCs showed significantly improved survival compared to those treated with control GFP/NP-hAMCSs. This study demonstrates that synthetic polymeric nanoparticles are a safe and effective approach for stem cell-based cancer-targeting therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Novel non-viral gene delivery systems composed of carbosilane dendron functionalized nanoparticles prepared from nano-emulsions as non-viral carriers for antisense oligonucleotides.

    Science.gov (United States)

    Fornaguera, Cristina; Grijalvo, Santiago; Galán, Marta; Fuentes-Paniagua, Elena; de la Mata, Francisco Javier; Gómez, Rafael; Eritja, Ramon; Calderó, Gabriela; Solans, Conxita

    2015-01-15

    The development of novel and efficient delivery systems is often the limiting step in fields such as antisense therapies. In this context, poly(d,l-lactide-co-glycolide) acid (PLGA) nanoparticles have been obtained by a versatile and simple technology based on nano-emulsion templating and low-energy emulsification methods, performed in mild conditions, providing good size control. O/W polymeric nano-emulsions were prepared by the phase inversion composition method at 25°C using the aqueous solution/polysorbate80/[4 wt% PLGA in ethyl acetate] system. Nano-emulsions formed at oil-to-surfactant (O/S) ratios between 10/90-90/10 and aqueous contents above 70 wt%. Nano-emulsion with 90 wt% of aqueous solution and O/S ratio of 70/30 was chosen for further studies, since they showed the appropriate characteristics to be used as nanoparticle template: hydrodynamic radii lower than 50 nm and enough kinetic stability. Nanoparticles, prepared from nano-emulsions by solvent evaporation, showed spherical shape, sizes about 40 nm, negative surface charges and high stability. The as-prepared nanoparticles were functionalized with carbosilane cationic dendrons through a carbodiimide-mediated reaction achieving positively charged surfaces. Antisense oligonucleotides were electrostatically attached to nanoparticles surface to perform gene-silencing studies. These complexes were non-haemolytic and non-cytotoxic at the concentrations required. The ability of the complexes to impart cellular uptake was also promising. Therefore, these novel nanoparticulate complexes might be considered as potential non-viral carriers in antisense therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives.

    Science.gov (United States)

    Khan, Mohammed A; Wu, Victoria M; Ghosh, Shreya; Uskoković, Vuk

    2016-06-01

    Despite the long history of nanoparticulate calcium phosphate (CaP) as a non-viral transfection agent, there has been limited success in attempts to optimize its properties for transfection comparable in efficiency to that of viral vectors. Here we focus on the optimization of: (a) CaP nanoparticle precipitation conditions, predominantly supersaturation and Ca/P molar ratios; (b) transfection conditions, mainly the concentrations of the carrier and plasmid DNA; (c) the presence of surface additives, including citrate anion and cationic poly(l-lysine) (PLL). CaP nanoparticles significantly improved transfection with plasmid DNA encoding enhanced green fluorescent protein (eGFP) in pre-osteoblastic MC3T3-E1 cells compared to a commercial non-viral carrier. At the same time they elicited significantly lesser cytotoxicity than the commercial carrier. Plasmid DNA acted as a nucleation promoter, decreasing the nucleation lag time of metastable CaP solutions and leading to a higher rate of nucleation and a lower size of the precipitated particles. The degree of supersaturation (DS) of 15 was found to be more optimal for transfection than that of 12.5 or 17.5 and higher. Because CaP particles precipitated at DS 15 were spherical, while DS 17.5 and 21 yielded acicular particles, it was concluded that spherical particle morphologies were more conducive to transfection than the anisotropic ones. Even though the yield at DS 15 was 10 and 100 times lower than that at DS 17.5 and 21, respectively, transfection rates were higher using CaP nanoparticle colloids prepared at DS 15 than using those made at higher or lower DS, indicating that the right particle morphology can outweigh the difference in the amount of the carrier, even when this difference is close to 100×. In contrast to the commercial carrier, the concentration of CaP-pDNA delivered to the cells was directly proportional to the transfection rate. Osteosarcoma K7M2 cells were four times more easily transfectable with

  14. Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

    Science.gov (United States)

    Pärn, Kalle; Viru, Liane; Lehto, Taavi; Oskolkov, Nikita; Langel, Ülo; Merits, Andres

    2013-01-01

    Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells. PMID:23861978

  15. Transfection of infectious RNA and DNA/RNA layered vectors of semliki forest virus by the cell-penetrating peptide based reagent PepFect6.

    Directory of Open Access Journals (Sweden)

    Kalle Pärn

    Full Text Available Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.

  16. Transfection of infectious RNA and DNA/RNA layered vectors of semliki forest virus by the cell-penetrating peptide based reagent PepFect6.

    Science.gov (United States)

    Pärn, Kalle; Viru, Liane; Lehto, Taavi; Oskolkov, Nikita; Langel, Ülo; Merits, Andres

    2013-01-01

    Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.

  17. RNAi in murine hepatocytes: the agony of choice--a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.

    Science.gov (United States)

    Böttger, Jan; Arnold, Katrin; Thiel, Carlo; Rennert, Christiane; Aleithe, Susanne; Hofmann, Ute; Vlaic, Sebastian; Sales, Susanne; Shevchenko, Andrej; Matz-Soja, Madlen

    2015-09-01

    Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4%), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

  18. Internalization of novel non-viral vector TAT-streptavidin into human cells

    Directory of Open Access Journals (Sweden)

    Kulomaa Markku S

    2007-01-01

    Full Text Available Abstract Background The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT47–57-streptavidin (TAT-SA, 60 kD. SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes. Results By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid (PPAA, nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs. Conclusion This study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells.

  19. Hydrodynamics based transfection in normal and fibrotic rats

    OpenAIRE

    Yeikilis, Rita; Gal, Shunit; Kopeiko, Natalia; Paizi, Melia; Pines, Mark; Braet, Filip; Spira, Gadi

    2006-01-01

    AIM: Hydrodynamics based transfection (HBT), the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated.

  20. Optimization of renal transfection using a renal suction-mediated transfection method in mice.

    Science.gov (United States)

    Taniguchi, Yota; Kawakami, Shigeru; Fuchigami, Yuki; Oyama, Natsuko; Yamashita, Fumiyoshi; Konishi, Satoshi; Shimizu, Kazunori; Hashida, Mitsuru

    2016-01-01

    We previously developed a suction-mediated transfection method in mice. The purpose of this study was to optimize the suction-mediated transfection conditions using a pressure-controlled computer system for efficient and safe kidney-targeted gene delivery in mice. Naked pCMV-Luc was injected into the tail vein in mice, and then the right kidney was suctioned by a device of the suction pressure-controlled system. The effects of renal transfection conditions, such as the suction pressure degree, suction pressure waveform and device area were evaluated by measuring luciferase expression. In addition, renal injury was examined. The renal suction-mediated transfection method at -30 kPa showed high transgene expression. The renal suction waveform did not affect the transfection activity. Under the optimized conditions, the high transgene expression was mostly observed at the renal suctioned site. The transfection conditions used did not induce histological defects or increases in two renal injury biomarkers (Kidney injury molecule-1 mRNA and Clusterin mRNA). We have clarified the transfection conditions for efficient and safe transfection in the kidney using the suction-mediated transfection method in mice.

  1. A Comparative Study of Non-Viral Gene Delivery Techniques to Human Adipose-Derived Mesenchymal Stem Cell

    Directory of Open Access Journals (Sweden)

    Nur Shuhaidatul Sarmiza Abdul Halim

    2014-08-01

    Full Text Available Mesenchymal stem cells (MSCs hold tremendous potential for therapeutic use in stem cell-based gene therapy. Ex vivo genetic modification of MSCs with beneficial genes of interest is a prerequisite for successful use of stem cell-based therapeutic applications. However, genetic manipulation of MSCs is challenging because they are resistant to commonly used methods to introduce exogenous DNA or RNA. Herein we compared the effectiveness of several techniques (classic calcium phosphate precipitation, cationic polymer, and standard electroporation with that of microporation technology to introduce the plasmid encoding for angiopoietin-1 (ANGPT-1 and enhanced green fluorescent protein (eGFP into human adipose-derived MSCs (hAD-MSCs. The microporation technique had a higher transfection efficiency, with up to 50% of the viable hAD-MSCs being transfected, compared to the other transfection techniques, for which less than 1% of cells were positive for eGFP expression following transfection. The capability of cells to proliferate and differentiate into three major lineages (chondrocytes, adipocytes, and osteocytes was found to be independent of the technique used for transfection. These results show that the microporation technique is superior to the others in terms of its ability to transfect hAD-MSCs without affecting their proliferation and differentiation capabilities. Therefore, this study provides a foundation for the selection of techniques when using ex vivo gene manipulation for cell-based gene therapy with MSCs as the vehicle for gene delivery.

  2. Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro

    NARCIS (Netherlands)

    Fielden, Matthew; Perrin, C.; Kremer, Andreas; Bergsma, M.; Stuart, M.C.A.; Camilleri, P.; Engberts, J.B.F.N.

    2001-01-01

    Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12-18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1).

  3. [Non-viral gene transfer results in therapeutic factor IX levels in haemophilia B mice].

    Science.gov (United States)

    Schüttrumpf, J; Milanov, P; Roth, S; Seifried, E; Tonn, T

    2008-10-01

    Safety issues concerning the risk of malignancy formation and immune response to viral vectors were raised in initial gene therapy trials. In contrast, non-viral gene delivery methods have long been offside. We therefore explore a non-viral gene transfer approach for the treatment of hemophilia B. First, we constructed a strong liver-specific expression plasmid for human factor IX (FIX). Next, we tested the vector by injecting two doses under hydrodynamic conditions into the tail veins of FIX knockout mice. A single injection resulted in an increase in FIX expression over 100% of normal plasma levels. The FIX resulted fully functional. Further, no anti-FIX antibodies were observed and expression levels were vector dose dependent. The high expression obtained in small animals give hope for further development of non-viral gene transfer for the treatment of hemophilia B in humans.

  4. Halting angiogenesis by non-viral somatic gene therapy alleviates psoriasis and murine psoriasiform skin lesions

    DEFF Research Database (Denmark)

    Zibert, John Robert; Wallbrecht, Katrin; Schön, Margarete

    2011-01-01

    with epidermal expression of human TGF-ß1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM...... in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders.......-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein-coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue...

  5. Optimized PEI-based Transfection Method for Transient Transfection and Lentiviral Production.

    Science.gov (United States)

    Yang, Shaozhe; Zhou, Xiaoling; Li, Rongxiang; Fu, Xiuhong; Sun, Pingnan

    2017-09-14

    Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI-based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI-based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI-based transfection has been optimized to improve its efficiency, reproducibility, and consistency. This protocol outlines the steps for ordinary transient transfection and lentiviral production using PEI. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  6. CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice

    Directory of Open Access Journals (Sweden)

    Oscar Meca-Cortés

    2017-09-01

    Full Text Available The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs, using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene, under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.

  7. pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

    Directory of Open Access Journals (Sweden)

    Ogris Manfred

    2010-03-01

    Full Text Available Abstract Background The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP and directed into a 2000 bp long matrix attachment region sequence (MARS derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression. Results Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P element that is known to be less affected by epigenetic silencing events. Conclusions The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

  8. Non-Viral Delivery and Therapeutic Application of Small Interfering RNAs

    National Research Council Canada - National Science Library

    Nikitenko, N A; Prassolov, V S

    2013-01-01

    .... This review summarizes various strategies for siRNA delivery, including chemical modification and non-viral approaches, such as the polymer-based, peptide-based, lipid-based techniques, and inorganic nanosystems. The advantages, disadvantages, and prospects for the therapeutic application of these methods are also examined in this paper.

  9. Synthesis of bifunctional molecules containing [12]aneN3 and coumarin moieties as effective DNA condensation agents and new non-viral gene vectors.

    Science.gov (United States)

    Yue, Pan; Zhang, Ying; Guo, Zhi-Fo; Cao, Ao-Cheng; Lu, Zhong-Lin; Zhai, Yong-Gong

    2015-04-21

    A series of bifunctional molecules with different combinations of macrocyclic polyamine [12]aneN3 and coumarin moieties, 4a/b and 5a/b, were synthesized by a two-step copper(I)-mediated alkyne–azide click reactions between 1,3,5-tris(azidomethyl)benzene and Boc-protected N-propynyl-[12]aneN3/7-propynyloxycoumarins. Agarose gel electrophoresis experiments indicated that bifunctional molecules 4b and 5b effectively induced complete plasmid DNA condensation at concentrations up to 40 μM. It was found that the structural variation had a major impact on the condensation behavior of these compounds. The electrostatic interaction involving the [12]aneN3 moiety can be compensated by the binding contribution of the coumarin units during the DNA condensation process. These two types of interaction showed different effects on the reversibility of DNA condensation. Results from studies using dynamic laser scattering, atomic force microscopy, and EB replacement assay further supported the above conclusion. Cytotoxicity assays on bifunctional compounds 4a/b and 5a/b indicated their low cytotoxicity. Results from cellular uptake and cell transfection experiments proved that bifunctional compounds 4b and 5b successfully served as non-viral gene vectors. Furthermore, methyl substituents attached to the coumarin unit (4b and 5b) greatly enhanced their DNA condensation capability and gene transfection. These bifunctional molecules, with the advantages of lower cytotoxicity, good water solubility, and potential structural modification, will have great potential for the development of new non-viral gene delivery agents.

  10. Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

    Directory of Open Access Journals (Sweden)

    Noura Abd Ellah

    Full Text Available Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1. Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

  11. Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

    Energy Technology Data Exchange (ETDEWEB)

    Kamau Chapman, Sarah W. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland); Hassa, Paul O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland); European Molecular Biology Laboratory (EMBL) Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Koch-Schneidemann, Sabine; Rechenberg, Brigitte von [Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich (Switzerland); Hofmann-Amtenbrink, Margarethe [MatSearch, Chemin Jean Pavillard 14, 1009 Pully (Switzerland); Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland); Hottiger, Michael O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland)], E-mail: hottiger@vetbio.uzh.ch

    2008-04-15

    Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

  12. Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

    Energy Technology Data Exchange (ETDEWEB)

    Wu Xuewen; Ding Dalian [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Jiang Haiyan [State University of New York at Buffalo, Center for Hearing and Deafness (United States); XingXiaowei [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Huang, Suping [Central South University, State Key Laboratory of Powder Metallurgy (China); Liu Hong [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Chen Zhedong [Central South University, State Key Laboratory of Powder Metallurgy (China); Sun Hong, E-mail: shjhaj@vip.163.com [Central South University, Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital (China)

    2012-01-15

    Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI-nHAT, diameter = 73.09 {+-} 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2-NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector-gene complex (PEI-nHAT-pEGFPC2-NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector-gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector-gene complexes. Considering the high transfection efficiency in the vestibular system, PEI-nHAT may be a potential vector for gene therapy of

  13. Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

    Science.gov (United States)

    Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

    2017-02-01

    The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

  14. Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach.

    Directory of Open Access Journals (Sweden)

    Katharina Debowski

    Full Text Available Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80 and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement

  15. Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers

    Directory of Open Access Journals (Sweden)

    Uludağ Hasan

    2008-02-01

    Full Text Available Abstract Background Efforts to improve the efficiency of non-viral gene delivery require a better understanding of delivery kinetics of DNA molecules into clinically relevant cells. Towards this goal, three DNA molecules were employed to investigate the effects of DNA properties on cellular delivery: a circular plasmid DNA (c-DNA, a linearized plasmid DNA (l-DNA formulated by single-site digestion of c-DNA, and smaller linear gene cassette generated by PCR (pcr-DNA. Four non-viral gene carriers were investigated for DNA delivery: polyethyleneimine (PEI, poly-L-Lysine (PLL, palmitic acid-grafted PLL (PLL-PA, and Lipofectamine-2000™. Particle formation, binding and dissociation characteristics, and DNA uptake by rat bone marrow stromal cells were investigated. Results For individual carriers, there was no discernible difference in the morphology of particles formed as a result of carrier complexation with different DNA molecules. With PEI and PLL carriers, no difference was observed in the binding interaction, dissociation characteristics, and DNA uptake among the three DNA molecules. The presence of serum in cell culture media did not significantly affect the DNA delivery by the polymeric carriers, unlike other lipophilic carriers. Using PEI as the carrier, c-DNA was more effective for transgene expression as compared to its linear equivalent (l-DNA by using the reporter gene for Enhanced Green Fluorescent Protein. pcr-DNA was the least effective despite being delivered into the cells to the same extent. Conclusion We conclude that the nature of gene carriers was the primary determinant of cellular delivery of DNA molecules, and circular form of the DNA was more effectively processed for transgene expression.

  16. Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach.

    Science.gov (United States)

    Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

    2015-01-01

    Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in

  17. Optical sorting and photo-transfection of mammalian cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-02-01

    Full Text Available , the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S...

  18. Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells.

    Science.gov (United States)

    Konopka, Krystyna; Overlid, Nathan; Nagaraj, Anitha C; Düzgüneş, Nejat

    2006-01-01

    Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett. 10 (2005) 455-470). Here, we examined the efficacy of these vectors to deliver beta-galactosidase (beta-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer. We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%) on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer are highly effective in transfecting SCCVII cells. Approximately 60-70% of SCCVII cells transfected with pCMV.lacZ were positive for beta-gal staining. The expression of beta-galactosidase was essentially not affected by serum. Mouse serum (20-60%) reduced both Metafectene-and GeneJammer-mediated luciferase expression by approximately 30-45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over 90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes is the

  19. DyNAvectors: dynamic constitutional vectors for adaptive DNA transfection.

    Science.gov (United States)

    Clima, Lilia; Peptanariu, Dragos; Pinteala, Mariana; Salic, Adrian; Barboiu, Mihail

    2015-12-25

    Dynamic constitutional frameworks, based on squalene, PEG and PEI components, reversibly connected to core centers, allow the efficient identification of adaptive vectors for good DNA transfection efficiency and are well tolerated by mammalian cells.

  20. Enhanced gene transfection by photochemical internalization of protomine sulfate/DNA complexes

    Science.gov (United States)

    Hirschberg, Henry; Mathews, Marlon B.; Shih, En-Chung; Madsen, Steen J.; Kwon, Young Jik

    2012-02-01

    Introduction: One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of the GFP indicator gene on the same plasmid as a tumor suppressor gene (PTEN) was investigated in monolayers of U251 human glioma cells. Materials and Methods: U251 monolayers were incubated in AlPcS2a for 18 h. The monolayers were incubated with non-viral vectors for either 4 or 18 hrs. In all cases, light treatment was performed with a diode laser at a wavelength of 670 nm. The non-viral transfection agents, branched PEI or protomine sulfate (PS), were used with the plasmid construct (GFP-PTEN). Results: PS was much less toxic to the gliomas cells compared to BPEI but was highly inefficient at gene transfection. PCI resulted in a 5-10 fold increase in GFP protein expression compared to controls. Conclusions: Collectively, the results suggest that AlPcS2a-mediated PCI can be used to enhance transfection of tumor suppressor genes in glioma cells.

  1. Transient high-level expression of ß-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA

    Directory of Open Access Journals (Sweden)

    R.C. Balestrin

    2008-04-01

    Full Text Available GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal. It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1 to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 µL lipofectamine 2000 and 1.5-2.0 µg DNA. Cells (2 x 10(5/well were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß-Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant. However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

  2. Non-viral reprogramming of fibroblasts into induced pluripotent stem cells by Sleeping Beauty and piggyBac transposons.

    Science.gov (United States)

    Talluri, Thirumala R; Kumar, Dharmendra; Glage, Silke; Garrels, Wiebke; Ivics, Zoltan; Debowski, Katharina; Behr, Rüdiger; Kues, Wilfried A

    2014-07-18

    The generation of induced pluripotent stem (iPS) cells represents a promising approach for innovative cell therapies. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity. Transposition of reprogramming cassettes represents a recent alternative to viral approaches. Since binary transposons can be produced as common plasmids they provide a safe and cost-efficient alternative to viral delivery methods. Here, we compared the efficiency of two different transposon systems, Sleeping Beauty (SB) and piggyBac (PB), for the generation of murine iPS. Murine fibroblasts derived from an inbred BL/6 mouse line carrying a pluripotency reporter, Oct4-EGFP, and fibroblasts derived from outbred NMRI mice were employed for reprogramming. Both transposon systems resulted in the successful isolation of murine iPS cell lines. The reduction of the core reprogramming factors to omit the proto-oncogene c-Myc was compatible with iPS cell line derivation, albeit with reduced reprogramming efficiencies. The transposon-derived iPS cells featured typical hallmarks of pluripotency, including teratoma growth in immunodeficient mice. Thus SB and PB transposons represent a promising non-viral approach for iPS cell derivation. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Repeated Aurora-A siRNA Transfection Results in Effective Apoptosis of A549 Cells Compared to Single Transfection.

    Science.gov (United States)

    Wang, Zhonghua; Sun, Wenwu; Cao, Jianping; Cui, Haiyang; Ma, Zhuang

    2016-01-01

    Suppression of Aurora kinase A (Aurora-A, AURKA) by Aurora-A siRNA has been proposed for lung tumor treatment. However, protocols using single administration have shown little benefit in some types of lung tumor. Given that transfection efficiency of Aurora-A siRNA is low due to tightly packed cells in the tumor, we hypothesized that repeated administration would result in efficient cell apoptosis. We compared single vs. repeated transfection (thrice) in A549 cells by transfecting Aurora-A siRNA (siA) on the 1st or 1st, 2nd and 3rd day after cell seeding. A random sequence was used as the negative siRNA control (siC). Cells in the single transfection group received only transfection reagent without siRNAs on the 2nd and 3rd day. Two days after the third transfection, both single and repeated siA administration decreased mRNA expression of Aurora-A and cell viability compared to no administration and siC single administration. However, the decrease in these two indices with repeated transfection was more obvious than that following single administration: cell viability decreased to 72.8 ± 3.05% (p transfection and to 64.2 ± 1.99% (p transfection, compared with normal control cells, respectively. Gene expression decreased to 17 ± 16.6% (p transfection and to 43.2 ± 13.0% (p transfection. Compared to single transfection, repeated Aurora-A siRNA transfection decreased Aurora-A, which, in turn, resulted in effective apoptosis of A549 cells.

  4. A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine

    Energy Technology Data Exchange (ETDEWEB)

    Oskuee, Reza Kazemi [Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Dosti, Fatemeh [School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Gholami, Leila [Targeted Drug Delivery Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Malaekeh-Nikouei, Bizhan, E-mail: malaekehb@mums.ac.ir [Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of)

    2015-04-01

    Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65 kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250 nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15 kDa had better transfection efficiency than the vectors made of PAA 65 kDa. In conclusion, results of the present study indicated that grafting PAA 15 kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity. - Highlights: • The modified polyallylamine was synthesized as a gene carrier. • Modification of polyallylamine (15 kDa) with high percentages of hexyl acrylate improved transfection activity remarkably. • Grafting of polymer with acrylate derivatives reduced polymer cytotoxicity especially at percentages of

  5. Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field.

    Science.gov (United States)

    He, Zhi-Yao; Men, Ke; Qin, Zhou; Yang, Yang; Xu, Ting; Wei, Yu-Quan

    2017-05-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

  6. A Comparison of Immune Functionality in Viral versus Non-Viral CFS Subtypes.

    Science.gov (United States)

    Porter, Nicole; Lerch, Athena; Jason, Leonard A; Sorenson, Matthew; Fletcher, Mary Ann; Herrington, Joshua

    2010-06-01

    Participants with CFS were grouped into viral and non-viral onset fatigue categories and assessed for differential immunological marker expression. Peripheral Blood Mononuclear Cells were assessed for differential phenotypic expression of surface adherence glycoproteins on circulating lymphocytes. The flow cytometric analysis employed fluorescent monoclonal antibody labeling. The viral in comparison to the non-viral group demonstrated significant elevations in several Th1 type subsets including: the percentage and number of CD4+ cells, the percentage and number of CD2+CD26+ cells, the percentage and number of CD2+CD4+CD26+ cells, the percentage and number of CD4+ CD26+ cells, and the percentage of Th2 naïve cells (CD4+ CD45RA+CD62L+). Of the remaining significant findings, the non viral group demonstrated significant elevations in comparison to the viral group for the following Th1 type subsets: the percentage of CD8+ cells, the percentage of T-cytotoxic suppressor cells (CD3+8+), and the percentage and number of Th1 memory cells (CD8+CD45RA-CD62L-). The viral group demonstrated a pattern of activation that differed from that of the group with a non-viral etiology, as evidenced by an elevated and out of range percentage and number of CD4+ cells, the percentage of CD2+CD26+, and the percentage of Th2 naïve cells (CD4+CD45RA+CD62L+). Both groups demonstrated reduced and out of range Natural Killer Cell Cytotoxicity and percentage of B-1 cells (CD5+CD19). In addition, both groups demonstrated an elevated and out of range percentage of CD2+CD8+CD26+, percentage of the Th1 memory subset (CD4+CD45RA-CD62L-), the percentage of Th1 memory and naïve cells (CD8+CD45RA-CD62L-, CD8+CD45RA+CD62L-), the percentage and number of Th2 memory cells (CD4+CD45RA-CD62L+), and the percentage of Th2 memory and naïve cells (CD8+CD45RA-CD62L+, CD8+CD45RA+CD62L+). These findings imply that the homeostatic mechanism responsible for the regulation of the Th1 (cell mediated) and Th2

  7. Effective photodynamic therapy in drug-resistant prostate cancer cells utilizing a non-viral antitumor vector (a secondary publication)

    Science.gov (United States)

    Yamauchi, Masaya; Honda, Norihiro; Hazama, Hisanao; Tachikawa, Shoji; Nakamura, Hiroyuki; Kaneda, Yasufumi; Awazu, Kunio

    2016-01-01

    Background and Aims: There is an urgent need to develop an efficient strategy for the treatment of drug-resistant prostate cancer. Photodynamic therapy (PDT), in which low incident levels of laser energy are used to activate a photosensitizer taken up by tumor cells, is expected as a novel therapy for the treatment of prostate cancer because of the minimal invasive nature of PDT. The present study was designed to assess the efficacy of a novel vector approach combined with a conventional porphyrin-based photosensitizer. Materials and Methods: Our group focused on a non-viral vector (hemagglutinating virus of Japan envelope; HVJ-E) combined with protoporphyrin IX (PpIX) lipid, termed the porphyrus envelope (PE). It has been previously confirmed that HVJ-E has drug-delivering properties and can induce cancer-specific cell death. The PE (HVJ-E contained in PpIX lipid) was developed as a novel photosensitizer. In this study, the antitumor and PDT efficacy of the PE against hormone-antagonistic human prostate cancer cells (PC-3) were evaluated. Results and Conclusions: Our results demonstrated that, under specific circumstances, PDT using the PE was very effective against PC-3 cells. A novel therapy for drug-resistant prostate cancer based on this vector approach is eagerly anticipated. PMID:27141155

  8. Transfection of Platyhelminthes

    Directory of Open Access Journals (Sweden)

    Bárbara Moguel

    2015-01-01

    Full Text Available Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  9. Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

    Science.gov (United States)

    Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Amino Acid-Modified Polyethylenimines with Enhanced Gene Delivery Efficiency and Biocompatibility

    Directory of Open Access Journals (Sweden)

    Qin-Fang Zhang

    2015-11-01

    Full Text Available The development of gene delivery vectors with high efficiency and biocompatibility is one of the key points of gene therapy. A series of polycations were prepared from polyethylenimine (PEI with several amino acids or their analogs. The target polymers have different charge and hydrophilic/hydrophobic properties, which may affect their performance in the gene transfection process. Gel retardation and DLS assays showed that these polymers may condense DNA into nanoparticles with positive zeta potentials and proper sizes for cellular uptake. Luciferase reporter gene transfection results revealed their higher transfection efficiency than PEI; especially in the presence of serum, in which up to 23 times higher efficiency was achieved by employing glycolic acid-grafted PEI. Moreover, it was found that the degree of substitution on PEI has an apparent influence on the transfection, and the balance between electron-positive/negative groups largely affects the delivery process. The higher serum tolerance was also proven by BSA adsorption, flow cytometry and confocal microscopy assays. Results demonstrate that such type of polycations may serve as promising non-viral gene delivery vectors.

  11. Non-viral causes of liver cancer: does obesity led inflammation play a role?

    Science.gov (United States)

    Alzahrani, Badr; Iseli, Tristan J; Hebbard, Lionel W

    2014-04-10

    Liver cancer is the fifth most common cancer worldwide and the third most common cause of cancer mortality. Hepatocellular carcinoma (HCC) accounts for around 90% of primary liver cancers. Chronic infection with hepatitis B and hepatitis C viruses are two of most common causes of liver cancer. However, there are non-viral factors that are associated with liver cancer development. Numerous population studies have revealed strong links between obesity and the development of liver cancer. Obesity can alter hepatic pathology, metabolism and promote inflammation, leading to nonalcoholic fatty liver disease (NAFLD) and the progression to the more severe form, non-alcoholic steatohepatitis (NASH). NASH is characterised by prominent steatosis and inflammation, and can lead to HCC. Here, we discuss the role of obesity in inflammation and the principal signalling mechanisms involved in HCC formation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

    Science.gov (United States)

    Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

    2015-08-07

    Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

  13. Poly(β-Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo

    Science.gov (United States)

    Bhutto, Imran; Handa, James T.; Green, Jordan J.

    2012-01-01

    A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer∶DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (ptransfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×103-fold and 1.5±0.7×103-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, ptransfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases. PMID:22629417

  14. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications.

    Science.gov (United States)

    Dalby, Brian; Cates, Sharon; Harris, Adam; Ohki, Elise C; Tilkins, Mary L; Price, Paul J; Ciccarone, Valentina C

    2004-06-01

    Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome-DNA complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors in Lipofectamine 2000 mediated transfection will be discussed together with some specific applications: transfection of primary neurons, high throughput transfection, and delivery of small interfering RNAs. Copyright 2003 Elsevier Inc.

  15. Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible and efficient gene delivery: a comparative study with protamine and lipofectamine.

    Science.gov (United States)

    Nematollahi, Mohammad Hadi; Torkzadeh-Mahanai, Masoud; Pardakhty, Abbas; Ebrahimi Meimand, Hossein Ali; Asadikaram, Gholamreza

    2017-10-28

    Non-viral gene delivery methods are considered due to safety and simplicity in human gene therapy. Since the use of cationic peptide and niosome represent a promising approach for gene delivery purposes we used recombinant fusion protein and cationic niosome as a gene carrier. A multi-domain fusion protein including nuclear localization motif (NLS) and two DNA-binding (Mu) domains, namely NLS-Mu-Mu (NMM) has been designed, cloned and expressed in E. coli DE3 strain. Afterward, the interested protein was purified by affinity chromatography. Binary vectors based on protein/DNA and ternary vectors based on protein/DNA/niosome were prepared. Protamine was used as a control. DNA condensing properties of NMM and protamine were evaluated by various experiments. Furthermore, we examined cytotoxicity, hemolysis and transfection potential of the binary and ternary complexes in HEK293T and MCF-7 cell lines. Protamine and Lipofectamine™2000 were used as positive controls, correspondingly. The recombinant NMM was expressed and purified successfully and DNA was condensed efficiently at charge ratios that were not harmful to cells. Peptidoplexes showed transfection efficiency (TE) but ternary complexes had higher TE. Additionally, NMM ternary complex was more efficient compared to protamine ternary vectors. Our results showed that niosomal ternary vector of NMM is a promising non-viral gene carrier to achieve an effective and safe carrier system for gene therapy.

  16. HVJ-E/importin-β hybrid vector for overcoming cytoplasmic and nuclear membranes as double barrier for non-viral gene delivery.

    Science.gov (United States)

    Kawazu, Takeshi; Kanzaki, Hiroyuki; Uno, Atsushi; Azuma, Hideki; Nagasaki, Takeshi

    2012-10-01

    In order to enhance the nuclear import of the transgene, we prepared plasmid DNA/importin-β conjugates consisting of biotinylated poly(ethylenimine)s and recombinant streptavidin-fused importin-β. Hemagglutinating virus of Japan-envelope vector containing the PEI polyplex/importin-β conjugate showed high transfection efficiency not only in vitro but also in vivo. We showed that novel HVJ-E/importin-β-conjugated PEI polyplex hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection. Copyright © 2012. Published by Elsevier SAS.

  17. Alkyl cross-linked low molecular weight polypropyleneimine dendrimers as efficient gene delivery vectors.

    Science.gov (United States)

    Ariaee, Faezeh Moghadam; Hashemi, Maryam; Farzad, Sara Amel; Abnous, Khalil; Ramezani, Mohammad

    2016-10-01

    In recent years, polypropyleneimine (PPI) dendrimers have attracted great interest as non-viral gene delivery systems because of their attractive features including highly branched architecture with number of reactive end groups. However, without being structurally modified, they are not efficient gene carriers. In the present study, generation 2 and 3 (G2 and G3) of PPI dendrimers were conjugated with alkylcarboxylate groups as linker to enhance the transfection efficiency while maintaining their low cell toxicity. First, 10-bromodecanoic acid was covalently attached to all available surface primary amines of PPI G2 and G3 to increase their lipophilicity. In the subsequent step, PPIs were conjugated to the alkylcarboxylate groups of alkylcarboxylate-PPI derivatives to increase the number of surface primary amines. Physicochemical properties of modified PPIs were determined. Transfection experiments (using both luciferase and green fluorescent protein (GFP)- expressing plasmids) and cytotoxicity assay were performed to evaluate the efficiency of the final derivatives. Fabricated vectors condensed DNA effectively so that polyplexes with appropriate size (below 155 nm) and positive surface charge were constructed. Cross-linked low molecular weight PPIs (G2 or G3) with decanoate linkage increased transfection efficiency significantly while maintaining the low cytotoxicity. PPI G2 derivative exhibited increased buffering capacity which is believed to be responsible for better proton sponge mechanism leading to higher transfection efficiency. Our results indicated that oligomerization of low molecular weight PPI (PPI G2-alkyl-PPI G2 conjugate) could be an approach to increase the transfection efficiency and to lower the cytotoxicity of low molecular weight polycations.

  18. In vitro studies of magnetically enhanced transfection in COS-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Ang, D.; Tay, C.Y.; Tan, L.P. [School of Materials Science and Engineering, Nanyang Technological University (Singapore); Preiser, P.R. [School of Biological Sciences, Nanyang Technological University (Singapore); Ramanujan, R.V., E-mail: Ramanujan@ntu.edu.sg [School of Materials Science and Engineering, Nanyang Technological University (Singapore)

    2011-10-10

    In the magnetically enhanced gene delivery technique, DNA complexed with polymer coated aggregated magnetic nanoparticles (AMNPs) is used for effecting transfection. The aim of this study is to examine the relationship between transfection efficiency and the physical characteristics of the polymer coated AMNPs. In vitro studies of transfection efficiency in COS-7 cells were carried out using pEGFP-N1 and pMIR-REPORT complexed polyethylenimine (PEI) coated iron oxide magnetic nanoparticles. PEI coated AMNPs (PEI-AMNPs) with average individual particle diameters in the range of 8 nm to 30 nm were studied and characterized by transmission electron microscopy, vibrating sample magnetometry, X-ray diffractometry, thermal gravimetric analysis and photon correlation spectroscopy methods. PEI-A8MNP and PEI-A30MNP yielded higher transfection efficiency compared to commercial polyMAG particles as well as PEI of equivalent molar ratio of nitrogen/phosphorous (N/P ratio). The transfection efficiency was related to the physical characteristics of the PEI-AMNPs and its complexes: transfection efficiency was strongly positively correlated with saturation magnetization (Ms) and susceptibility ({chi}), strongly negatively correlated with N/P ratio, moderately positively correlated to zeta potential and moderately negatively correlated to hydrodynamic diameter of the complex. PEI-A8MNP and PEI-A30MNP possessing higher Ms, {chi}, lower N/P ratio and smaller complex size exhibited higher transfection efficiency compared to PEI-A16MNP which have weaker magnetic properties, higher N/P ratio and larger complex size. We have demonstrated that optimization of the physical properties of PEI-AMNPs is needed to maximize transfection efficiency. - Research highlights: {yields}The transfection efficiency in magnetically enhanced gene delivery was studied. {yields}Transfection efficiency was strongly positively correlated to magnetic properties. {yields}Transfection efficiency was strongly

  19. Delivery of episomal vectors into primary cells by means of commercial transfection reagents.

    Science.gov (United States)

    Han, Na Rae; Lee, Hyun; Baek, Song; Yun, Jung Im; Park, Kyu Hyun; Lee, Seung Tae

    2015-05-29

    Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors.

    Science.gov (United States)

    Monjezi, R; Miskey, C; Gogishvili, T; Schleef, M; Schmeer, M; Einsele, H; Ivics, Z; Hudecek, M

    2017-01-01

    Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

  1. Acoustic Liquid Handling for Rapid siRNA Transfection Optimization.

    Science.gov (United States)

    Xiao, Andrew S; Lightcap, Eric S; Bouck, David C

    2015-09-01

    Gene knockdown by small interfering RNA (siRNA) has been used extensively to investigate the function of genes in targeted and genome-wide studies. One of the primary challenges of siRNA studies of any scale is to achieve sufficient gene knockdown to produce the biological changes that lead to measurable phenotypes. Reverse, lipid-based transfection efficiency minimally requires the optimization of the following parameters: cell number, knockdown duration, siRNA oligonucleotide concentration, type/brand of transfection lipid, and transfection lipid concentration. In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues. © 2015 Society for Laboratory Automation and Screening.

  2. Toward Contactless Biology: Acoustophoretic DNA Transfection

    Science.gov (United States)

    Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors. PMID:26828312

  3. [Optimization of triple plasmids transfection into HEK293 cells mediated by polyethylenimine].

    Science.gov (United States)

    Fu, Qiang; Li, Yan; Zheng, Zhaofen; Liu, Aizhong; Yuan, Zhenhua; Peng, Jianqiang; He, Jin

    2015-02-01

    In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.

  4. Biodegradable Poly(Amino Ester with Aromatic Backbone as Efficient Nonviral Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    2017-03-01

    Full Text Available The development of gene delivery vectors with high efficiency and biocompatibility is one of the critical points of gene therapy. Two biodegradable poly(amino esters were synthesized via ring-opening polymerization between low molecular weight (LMW PEI and diepoxide. The molecular weights of poly(amino esters were measured by GPC. Agarose gel retardation assays showed that these materials have good DNA-binding ability and can retard the electrophoretic mobility of plasmid DNA (pDNA at a weight ratio of 1. The formed polyplexes have proper sizes of around 200 nm and zeta-potential values of about 30–40 mV for cellular uptake. In vitro experiments revealed that polymer P2 gave higher transfection efficiency than PEI 25KDa and Lipofectamine 2000 with less toxicity, especially in 293 cells. Results demonstrate that such a type of degradable poly(amino ester may serve as a promising non-viral gene vector.

  5. The Study of Non-Viral Nanoscale Delivery Systems for Islet Transplantation

    Science.gov (United States)

    Gutierrez, Diana

    Due to safety concerns associated with using viral systems clinically to expand islet cells and make them available to many more patients, significant emphasis has been placed on producing a safe and effective non-viral delivery system for biological research and gene therapy. To obtain this goal, we propose the use of an innovative technology that utilizes gold nanoparticles (AuNPs) as a non-viral method of delivery. Our laboratory was one of the first to describe the use of AuNPs in human islets and observe AuNPs can penetrate into the core of islets to deliver a gene to the vast majority of the cells, without damaging the cell. Gold nanoparticles proved to be a biocompatible delivery system both in vitro and in vivo. Thus far, gene therapy and molecular biology have focused primarily on delivering DNA of a specific gene into cells. The risk of this approach is that the DNA can be permanently incorporated into the genome and lead to damages in the cell that could result in overexpression of cancerous tumor cells. This risk does not exist with the use of mRNA. Many researchers believe mRNA is too unstable to be used as a molecular tool to overexpress specific proteins. With advances in nanotechnology, and better understanding of the translation process, methods have been developed that allow for expression of specific proteins by intracellular delivery of protein-encoding mRNA. We used AuNPs conjugated to mCherry mRNA to establish a proof of concept of the feasibility of using AuNP-mRNA to achieve increased expression of a specific protein within cells. To do this, we conjugated mCherry mRNA to AuNPs and tested the feasibility for increasing delivery efficacy and preserve functionality of human pancreatic islets. We believe that with this novel technology we can create AuNPs that allow specific mRNA to enter islets and lead to the production of a specific protein within the cell, with the aim to induce beta cell proliferation. In a previous experiment with single

  6. Poly(β-amino ester-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Joel C Sunshine

    Full Text Available A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino esters (PBAEs have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19 cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer:DNA ratio transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7% and X-tremeGENE HP DNA (22±6%; (p0.05 for both. Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI, which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10(3-fold and 1.5±0.7×10(3-fold increased GFP expression in the retinal pigment epithelium (RPE/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, p<0.001 for neural retina. The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.

  7. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture

    Directory of Open Access Journals (Sweden)

    M Majumdar

    2014-01-01

    Full Text Available The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001, even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  8. Oligopeptide complex for targeted non-viral gene delivery to adipocytes

    Science.gov (United States)

    Won, Young-Wook; Adhikary, Partho Protim; Lim, Kwang Suk; Kim, Hyung Jin; Kim, Jang Kyoung; Kim, Yong-Hee

    2014-12-01

    Commercial anti-obesity drugs acting in the gastrointestinal tract or the central nervous system have been shown to have limited efficacy and severe side effects. Anti-obesity drug development is thus focusing on targeting adipocytes that store excess fat. Here, we show that an adipocyte-targeting fusion-oligopeptide gene carrier consisting of an adipocyte-targeting sequence and 9-arginine (ATS-9R) selectively transfects mature adipocytes by binding to prohibitin. Injection of ATS-9R into obese mice confirmed specific binding of ATS-9R to fat vasculature, internalization and gene expression in adipocytes. We also constructed a short-hairpin RNA (shRNA) for silencing fatty-acid-binding protein 4 (shFABP4), a key lipid chaperone in fatty-acid uptake and lipid storage in adipocytes. Treatment of obese mice with ATS-9R/shFABP4 led to metabolic recovery and body-weight reduction (>20%). The ATS-9R/shFABP4 oligopeptide complex could prove to be a safe therapeutic approach to regress and treat obesity as well as obesity-induced metabolic syndromes.

  9. Optimization of in vitro culture and transfection condition of bovine ...

    African Journals Online (AJOL)

    The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase ...

  10. Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection.

    Science.gov (United States)

    Martin, Timothy M; Plautz, Sarah A; Pannier, Angela K

    2015-01-01

    Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection. Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid-encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors. Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2 h; RAP1A and ACTA1 after 8 h; RAP1A, WDR78 and ACTA1 after 16 h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24 h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells. The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered. Copyright © 2015 John Wiley & Sons, Ltd.

  11. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  12. Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles

    NARCIS (Netherlands)

    van Leeuwen, E B; van der Veen, A Y; Hoekstra, D; Engberts, J B; Halie, M R; van der Meer, J; Ruiters, M H

    OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a

  13. Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

    Science.gov (United States)

    Green, David W; Kim, Eun-Jung; Jung, Han-Sung

    2015-09-01

    The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. © 2015 Wiley Periodicals, Inc.

  14. Monitoring and visualizing microRNA dynamics during live cell differentiation using microRNA-responsive non-viral reporter vectors.

    Science.gov (United States)

    Nakanishi, Hideyuki; Miki, Kenji; Komatsu, Kaoru R; Umeda, Masayuki; Mochizuki, Megumi; Inagaki, Azusa; Yoshida, Yoshinori; Saito, Hirohide

    2017-06-01

    MicroRNA (miRNA) activity differs with cell type, suggesting it can be used as a cell marker. In this study, we developed novel miRNA-responsive non-viral reporter vectors to continuously monitor and visualize miRNA dynamics during differentiation and to efficiently purify target living cells. Each vector codes miRNA-responsive and reference reporter genes in a single mRNA. These two genes are independent modules but transcribed by a single promoter, which enables us to distinguish miRNA-mediated post-transcriptional repression from transcriptional repression. We generated stable, miRNA-responsive vector-containing human induced pluripotent stem cells (hiPSCs) using the piggyBac transposon or episomal vectors. We could continuously monitor the differentiation status of living hiPSCs by detecting the activity of hiPSC-specific miRNA (miR-302a*). In addition, we could selectively sort hiPSC-derived cardiomyocytes using cardiomyocyte-specific miRNA (miR-208a or miR-1)-reporter vectors. Our miRNA reporter system provides a simple way to quantitatively and continuously monitor and visualize changes in the cellular state and should facilitate a broad range of studies that depend on cellular changes including drug discovery and cell-fate conversion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Koyama

    2015-07-01

    Full Text Available We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”, is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

  16. Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells

    National Research Council Canada - National Science Library

    Konopka, Krystyna; Overlid, Nathan; Nagaraj, Anitha C; Düzgüneş, Nejat

    2006-01-01

    .... A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60...

  17. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    Science.gov (United States)

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons.

    Science.gov (United States)

    Wang, Shiyao; Cho, Yong Ku

    2016-01-01

    In order to characterize genetically encoded tools under the most relevant conditions, the constructs need to be expressed in the cell type in which they will be used. This is a major hurdle in developing optogenetic tools for neuronal cells, due to the difficulty of gene transfer to these cells. Several protocols have been developed for transfecting neurons, focusing on improved transfection efficiency. However, obtaining healthy cells is as important. We monitored transfected cell health by measuring electrophysiological parameters, and used them as a guideline to optimize transfection. Here we describe an optimized transfection protocol that achieves reasonably high efficiency (10-20 %) with no discernable impact on cell health, as characterized by electrophysiology.

  19. Integrated application of uniform design and least-squares support vector machines to transfection optimization

    Directory of Open Access Journals (Sweden)

    Pan Jin-Shui

    2009-05-01

    Full Text Available Abstract Background Transfection in mammalian cells based on liposome presents great challenge for biological professionals. To protect themselves from exogenous insults, mammalian cells tend to manifest poor transfection efficiency. In order to gain high efficiency, we have to optimize several conditions of transfection, such as amount of liposome, amount of plasmid, and cell density at transfection. However, this process may be time-consuming and energy-consuming. Fortunately, several mathematical methods, developed in the past decades, may facilitate the resolution of this issue. This study investigates the possibility of optimizing transfection efficiency by using a method referred to as least-squares support vector machine, which requires only a few experiments and maintains fairly high accuracy. Results A protocol consists of 15 experiments was performed according to the principle of uniform design. In this protocol, amount of liposome, amount of plasmid, and the number of seeded cells 24 h before transfection were set as independent variables and transfection efficiency was set as dependent variable. A model was deduced from independent variables and their respective dependent variable. Another protocol made up by 10 experiments was performed to test the accuracy of the model. The model manifested a high accuracy. Compared to traditional method, the integrated application of uniform design and least-squares support vector machine greatly reduced the number of required experiments. What's more, higher transfection efficiency was achieved. Conclusion The integrated application of uniform design and least-squares support vector machine is a simple technique for obtaining high transfection efficiency. Using this novel method, the number of required experiments would be greatly cut down while higher efficiency would be gained. Least-squares support vector machine may be applicable to many other problems that need to be optimized.

  20. Cellular processing and nuclear targeting of non-viral gene delivery systems

    NARCIS (Netherlands)

    Aa, M.A.E.M. van der

    2005-01-01

    Gene therapy utilizes genetic material in order to cure patients either by DNA vaccines or by replacement of a defective gene with a normal one. For successful gene therapy certain elements are required: gene delivery systems with low toxicity and immunity, with efficient gene transfer and high gene

  1. Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering

    Science.gov (United States)

    2017-01-01

    The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types

  2. [Preliminary study of primarily cultured C57 articular cartilage transfected with plasmid IDO-EGFP by lipofectamine].

    Science.gov (United States)

    Duan, Xiao-Hong; He, Xian-Hui; Cui, Peng-Cheng; Wang, Xiao-Yan; Wu, Ming-Ming; Shi, Jian-Bo; Xu, Geng; Jiang, Xun

    2007-12-01

    To determine the transfection efficiency and transient expression of pIDO-EGFP gene in primarily cultured C57 articular cartilage of mice, and to establish a transfection method of the primarily cultured articular cartilage in mice. Plasmid IDO-EGFP was amplified in Escherichia coli. The primarily cultured mouse chondrocytes which were initially obtained from articular cartilage were cultured in vitro and transfected with pIDO-EGFP by lipofectamine2000 reagent under optimized condition. Transfection process and transient expression were evaluated by fluorescent microscopy and laser scanning confocal microscopy (LSCM), and transfection efficiency was determined by flow cytometry. There was obvious expression of EGFP at 24 h after transfection. The transfection efficiency of pIDO-EGFP into primarily cultured mouse chondrocytes reached 36.43% at 48 hours and the transfection did not affect the process of cell adherence. IDO gene has been successfully transfected into primarily cultured chondrocytes by means of lipofectamine2000 reagent and the chondrocytes can survive in vitro. Satisfactory efficiency of transient transfection can be reached under optimized condition, which will provide a basis for gene introduction and modification of tissue engineered cartilage.

  3. Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stiehler, Maik; Duch, Mogens R.; Mygind, Tina

    2006-01-01

    and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized......INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors...... were evaluated by realtime quantitative RT-PCR and histochemical detection of alkaline phosphatase activity, respectively. RESULTS: Non-viral gene delivery methods resulted in transient eGFP expression by less than 2% of the cells. Using high titer rAAV-based vector up to 90% of the cells were...

  4. Is passive transmission of non-viral vectors through artificial insemination of sperm-DNA mixtures sufficient for chicken transgenesis?

    Science.gov (United States)

    Chaparian, Shahram; Abdulahnejad, Ahad; Rashidi, Farzad; Toghyani, Majid; Gheisari, Abbasali; Eghbalsaied, Shahin

    2016-06-17

    DNA uptake in the post-acrosomal region of the spermatozoa takes place exclusively in immotile spermatozoa that are naturally unable to fertilize eggs. The present study aimed to assess whether passive transmission of non-viral vectors to the surrounding areas of chicken embryos could be an alternate mechanism in chicken sperm-mediated gene transfer. First, the presence of nucleases in rooster seminal plasma was evaluated. Semen ejaculates from five roosters were centrifuged and the supernatant was incubated with pBL2 for 1 h. A robust nuclease cocktail was detected in the rooster semen. To overcome these nucleases, plasmid-TransIT combinations were incubated with semen for 1 h. Incubation of exogenous DNA in the lipoplex structure could considerably bypass the semen nuclease effect. Then, intravaginal insemination of 1 × 10(9) sperm mixed with lipoplexes (40 µg pBL2:40 µl TransIT) was carried out in 15 virgin hens. Neither the epithelial tissue from the inseminated female reproductive tracts nor the produced embryos following artificial insemination showed the transgene. To remove any bias in the transgene transmission possibility, the plasmid-TransIT admixture was directly injected in close vicinity of the embryos in newly laid eggs. Nonetheless, none of the produced fetuses or chicks carried the transgene. In conclusion, the results of the present study revealed a nuclease admixture in rooster seminal plasma, and passive/active transmission of the non-viral vector into close vicinity of the chicken embryo was inefficient for producing transgenic chicks.

  5. Amniotic membrane graft to conjunctival flap in treatment of non-viral resistant infectious keratitis: a randomised clinical study.

    Science.gov (United States)

    Abdulhalim, Bahaa-Eldin Hasan; Wagih, Mostafa Mohamed; Gad, Ahmed A M; Boghdadi, Ghada; Nagy, Ragy R S

    2015-01-01

    To evaluate and compare the results of bipedicle conjunctival flap (CF) and cryopreserved amniotic membrane graft (AMG) in the treatment of non-viral infectious keratitis resistant to medical treatment. This prospective randomised interventional study included 40 eyes of 40 patients with resistant non-viral infectious keratitis. Twenty eyes received CF and 20 eyes received AMG. In the CF group, there were 12 eyes with fungal keratitis, 7 eyes with bacterial keratitis and 1 eye with Acanthamoeba keratitis. In the AMG group there were 13 eyes with fungal keratitis, 5 eyes with bacterial keratitis and 2 eyes with Acanthamoeba keratitis. In the CF group, three ulcers had descemetocele and four ulcers were perforated. In the AMG group, four ulcers had descemetocele and two ulcers were perforated. In CF, 360° peritomy was done and a bipedicle CF from the upper conjunctiva was dissected from Tenon's capsule, mobilised to cover the cornea and sutured to episclera. In AMG, one or two layers of AM were trimmed to fit the ulcer and sutured to the cornea. The follow-up period was 6 months. Successful results were observed in 18/20 eyes (90%) in each group. Postoperatively, no significant differences between the two groups were found regarding success rate (p=1.0), epithelialisation time (p=0.75) or visual acuity improvement (p=0.84). CF and AMG are effective in treatment-resistant infectious keratitis. They could restore ocular surface integrity and provide metabolic and mechanical support for corneal healing. For large corneal perforation, it may be better to use another procedure such as penetrating keratoplasty to restore ocular integrity. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  6. Synergistic effect of electrical and chemical factors on endocytosis in micro-discharge plasma gene transfection

    Science.gov (United States)

    Jinno, M.; Ikeda, Y.; Motomura, H.; Isozaki, Y.; Kido, Y.; Satoh, S.

    2017-06-01

    We have developed a new micro-discharge plasma (MDP)-based gene transfection method, which transfers genes into cells with high efficiency and low cytotoxicity; however, the mechanism underlying the method is still unknown. Studies revealed that the N-acetylcysteine-mediated inhibition of reactive oxygen species (ROS) activity completely abolished gene transfer. In this study, we used laser-produced plasma to demonstrate that gene transfer does not occur in the absence of electrical factors. Our results show that both electrical and chemical factors are necessary for gene transfer inside cells by microplasma irradiation. This indicates that plasma-mediated gene transfection utilizes the synergy between electrical and chemical factors. The electric field threshold required for transfection was approximately 1 kV m-1 in our MDP system. This indicates that MDP irradiation supplies sufficient concentrations of ROS, and the stimulation intensity of the electric field determines the transfection efficiency in our system. Gene transfer by plasma irradiation depends mainly on endocytosis, which accounts for at least 80% of the transfer, and clathrin-mediated endocytosis is a dominant endocytosis. In plasma-mediated gene transfection, alterations in electrical and chemical factors can independently regulate plasmid DNA adhesion and triggering of endocytosis, respectively. This implies that plasma characteristics can be adjusted according to target cell requirements, and the transfection process can be optimized with minimum damage to cells and maximum efficiency. This may explain how MDP simultaneously achieves high transfection efficiency with minimal cell damage.

  7. Metafectene is superior to lipofectamine in the transfection of G(s) alpha prostate cancer cells.

    Science.gov (United States)

    Iczkowski, Kenneth A; Omara-Opyene, A Levi; Klösel, Roland

    2004-10-01

    Transfection efficiency of the novel reagent metafectene has not been compared with that of lipofectamine in the published English literature. We used these agents to transfect two prostate cancer cell lines, PC3 and G(s) alpha, with a deoxyribonucleic acid (DNA) expression vector that generates double-stranded ribonucleic acid (RNA) for RNA interference (RNAi). Cotransfection of the green fluorescent protein (GFP) reporter gene revealed that the mean (+/- standard deviation) transfection efficiencies with lipofectamine were 5.8+/-0.4% for PC3 cells and 3.6+/-1.5% for G(s) alpha cells. Mean transfection efficiency with metafectene declined to 0.1+/-0% for PC3 cells but improved to 54.6+/-5.5% for G(s) alpha cells. With G(s) alpha cells, metafectene transfection of GFP plasmid alone yielded 46.9% positive cells, and cotransfection with CD44v9 expression vector yielded 45.9% positive cells. The visual impact of the transfected RNAi construct was detectable at the protein level 4 to 6 d posttransfection and was more dramatic after using metafectene than after using lipofectamine. Thus, in vitro, metafectene transfection efficiency was sufficient to allow us to assess the functional significance of our RNAi construct, suggesting metafectene as an excellent candidate for RNAi-mediated anticancer gene therapy.

  8. Comparison of ethanolamine/ethylenediamine-functionalized poly(glycidyl methacrylate) for efficient gene delivery.

    Science.gov (United States)

    Xu, F J; Zhu, Y; Chai, M Y; Liu, F S

    2011-08-01

    Cationic polymers with low cytotoxicity and high transfection efficiency have attracted considerable attention as non-viral carriers for gene delivery. Recently we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) (termed PGEA) vectors can have excellent transfection efficiency, while exhibiting very low toxicity. Herein different EA- and ethylenediamine (ED)-functionalized PGMA (termed PGEAED) vectors, as well as ED-functionalized PGMA (termed PGED) vectors, are proposed and compared for efficient gene delivery. In addition to the cationic pendant secondary amine and hydroxyl groups of PGEA, PGEAED, and PGED also contain flanking primary amine groups. PGEAED and PGED exhibited a substantially enhanced ability to condense pDNA into complex nanoparticles at the 100 nm level with positive zeta potentials of about 30 mV at nitrogen/phosphate (N/P) ratios of 10 or higher. More interestingly, no obvious change in the cytotoxicity of PGEAED was observed with a substantial increase in ED content. Moreover, the flanking primary amine groups induced by ED could be readily functionalized by glycyrrhetinic acid or cholic acid to improve the biophysical properties of the gene vectors. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  9. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis

    NARCIS (Netherlands)

    Zuhorn, IS; Kalicharan, Ruby; Hoekstra, D

    2002-01-01

    Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often

  10. Optical transfection using an endoscope-like system

    Science.gov (United States)

    Ma, Nan; Gunn-Moore, Frank; Dholakia, Kishan

    2011-02-01

    Optical transfection is a powerful method for targeted delivery of therapeutic agents to biological cells. A tightly focused pulsed laser beam may transiently change the permeability of a cell membrane to facilitate the delivery of foreign genetic material into cells. We report the first realization of an endoscope-like integrated system for optical transfection. An imaging fiber (coherent optical fiber bundle) with ~6000 cores (pixels) embedded in a fiber cladding of ~300 μm in diameter, produces an image circle (area) of ~270 μm diam. This imaging fiber, with an ordered axicon lens array chemically etched at its exit face, is used for the delivery of a femtosecond laser to the cell membrane for optical transfection along with subcellular resolution imaging. A microcapillary-based microfluidic system for localized drug delivery was also combined in this miniature, flexible system. Using this novel system, a plasmid transfection efficiency up to ~72% was obtained for CHO-K1 cells. This endoscope-like system opens a range of exciting applications, in particular, in the targeted in vivo optical microsurgery area.

  11. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    Science.gov (United States)

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  12. Minimally-Invasive Gene Transfection by Chemical and Physical Interaction of Atmospheric Pressure Plasma Flow

    Science.gov (United States)

    Kaneko, Toshiro

    2014-10-01

    Non-equilibrium atmospheric pressure plasma irradiated to the living-cell is investigated for medical applications such as gene transfection, which is expected to play an important role in molecular biology, gene therapy, and creation of induced pluripotent stem (iPS) cells. However, the conventional gene transfection using the plasma has some problems that the cell viability is low and the genes cannot be transferred into some specific lipid cells, which is attributed to the unknown mechanism of the gene transfection using the plasma. Therefore, the time-controlled atmospheric pressure plasma flow is generated and irradiated to the living-cell suspended solution for clarifying the transfection mechanism toward developing highly-efficient and minimally- invasive gene transfection system. In this experiment, fluorescent dye YOYO-1 is used as the simulated gene and LIVE/DEAD Stain is simultaneously used for cell viability assay. By the fluorescence image, the transfection efficiency is calculated as the ratio of the number of transferred and surviving cells to total cell count. It is clarified that the transfection efficiency is significantly increased by the short-time (90%). This result indicates that the physical effects such as the electric field caused by the charged particles arriving at the surface of the cell membrane, and chemical effects associated with plasma-activated products in solution act synergistically to enhance the cell-membrane transport with low-damage. This work was supported by JSPS KAKENHI Grant Number 24108004.

  13. Structure-activity correlation in transfection promoted by pyridinium cationic lipids.

    Science.gov (United States)

    Parvizi-Bahktar, P; Mendez-Campos, J; Raju, L; Khalique, N A; Jubeli, E; Larsen, H; Nicholson, D; Pungente, M D; Fyles, T M

    2016-03-21

    The efficiency of the transfection of a plasmid DNA encoding a galactosidase promoted by a series of pyridinium lipids in mixtures with other cationic lipids and neutral lipids was assessed in CHO-K1 cells. We identify key molecular parameters of the lipids in the mixture - clog P, lipid length, partial molar volume - to predict the morphology of the lipid-DNA lipoplex and then correlate these same parameters with transfection efficiency in an in vitro assay. We define a Transfection Index that provides a linear correlation with normalized transfection efficiency over a series of 90 different lipoplex compositions. We also explore the influence of the same set of molecular parameters on the cytotoxicity of the formulations.

  14. A comparative study of transfection methods for RNA interference in bone marrow-derived murine dendritic cells

    DEFF Research Database (Denmark)

    Pedersen, Charlotte Demuth; Fang, J J; Pedersen, Anders Elm

    2009-01-01

    Selective gene silencing using RNA interference (RNAi) has been shown to be an efficient method for manipulation of cellular functions. In this study, we compare three previously established methods for transfection of murine bone marrow-derived DC (BM-DC). We tested the efficacy of electroporation...... with the Mouse Nucleofector kit((R)) from Amaxa Biosystems and lipid-based transfection methods using transfection reagents from Santa Cruz Biotechnology or Genlantis. To analyse the transfection efficacy we used FITC-conjugated siRNA as a positive control together with CD80 and CD86 specific siRNA. We show...... that electroporation using the Mouse Nucleofector kit((R)) from Amaxa Biosystems was not an efficient method to transfect BM-DC with siRNA in our hands. Transfection with Santa Cruz Biotechnology reagents resulted in up to 59% FITC-siRNA positive cells, but did not result in effective silencing of CD80 surface...

  15. Highly Effective Gene Transfection In Vivo by Alkylated Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Jennifer A. Fortune

    2011-01-01

    Full Text Available We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp. while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.

  16. Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics.

    Science.gov (United States)

    Wilmes, Gwendolyn M; Carey, Kimberly L; Hicks, Stuart W; Russell, Hugh H; Stevenson, Jesse A; Kocjan, Paulina; Lutz, Stephen R; Quesenberry, Rachel S; Shulga-Morskoy, Sergey V; Lewis, Megan E; Clark, Ethan; Medik, Violetta; Cooper, Anthony B; Reczek, Elizabeth E

    2014-01-01

    Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.

  17. Single-Tailed Lipidoids Enhance the Transfection Activity of Their Double-Tailed Counterparts.

    Science.gov (United States)

    Wu, Yihang; Li, Linxian; Chen, Qing; Su, Yi; Levkin, Pavel A; Davidson, Gary

    2016-01-11

    Cationic lipid-like molecules (lipidoids) are widely used for in vitro and in vivo gene delivery. Nearly all lipidoids developed to date employ double-tail or multiple-tail structures for transfection. Single-tail lipidoids are seldom considered for transfection as they have low efficiency in gene delivery. So far, there is no detailed study on the contribution to transfection efficiency of single-tail lipidoids when combined with standard double-tail lipidoids. Here, we use combinatorial chemistry to synthesize 17 double-tail and 17 single-tail lipidoids using thiol-yne and thiol-ene click chemistry, respectively. HEK 293T cells were used to analyze transfection efficiency by fluorescence microscopy and calculated based on the percentage of cells transfected. The size and zeta potential of liposomes and lipoplexes were characterized by dynamic light scattering (DLS). Intracellular DNA delivery and trafficking was further examined using confocal microscopy. Our study shows that combining single with double-tail lipidoids increases uptake of lipoplexes, as well as cellular transfection efficiency.

  18. A study on the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles.

    Science.gov (United States)

    Li, Huiling; Chen, Jinwen; Xu, Xuan; Yang, Ruhao; Xiang, Xudong; Zhang, Dongshan

    2016-02-01

    To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (Ptransfection of CTGF-antisense-ODN into kidney. The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.

  19. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

    DEFF Research Database (Denmark)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

    2017-01-01

    Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage......, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain...

  20. Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum

    Science.gov (United States)

    2012-01-01

    Genetic manipulation of malaria parasites remains an inefficient, time-consuming and resource-intensive process. Presented here is a set of methods for 96-well plate-based transfection and culture that improve the efficiency of genetic manipulation of Plasmodium falciparum. Compared to standard protocols plate-based transfection requires 20-fold less DNA, transient transfection efficiency achieved is approximately seven-fold higher, whilst stable transfection success rate is above 90%. Furthermore the utility of this set of protocols to generate a knockout of the PfRH3 pseudogene, screened by whole-cell PCR, is demonstrated. The methods and tools presented here will facilitate genome-scale genetic manipulation of P. falciparum. PMID:22257490

  1. Experimental design optimization of filamentous phage transfection into mammalian cells by cationic lipids.

    Science.gov (United States)

    Aujame, L; Seguin, D; Droy, C; Hessler, C

    2000-06-01

    A previous study showed that filamentous phage could be efficiently transfected into mammalian cells in the presence of the cationic lipid Transfectam. In the present study, we used an experimental plan based on a uniform network (Doehlert) matrix to estimate optimal transfection conditions in two different cell lines, CHO and Cos-7. Using the cationic lipid RPR120535b as a model, we show that optimal conditions can be determined much more readily than with standard response curves. Under optimal conditions as analyzed by FACS, up to 60% of Cos-7 and 50% of CHO cells can be transfected. Furthermore, a comparison of different lipids (Transfectam, RPR120535b, TC1-12 and GAP-DLRIE/DOPE) suggests that lipids with multiple amine groups are more efficient for the transfection of filamentous phage.

  2. pH and reduction dual-responsive dipeptide cationic lipids with α-tocopherol hydrophobic tail for efficient gene delivery.

    Science.gov (United States)

    Liu, Qiang; Su, Rong-Chuan; Yi, Wen-Jing; Zheng, Li-Ting; Lu, Shan-Shan; Zhao, Zhi-Gang

    2017-03-31

    A series of tocopherol-based cationic lipid 3a-3f bearing a pH-sensitive imidazole moiety in the dipeptide headgroup and a reduction-responsive disulfide linkage were designed and synthesized. Acid-base titration of these lipids showed good buffering capacities. The liposomes formed from 3 and co-lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) could efficiently bind and condense DNA into nanoparticles. Gel binding and HPLC assays confirmed the encapsulated DNA could release from lipoplexes 3 upon addition of 10 mM glutathione (GSH). MTT assays in HEK 293 cells demonstrated that lipoplexes 3 had low cytotoxicity. The in vitro gene transfection studies showed cationic dipeptide headgroups clearly affected the transfection efficiency (TE), and arginine-histidine based dipeptide lipid 3f give the best TE, which was 30.4 times higher than Lipofectamine 3000 in the presence of 10% serum. Cell-uptake assays indicated that basic amino acid containing dipeptide cationic lipids exhibited more efficient cell uptake than serine and aromatic amino acids based dipeptide lipids. Confocal laser scanning microscopy (CLSM) studies corroborated that 3 could efficiently deliver and release DNA into the nuclei of HeLa cells. These results suggest that tocopherol-based dipeptide cationic lipids with pH and reduction dual-sensitive characteristics might be promising non-viral gene delivery vectors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. Insulin receptor activation and down-regulation by cationic lipid transfection reagents

    Directory of Open Access Journals (Sweden)

    Renström Ing-Marie

    2004-01-01

    Full Text Available Abstract Background Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000™ and FuGENE-6™. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types Results We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000™ on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000™ can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells. Conclusion Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.

  4. A novel dendrimer based on poly (L-glutamic acid) derivatives as an efficient and biocompatible gene delivery vector

    Energy Technology Data Exchange (ETDEWEB)

    Zeng Xin; Pan Shirong; Wang Chi; Wen Yuting; Wu Hongmei; Wang Cuifeng; Wu Chuanbin; Feng Min [School of Pharmaceutical Sciences and the First Affiliated Hospital, Sun Yat-sen University, 80 Zhongshan Road II, Guangzhou 510080 (China); Li Jie, E-mail: gzpshr@163.com, E-mail: fengmin@mail.sysu.edu.cn [Department of Gastrointestinal Surgery, The Third Affiliated Hospital of Sun Yat-sen University, 80 Zhongshan Road II, Guangzhou 510080 (China)

    2011-09-16

    Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.

  5. A novel dendrimer based on poly (L-glutamic acid) derivatives as an efficient and biocompatible gene delivery vector

    Science.gov (United States)

    Zeng, Xin; Pan, Shirong; Li, Jie; Wang, Chi; Wen, Yuting; Wu, Hongmei; Wang, Cuifeng; Wu, Chuanbin; Feng, Min

    2011-09-01

    Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.

  6. Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection.

    Science.gov (United States)

    Jinno, Masafumi; Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu

    2016-09-01

    This study has been done to know what kind of factors in plasmas and processes on cells induce plasma gene transfection. We evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones, inducing gene transfection. First, the laser produced plasma (LPP) was employed to estimate the contribution of the chemical factors. Second, liposomes were fabricated and employed to evaluate the effects of plasma irradiation on membrane under the condition without biochemical reaction. Third, the clathrin-dependent endocytosis, one of the biochemical processes was suppressed. It becomes clear that chemical factors (radicals and reactive oxygen/nitrogen species) do not work by itself alone and electrical factors (electrical current, charge and field) are essential to plasma gene transfection. It turned out the clathrin-dependent endocytosis is the process of the transfection against the 60% in all the transfected cells. The endocytosis and electrical poration are dominant in plasma gene transfection, and neither permeation through ion channels nor chemical poration is dominant processes. The simultaneous achievement of high transfection efficiency and high cell survivability is attributed to the optimization of the contribution weight among three groups of processes by controlling the weight of electrical and chemical factors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Impact of plasmid quality on lipoplex-mediated transfection.

    Science.gov (United States)

    De La Vega, Jonathan; Braak, Bas Ter; Azzoni, Adriano R; Monteiro, Gabriel A; Prazeres, Duarte Miguel F

    2013-11-01

    This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

  8. Ultrasound-assisted non-viral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion

    Science.gov (United States)

    Wang, Z; Zourelias, L; Wu, C; Edwards, PC; Trombetta, M; Passineau, MJ

    2015-01-01

    Rationale Xerostomia is a common side effect of ionizing radiation used to treat head and neck cancer. A groundbreaking Phase I human clinical trial utilizing Adenoviral gene transfer of Aquaporin-1 (AQP1) to a single salivary gland of individuals suffering from radiation-induced xerostomia has recently been reported. Unfortunately, the limitations of the Adenoviral vector system utilized in this pioneering trial preclude its advancement to a Phase II trial and we have thus undertaken to evaluate the therapeutic potential of ultrasound-assisted non-viral gene transfer (UAGT) as an alternative means of delivering AQP1 gene therapy to the salivary gland by comparing head-to-head with the canonical Adenoviral vector in a swine model. Findings Swine irradiated unilaterally with a 10Gy electron beam targeted at the parotid gland suffered from significant, sustained hyposalivation that was bilateral, despite irradiation being confined to the targeted gland. Unilateral AQP1 gene therapy with UAGT resulted in bilateral restoration of stimulated salivary flow at 48 hours and one week post-treatment (1.62+/−0.48ml, 1.87+/−0.45ml) to pre-injury levels (1.34+/−0.14ml) in a manner comparable to Adenoviral delivery (2.32+/−0.6ml, 1.33+/−0.97ml). Conclusions UAGT can replace the Adenoviral vector as a means of delivering AQP1 gene therapy in the irradiated swine model and is a candidate for advancement to a Phase I human clinical trial. PMID:25871828

  9. Trichomoniasis - are we giving the deserved attention to the most common non-viral sexually transmitted disease worldwide?

    Science.gov (United States)

    Menezes, Camila Braz; Frasson, Amanda Piccoli; Tasca, Tiana

    2016-01-01

    Etiology: Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world. Transmission: Trichomoniasis is transmitted by sexual intercourse and transmission via fomites is rare. Epidemiology, incidence and prevalence: The WHO estimates an incidence of 276 million new cases each year and prevalence of 187 million of infected individuals. However, the infection is not notifiable. Pathology/Symptomatology: The T. vaginalis infection results in a variety of clinical manifestations - in most cases the patients are asymptomatic, but some may develop signs typically associated to the disease. Importantly, the main issue concerning trichomoniasis is its relationship with serious health consequences such as cancer, adverse pregnancy outcomes, infertility, and HIV acquisition. Molecular mechanisms of infection: To achieve success in parasitism trichomonads develop a complex process against the host cells that includes dependent- and independent-contact mechanisms. This multifactorial pathogenesis includes molecules such as soluble factors, secreted proteinases, adhesins, lipophosphoglycan that culminate in cytoadherence and cytotoxicity against the host cells. Treatment and curability: The treatment with metronidazole or tinidazole is recommended; however, cure failures remain problematic due to noncompliance, reinfection and/or lack of treatment of sexual partners, inaccurate diagnosis, or drug resistance. Therefore, new therapeutic alternatives are urgently needed. Protection: Strategies for protection including sexual behavior, condom usage, and therapy have not contributed to the decrease on disease prevalence, pointing to the need for innovative approaches. Vaccine development has been hampered by the lack of long-lasting humoral immunity associated to the absence of good animal models. PMID:28357378

  10. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B

    1996-01-01

    transfected. Parvalbumin-transfected and mock-transfected cells were loaded with the calcium indicator fura-2 and were exposed, in the same dish, to different concentrations of the calcium ionophore A23187 or to KCI. The results show that parvalbumin-transfected PCC7 cells had much better calcium buffering...... capacity than control cells....

  11. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B

    1996-01-01

    transfected. Parvalbumin-transfected and mock-transfected cells were loaded with the calcium indicator fura-2 and were exposed, in the same dish, to different concentrations of the calcium ionophore A23187 or to KCI. The results show that parvalbumin-transfected PCC7 cells had much better calcium buffering...

  12. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

    Science.gov (United States)

    2010-01-01

    Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival. PMID:20144189

  13. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

    Directory of Open Access Journals (Sweden)

    Bedayat Babak

    2010-02-01

    Full Text Available Abstract Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts. With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.

  14. Gene Transfection Method Using Atmospheric Pressure Dielectric-Barrier Discharge Plasmas

    Science.gov (United States)

    Sasaki, Shota; Kanzaki, Makoto; Kaneko, Toshiro

    2013-09-01

    Gene transfection which is the process of deliberately introducing nucleic acids into cells is expected to play an important role in medical treatment because the process is necessary for gene therapy and creation of induced pluripotent stem (iPS) cells. However, the conventional transfection methods have some problems, so we focus attention on promising transfection methods by atmospheric pressure dielectric-barrier discharge (AP-DBD) plasmas. AP-DBD He plasmas are irradiated to the living cell covered with genes. Preliminarily, we use fluorescent dye YOYO-1 instead of the genes and use LIVE/DEAD Stain for cell viability test, and we analyze the transfection efficiency and cell viability under the various conditions. It is clarified that the transfection efficiency is strongly dependence on the plasma irradiation time and cell viability rates is high rates (>90%) regardless of long plasma irradiation time. These results suggest that ROS (Reactive Oxygen Species) and electric field generated by the plasma affect the gene transfection. In addition to this (the plasma irradiation time) dependency, we now investigate the effect of the plasma irradiation under the various conditions.

  15. [Effect of different approaches of lentiviral vector transfection on target gene expression in rat liver].

    Science.gov (United States)

    Zhao, Yingpeng; Li, Li; Ma, Jingpan; Bai, Jianhua; Liu, Qiyu

    2014-01-01

    To investigate the optimal approach of lentiviral vector transfection for effective delivery of exogenous gene into the liver. The lentiviral vector was delivered via the ileocolic vein of the ileocecus (portal vein group) or via the caudal vein of SD rats. The effect gene transfection into the liver was assessed by observing the expression of green fluorescence protein expression carried by the lentiviral vector, silencing of LXRα mRNA expression mediated by RNA interference, and liver transaminase changes. The efficiency and safety of the two approaches of transfection were evaluated. All the rats receiving lentiviral transfection survived. In the portal vein group, abundant green fluorescence was detected in the liver at 96 h following the transfection and lasted till 14 days, whereas only weak fluorescence was observed in the caudal vein group. The results of RT-PCR demonstrated a significant higher rate of LXRα knock-down in portal vein group than in caudal vein group (0.135∓0.002 vs 0.713∓0.036, Ptransfection into the liver, and puncture from the ileocolic vein of ileocecus can guarantee the survival of rats and improve the transfection efficiency without causing liver injury.

  16. Novel mechanism of gene transfection by low-energy shock wave.

    Science.gov (United States)

    Ha, Chang Hoon; Lee, Seok Cheol; Kim, Sunghyen; Chung, Jihwa; Bae, Hasuk; Kwon, Kihwan

    2015-08-05

    Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

  17. Modulating polyplex-mediated gene transfection by small-molecule regulators of autophagy.

    Science.gov (United States)

    Zhong, Xiao; Panus, David; Ji, Weihang; Wang, Chun

    2015-03-02

    Nonviral gene transfection mediated by cationic polymer/DNA polyplexes often imposes stress and toxicity to cells. To better understand the relationship between cellular stress responses and polyplex-mediated transfection, polyplex-induced early autophagy in mouse fibroblasts was characterized and the impact of autophagy modulation on transgene expression evaluated. Transmission electron microscopy revealed the formation of double-membraned autophagosome in the cytoplasm of polyplex-transfected cells. Immunofluorescence staining and microscopy revealed intracellular LC3 punctation that was characteristic of early autophagy activation. Elevated expression of autophagosome-associated LC3 II protein was also detected by Western blot. When cells were treated with small-molecule modulators of autophagy, polyplex-mediated gene transfection efficiency was significantly affected. 3-Methyladenine (3-MA), an early autophagy inhibitor, reduced transfection efficiency, whereas rapamycin, an autophagy inducer, enhanced transgene expression. Importantly, the observed functional impact on gene transfection by autophagy modulation was decoupled from that of other modes of cellular stress response (apoptosis/necrosis). Treatment of cells by 3-MA or rapamycin did not affect the level of intracellular reactive oxygen species (ROS) but did decrease or increase, respectively, nuclear localization of polyplex-delivered plasmid DNA. These findings suggest new possibilities of enhancing polyplex-mediated gene delivery by codelivery of small-molecule regulators of autophagy.

  18. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite

    KAUST Repository

    Zhang, G.

    2015-06-25

    RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in ‘double transfections’, in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments.

  19. Oscillating magnet array-based nanomagnetic gene transfection of human mesenchymal stem cells.

    Science.gov (United States)

    Fouriki, Angeliki; Dobson, Jon

    2014-05-01

    In this work, the potential of nanomagnetic transfection of primary human mesenchymal stem cells (hMSCs) and the effects of a novel nonviral oscillating magnet array system in enhancing transfection efficiency were investigated. Green fluorescent protein plasmids coupled to magnetic nanoparticles (MNPs) were introduced onto hMSCs in culture. Magnetic fields generated by arrays of neodymium iron boron magnets positioned below the culture plates direct the MNP/DNA complexes into contact with the cells. The magnet arrays were oscillated, promoting more efficient endocytosis via mechanical stimulation. Green fluorescent protein expression, cell viability and stem cell surface markers were assayed. MNP/DNA complexes were delivered into hMSCs, and the oscillating magnet array system appears to improve transfection efficiency as well as cell viability. The expression of hMSC-specific cell surface markers was unaffected. Nonviral transfection using MNPs and oscillating magnet arrays offers a more efficient and 'cell-friendly' method of transfecting hMSCs than other nonviral techniques, while preserving their stem cell characteristics.

  20. Cationic, amphiphilic copolymer micelles as nucleic acid carriers for enhanced transfection in rat spinal cord.

    Science.gov (United States)

    Gwak, So-Jung; Nice, Justin; Zhang, Jeremy; Green, Benjamin; Macks, Christian; Bae, Sooneon; Webb, Ken; Lee, Jeoung Soo

    2016-04-15

    Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1%, and 8.1%, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration. Gene therapy is a promising strategy to overcome barriers to axonal regeneration in the injured central nervous system. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to cytotoxicity and low transfection efficiency in the presence of serum proteins. Here, we report cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) that are capable of efficiently transfecting reporter

  1. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    Science.gov (United States)

    Wu, Kaimin; Xu, Jie; Liu, Mengyuan; Song, Wen; Yan, Jun; Gao, Shan; Zhao, Lingzhou; Zhang, Yumei

    2013-01-01

    MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and −20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials. PMID:23662054

  2. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate.

    Science.gov (United States)

    Wu, Kaimin; Xu, Jie; Liu, Mengyuan; Song, Wen; Yan, Jun; Gao, Shan; Zhao, Lingzhou; Zhang, Yumei

    2013-01-01

    MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.

  3. Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva.

    Science.gov (United States)

    De Goeyse, Ine; Jansen, Famke; Madder, Maxime; Hayashida, Kyoko; Berkvens, Dirk; Dobbelaere, Dirk; Geysen, Dirk

    2015-03-15

    Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host-parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1α promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Selective transfection with osmotically active sorbitol modified PEI nanoparticles for enhanced anti-cancer gene therapy.

    Science.gov (United States)

    Nguyen, Kim Cuc Thi; Muthiah, Muthunarayanan; Islam, Mohammad Ariful; Kalash, R Santhosh; Cho, Chong-Su; Park, Hansoo; Lee, Il-Kwon; Kim, Hyeoung-Joon; Park, In-Kyu; Cho, Kyung A

    2014-07-01

    Polysorbitol-mediated transporter (PSMT) has been previously shown to achieve high transfection efficiency with minimal cytotoxicity. Polysorbitol backbone possesses osmotic properties and leads to enhanced cellular uptake. The PSMT/pDNA nanoparticles were prepared and the particle size, surface charge of the nanoparticles was determined for the study. PSMT delivers genes into cells by the caveolae mediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Transfection through the caveolae may help PSMT to selectively transfect cancer cells rather than normal cells. Transfection of the luciferase gene by PSMT was tested in various cell types including cancer cell lines, primary cells, and immortalized cells. Luciferase transgene expression mediated by PSMT was remarkably increased in HeLa cells compared to expression using the control carrier Lipofectamine. Moreover, the toxicity of PSMT was comparable to the control carrier (Lipofectamine) in the same cells. Selective transfection of cancer cells using PSMT was further confirmed by co-culture of cancer and normal cells, which showed that transgene expression was pre-dominantly achieved in cancer cells. A functional p53 gene was also delivered into HeLa cells using PSMT and the selective transgene expression of p53 protein in cancer cells was analyzed through western blotting and confocal microscopy. HeLa cells transfected with PSMT/p53 plasmid nanoparticles showed cellular damage and apoptosis, which was confirmed through propidium iodide staining. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. [EFFECT OF Akt1 GENE TRANSFECTION ON HYPOXIA TOLERANCE OF BONE MARROW MESENCHYMAL STEM CELLS].

    Science.gov (United States)

    Yu, Fengxu; Chen, Yongen; Chen, Feng; Xia, Jiyi; Liu, Hongduan; Fu, Yong; Li, Miaoling; Liao, Bin

    2016-04-01

    To investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. LVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3PLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94% N₂, 1% O₂, and 5% CO₂ for 0, 3, 6, 9, and 12 hours (group B1 and group C1). The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t = 17.525, P = 0.013). The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.

  6. Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Alexander Raup

    2016-06-01

    Full Text Available Genetic modification (“transfection” of mammalian cells using non-viral, synthetic agents such as polycations, is still a challenge. Polyplex formation between the DNA and the polycation is a decisive step in such experiments. Star-shaped polycations have been proposed as superior transfection agents, yet have never before been compared side-by-side, e.g., in view of structural effects. Herein four star-shaped polycationic structures, all based on (2-dimethylamino ethyl methacrylate (DMAEMA building blocks, were investigated for their potential to deliver DNA to adherent (CHO, L929, HEK-293 and non-adherent (Jurkat, primary human T lymphocytes mammalian cells. The investigated vectors included three structures where the PDMAEMA arms (different arm length and grafting densities had been grown from a center silsesquioxane or silica-coated γ-Fe2O3-core and one micellar structure self-assembled from poly(1,2-butadiene-block PDMAEMA polymers. All nano-stars combined high transfection potential with excellent biocompatibility. The micelles slightly outperformed the covalently linked agents. For method development and optimization, the absolute amount of polycation added to the cells was more important than the N/P-ratio (ratio between polycation nitrogen and DNA phosphate, provided a lower limit was passed and enough polycation was present to overcompensate the negative charge of the plasmid DNA. Finally, the matrix (NaCl vs. HEPES-buffered glucose solution, but also the concentrations adjusted during polyplex formation, affected the results.

  7. Gene carriers and transfection systems used in the recombination of dendritic cells for effective cancer immunotherapy.

    Science.gov (United States)

    Chen, Yu-Zhe; Yao, Xing-Lei; Tabata, Yasuhiko; Nakagawa, Shinsaku; Gao, Jian-Qing

    2010-01-01

    Dendritic cells (DCs) are the most potent antigen-presenting cells. They play a vital role in the initiation of immune response by presenting antigens to T cells and followed by induction of T-cell response. Reported research in animal studies indicated that vaccine immunity could be a promising alternative therapy for cancer patients. However, broad clinical utility has not been achieved yet, owing to the low transfection efficiency of DCs. Therefore, it is essential to improve the transfection efficiency of DC-based vaccination in immunotherapy. In several studies, DCs were genetically engineered by tumor-associated antigens or by immune molecules such as costimulatory molecules, cytokines, and chemokines. Encouraging results have been achieved in cancer treatment using various animal models. This paper describes the recent progress in gene delivery systems including viral vectors and nonviral carriers for DC-based genetically engineered vaccines. The reverse and three-dimensional transfection systems developed in DCs are also discussed.

  8. A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios

    Science.gov (United States)

    Wang, Mingming; Liu, Hongmei; Li, Lei; Cheng, Yiyun

    2014-01-01

    Polymers have shown great promise in the design of high efficient and low cytotoxic gene vectors. Here we synthesize fluorinated dendrimers for use as gene vectors. Fluorinated dendrimers achieve excellent gene transfection efficacy in several cell lines (higher than 90% in HEK293 and HeLa cells) at extremely low N/P ratios. These polymers show superior efficacy and biocompatibility compared with several commercial transfection reagents such as Lipofectamine 2000 and SuperFect. Fluorination enhances the cellular uptake of the dendrimer/DNA polyplexes and facilitates their endosomal escape. In addition, the fluorinated dendrimer shows excellent serum resistance and exhibits high gene transfection efficacy even in medium containing 50% FBS. The results suggest that fluorinated dendrimers are a new class of highly efficient gene vectors and fluorination is a promising strategy to design gene vectors without involving sophisticated syntheses.

  9. Combining polyethylenimine and Fe(III) for mediating pDNA transfection.

    Science.gov (United States)

    Jorge, Andreia F; Röder, Ruth; Kos, Petra; Dias, Rita S; Wagner, Ernst; Pais, Alberto A C C

    2015-06-01

    The potential use of Fe(III) ions in biomedical applications may predict the interest of its combination with pDNA-PEI polyplexes. The present work aims at assessing the impact of this metal on pDNA complex properties. Variations in the formation of complexes were imposed by using two types of biological buffers at different salt conditions. The incorporation of pDNA in complexes was characterised by gel electrophoresis and dynamic light scattering. Transfection efficiency and cytotoxicity were evaluated in HeLa and HUH-7 cell lines, supported by flow cytometry assays. Fe(III) enhances pDNA incorporation in the complex, irrespective of the buffer used. Transfection studies reveal that the addition of Fe(III) to complexes at low ionic strength reduces gene transfection, while those prepared under high salt content do not affect or, in a specific case, increase gene transfection up to 5 times. This increase may be a consequence of a favoured interaction of polyplexes with cell membrane and uptake. At low salt conditions, results attained with chloroquine indicate that the metal may inhibit polyplex endosomal escape. A reduction on the amount of PEI (N/P 5) formed at intermediary ionic strength, complemented by Fe(III), reduces the size of complexes while maintaining a transfection efficiency similar to that obtained to N/P 6. Fe(III) emerges as a good supporting condensing agent to modulate pDNA-PEI properties, including condensation, size and cytotoxicity, without a large penalty on gene transfection. This study highlights important aspects that govern pDNA transfection and elucidates the benefits of incorporating the versatile Fe(III) in a gene delivery system. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

    Directory of Open Access Journals (Sweden)

    Lummis Sarah CR

    2011-06-01

    Full Text Available Abstract Background Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles. Results Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles. Conclusions We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.

  11. Persistent gene expression in rat liver in vivo by repetitive transfections using HVJ-liposome.

    Science.gov (United States)

    Hirano, T; Fujimoto, J; Ueki, T; Yamamoto, H; Iwasaki, T; Morisita, R; Sawa, Y; Kaneda, Y; Takahashi, H; Okamoto, E

    1998-04-01

    Most viral vectors are highly immunogenic and are of limited use for somatic gene therapy that requires repetitive administrations. We have developed a highly efficient gene transduction procedure useful for repetitive transfections using liposome containing hemagglutinating virus of Japan (HVJ-liposome). The Escherichia coli beta-galactosidase (beta-gal) gene was embodied in HVJ-liposome, and introduced directly into the caudal lobe of rat liver that was transiently isolated from a systemic circulation. A 116 kDa beta-gal protein was detected in transfected rat liver tissues by Western blot analysis and it was expressed in more than two-thirds of the liver by histological staining. It was found that the transfection efficiency was not affected by repetitive transfections. In support of these findings, antibody response to HVJ-liposome detected in the rat sera was weak and transient. Furthermore, cytotoxic T lymphocytes were not elicited against autologous rat hepatocytes that were transfected in vivo using HVJ-liposome. Thus, our results demonstrate that the isolation of a target liver from systemic circulation and the direct administration of foreign genes using HVJ-liposomes are useful for high gene transduction and persistent gene expression in the liver.

  12. Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells

    Science.gov (United States)

    Kelly, Abby M; Plautz, Sarah A; Zempleni, Janos; Pannier, Angela K

    2016-01-01

    Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4–15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6–10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells. PMID:26478250

  13. Optimizing conditions for calcium phosphate mediated transient transfection

    Directory of Open Access Journals (Sweden)

    Ling Guo

    2017-03-01

    Conclusions: Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.

  14. Graphene for enhanced embryonic stem cell photo-transfection efficiency

    CSIR Research Space (South Africa)

    Mthunzi, P

    2013-04-01

    Full Text Available Due to their pluripotency properties, embryonic stem (ES) cells possess great potential in regenerative therapy. Since reported a promising tissue engineering scaffold material, here, graphene is demonstrated to significantly improve the ES cell...

  15. Graphene for improved femtosecond laser based pluripotent stem cell transfection.

    Science.gov (United States)

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Khanyile, Thulile; Warner, Jamie H

    2014-05-01

    Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self-renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non-invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO-K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO-K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up-regulation of cell adhesion promoting peptides or laminin-related receptors of the extracellular matrix (ECM) in cell samples

  16. Optical and Acoustical Techniques for Non-viral Gene Delivery to Mammalian Cells and In-situ Study of Cytoskeletal Mechanics

    Science.gov (United States)

    Ma, Zili

    surface acoustic waves, which not only achieved a high efficiency of cells permeabilization in a quick speed, but also allowed us to observe the permeabilization process in real time by microscope. This device is also compatible with biophotonics studies based on fs laser, which can be further developed as a powerful tool for optical gene delivery with the capability of precisely controlling the fluid on-chip by SAW. SAW devices could also achieve exogenous gene delivery through the cell membrane without the need of adding chemical agents. Our results showed that the membrane of mammalian adherent cells could be effectively perforated transiently by applying a SAW. The transfection of pEGFP plasmids into endothelial cells was carried out successfully via this SAW-induced cell perforation. The expression of GFP was observed after 24-hour incubation subsequent to the SAW treatment. In regard to the application of fs lasers in cellular and subcellular level studies, we applied the optical nanoscissoring technique based on fs lasers in biomechanical studies to study the mechanical properties of single SF in-situ. Integrated into a confocal microscope, the fs laser showed great power in manipulating targeted in-situ subcellular structures under real-time imaging without damaging nearby regions. Here, how oxidative challenges would alter the mechanical properties of SFs in myoblasts was firstly investigated using the optical nanoscissoring technique to comprehend the whole picture of muscle tissue injury and repair from the basics. The prestress of stress fibers after the oxidative challenges was found through our modified viscoelastic retraction model and experiment result.

  17. [EFFECT OF TRITON X-100 ON LIPOSOME MEDIATED BONE MORPHOGENETIC PROTEIN 2 BY TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS].

    Science.gov (United States)

    Xia, Delin; Huang, Mingke; Fu, Guangxing; Ma, Zheng; Wu, Shuangjiang; Zhou, Hangyu

    2015-01-01

    To study the effect of Triton X-100 promoting liposome-mediated bone morphogenetic protein 2 (BMP-2) gene transfection of rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were separated and cultured from the femur and tibia of healthy Wistar rats (8-week-old, male). The 3rd passage BMSCs identified by detecting the surface antigen were used to transfect. The optimum concentration of Triton X-100 for liposome mediated gene transfection was determined with ELISA meter by the way of MTT. In optimum concentration of Triton X-100, liposome mediated BMP-2 gene was transfected to BMSCs. The experiment was divided into 3 groups according to types of trasfection agents: BMSCs were transfected with Triton X-100+liposome+BMP-2 (experimental group), with liposome+ BMP-2 (conventional transfection group), and untransfected BMSCs served as blank control group. After 48 hours of transfecting, the green fluorescent protein (GFP) in cells was detected through inverted fluorescence microscope. After 72 hours of transfection, real-time fluorescence quantitative PCR was applied to measure the mRNA expression of BMP-2. 0.01% Triton X-100 was determined to be the optimum concentration for not only making the BMSCs maintain vitality, but also achieving a certain effect on BMSCs. After trasfecting for 48 hours, GFP was observed through inverted fluorescence microscope in the experimental group and conventional transfection group, but was not observed in the blank control group. After trasfecting for 72 hours, the relative BMP-2 mRNA expression level was 5.94 ± 0.12 in the experimental group, and was 4.99 ± 0.08 in the conventional transfection group, showing significant difference (t = 360.28, P = 0.02). The transfection efficiency was increased by 19% in the experimental group. 0.010% Triton X-100 can promote the liposome mediated BMP-2 gene transfection of rat BMSUs, and can improve the transfection efficiency.

  18. Transfection mediated by gemini surfactants : Engineered escape from the endosomal compartment

    NARCIS (Netherlands)

    Bell, PC; Bergsma, M; Dolbnya, IP; Bras, W; Stuart, MCA; Rowan, AE; Feiters, MC; Engberts, JBFN

    2003-01-01

    The structure of the lipoplex formed from DNA and the sugar-based cationic gemini surfactant 1, which exhibits excellent transfection efficiency, has been investigated in the pH range 8.8-3.0 utilizing small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-TEM). Uniquely, three

  19. Comparison of nonviral transfection and adeno-associated viral transduction on cardiomyocytes.

    Science.gov (United States)

    Djurovic, Srdjan; Iversen, Nina; Jeansson, Stig; Hoover, Frank; Christensen, Geir

    2004-09-01

    Cardiomyocytes are terminally differentiated cells that to date have been characterized as poor targets for nonviral gene transfer. This study was therefore designed to determine the optimal nonviral gene transfer parameters in cell cultures of neonatal rat cardiomyocytes and to compare them with the efficiency of gene transfer using adeno-associated viral vectors (AAV). Transfection efficiency was measured by quantitative chloramphenicol acetyltransferase type I (CAT)-enzyme-linked immunosorbent assay and beta-galactosidase staining, based on overexpression of reporter genes (CAT and LacZ). The efficiency of CAT/LacZ overexpression was assessed using the following techniques: (1) liposomal reagents, such as: FuGENE 6, LipofectAMINE 2000, LipofectAMINE PLUS, GenePORTER, Metafectene, and LipoGen; (2) electroporation and nucleofector techniques; and (3) an AAV2 vector harboring a lacZ reporter gene. Toxicity was monitored by total protein measurement and by analyzing cell metabolism. On average, Lipofectamine 2000 was the most effective nonviral method examined yielding consistently high transfection rates (8.1% beta-galactosidase-positive cells) combined with low toxicity. Electroporation also resulted in high transfection values (7.5%); however, cellular toxicity was higher than that of Lipofectamine 2000. Finally, transduction with AAV2 vectors provided the highest levels of transduction (88.1%) with no cellular toxicity. We conclude that although transduction with AAV is more efficient (88.1%), transfections with nonviral techniques, when optimized, may provide a useful alternative for overexpression of therapeutic genes in neonatal cardiomyocytes.

  20. Nano-flares: Probes for Transfection and mRNA Detection in Living Cells

    Science.gov (United States)

    Seferos, Dwight S.; Giljohann, David A.; Hill, Haley D.; Prigodich, Andrew E.

    2011-01-01

    We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular “nano-flares” for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells. PMID:18034495

  1. Spacer structure and hydrophobicity influences transfection activity of novel polycationic gemini amphiphiles.

    Science.gov (United States)

    Puchkov, Pavel A; Kartashova, Irina A; Shmendel, Elena V; Luneva, Anastasya S; Morozova, Nina G; Zenkova, Marina A; Maslov, Mikhail A

    2017-08-01

    Three novel polycationic gemini amphiphiles with different spacers were developed and evaluated in terms of their physiochemical properties and transfection efficiencies. Cationic liposomes formed by these amphiphiles and the helper lipid DOPE were able to successfully condense DNA, as shown by gel mobility shift and ethidium bromide intercalation assays. Transfection activity of the liposomes was superior to Lipofectamine(®) 2000 and was dependent on spacer structure, hydrophobicity, and nucleic acid type (pDNA or siRNA). We demonstrated that the cationic liposomes 2X6/DOPE and 2X7/DOPE are potential non-toxic vehicles for gene delivery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Growth of avian adeno-associated virus in chicken cells transfected with fowl adenovirus serotype 1 DNA.

    Science.gov (United States)

    Bauer, A; Monreal, G; Bauer, H J

    1990-09-01

    Using the chloroquine-modified calcium phosphate coprecipitation technique, fowl adenovirus serotype 1 (FAV 1) DNA transfects efficiently chicken cell cultures. Infection of FAV 1 DNA transfected cells with helper dependent avian adeno-associated virus (AAAV) results in the production of AAAV progeny, being detected by an indirect immunofluorescence assay. These findings indicate that FAV 1 DNA introduced into the host cell promotes actively the growth of AAAV.

  3. Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors.

    Science.gov (United States)

    Benet, Marta; Jover, Ramiro; Bort, Roque

    2015-01-01

    Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infecting primary cultured hepatocytes. This recombinant adenovirus induces robust expression of the protein of interest in hepatocytes within a wide range of doses.

  4. Plasmid transfection in bovine cells: Optimization using a realtime monitoring of green fluorescent protein and effect on gene reporter assay.

    Science.gov (United States)

    Osorio, Johan S; Bionaz, Massimo

    2017-08-30

    Gene reporter technology (GRT) has opened several new avenues for monitoring biological events including the activation of transcription factors, which are central to the study of nutrigenomics. However, this technology relies heavily on the insertion of foreign plasmid DNA into the nuclei of cells (i.e., transfection), which can be very challenging and highly variable among cell types. The objective of this study was to investigate the optimal conditions to generate reliable GRT assay data on bovine immortalized cell lines, Madin Darby Bovine Kidney (MDBK) and bovine mammary epithelial alveolar (MACT) cells. Results are reported for two experiments. In Experiment 1, using 96 well-plate and a robotic inverted fluorescent microscope, we compared transfection efficiency among commercially available transfection reagents (TR) Lipofectamine® 3000 (Lipo3), Lipofectamine® LTX (LipoLTX), and TransIT-X2® (TransX2), three doses of TR (i.e., 0.15, 0.3, and 0.4μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20h post-transfection) at which point MACT had greater transfection than MDBK cells (16.3% vs. 2.2%). It is unclear the reason for the low transfection in MDBK cells. Maximal transfection efficiency was obtained with 0.3μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 29.5±1.9%) and 0.15μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 4.0±0.4%) for MACT and MDBK cells, respectively. The higher amount of TR and DNA was generally associated with higher cell mortality. Using high, medium, and low transfection efficiency conditions determined in Experiment 1, we performed a GRT assay for peroxisome proliferator-activated response element (PPRE) luciferase in MACT and MDBK cells treated with 10nM or 100nM of synthetic Peroxisome Proliferator-activated Receptor

  5. The development of mechanically formed stable nanobubbles intended for sonoporation-mediated gene transfection.

    Science.gov (United States)

    Abdalkader, Rodi; Kawakami, Shigeru; Unga, Johan; Higuchi, Yuriko; Suzuki, Ryo; Maruyama, Kazuo; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2017-11-01

    In this study, stable nano-sized bubbles (nanobubbles [NBs]) were produced using the mechanical agitation method in the presence of perfluorocarbon gases. NBs made with perfluoropropane had a smaller size (around 400 nm) compared to that of those made with perfluorobutane or nitrogen gas. The lipid concentration in NBs affected both their initial size and post-formulation stability. NBs formed with a final lipid concentration of 0.5 mg/ml tended to be more stable, having a uniform size distribution for 24 h at room temperature and 50 h at 4 °C. In vitro gene expression revealed that NBs/pDNA in combination with ultrasound (US) irradiation had significantly higher transfection efficacy in colon C26 cells. Moreover, for in vivo gene transfection in mice left limb muscles, there was notable local transfection activity by NBs/pDNA when combined with US irradiation. In addition, the aged NBs kept at room temperature or 4 °C were still functional at enhancing gene transfection in mice. We succeeded in preparing stable NBs for efficient in vivo gene transfection, using the mechanical agitation method.

  6. Improved transfection of spleen-derived antigen-presenting cells in culture using TATp-liposomes.

    Science.gov (United States)

    Pappalardo, Juan Sebastián; Quattrocchi, Valeria; Langellotti, Cecilia; Di Giacomo, Sebastián; Gnazzo, Victoria; Olivera, Valeria; Calamante, Gabriela; Zamorano, Patricia I; Levchenko, Tatyana S; Torchilin, Vladimir P

    2009-02-20

    Antigen presenting cells (APC) are among the most important cells of the immune system since they link the innate and the adaptative immune responses, directing the type of immune response to be elicited. To modulate the immune response in immune preventing or treating therapies, gene delivery into immunocompetent cells could be used. However, APC are very resistant to transfection. To increase the efficiency of APC transfection, we have used liposome-based lipoplexes additionally modified with cell-penetrating TAT peptide (TATp) for better intracellular delivery of a model plasmid encoding for the enhanced-green fluorescent protein (pEGFP). pEGFP-bearing lipoplexes made of a mixture of PC:Chol:DOTAP (60:30:10 molar ratio) with the addition of 2% mol of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate (plain-L) or TATp-PEG-PE (TATp-L) were shown to effectively protect the incorporated DNA from degradation. Uptake assays of rhodamine-labeled lipoplexes and transfections with the EGFP reporter gene were performed with APC derived from the mouse spleen. TATp-L-based lipoplexes allowed for significantly enhanced both, the uptake and transfection in APC. Such a tool could be used for the APC transfection as a first step in immune therapy.

  7. Transfection Agent Induced Nanoparticle Cell Loading

    Directory of Open Access Journals (Sweden)

    Karin Montet-Abou

    2005-07-01

    Full Text Available Loading cells with magnetic nanoparticles, and tracking their fate in vivo by high resolution MRI, is an attractive approach for enhancing the efficacy of cell-based therapies including those utilizing hematopoietic stem cells, neuroprogenitor cells, and T cells. The transfection agent (internalization agent assisted loading with the Feridex IV® nanoparticle is an attractive method of loading because of the low cost of materials, and possible low regulatory barriers for eventual clinical use. We therefore explored the interaction between Feridex IV® and three internalization agents protamine (PRO, polylysine (PLL, and lipofectamine (LFA. Feridex reacted with internalization agents to form aggregates, except when either the internalization agent or Feridex was present in large excess. When Jurkat T cells were incubated with Feridex/LFA or Feridex/PRO mixtures, and washed by centrifugation, nanoparticle aggregates co-purified with cells. With C17.2 cells large iron oxide particles adhered to the cell surface. At 30 μg/mL Feridex and 3 μg/mL LFA, internalization was largely mediated by LFA and was largely cytoplasmic. However, we found that the conditions used to label cells with Feridex and transfection agents need to be carefully selected to avoid the problems of surface adsorption and nanoparticle precipitation.

  8. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    DEFF Research Database (Denmark)

    Wu, Kaimin; Xu, Jie; Liu, Mingzhe

    2013-01-01

    MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes...

  9. [The preparation of recombinant adenovirus Ad-Rad50-GFP and detection of the optimal multiplicity of infection in CNE1 transfected hv Ad-Rad50-GFP].

    Science.gov (United States)

    Yan, Ruicheng; Huang, Jiancong; Zhu, Ling; Chang, Lihong; Li, Jingjia; Wu, Xifu; Ye, Jin; Zhang, Gehua

    2015-12-01

    The optimal multiplicity of infection (MOI) of the recombinant adenovirus Ad-Rad50-GFP carrying a mutant Rad50 gene expression region on the cell growth of nasopharyngeal carcinoma and the viral amplification efficiency of CNE1 cell infected by this adenovirus were studied. The biological titer of Ad-Rad50-GFP was measured by end point dilution method. The impact of recombinant adenoviral vector transfection on the growth of CNE1 cells was observed by cell growth curve. Transfection efficacy of recombinant adenoviral vector was observed and calculated through fluorescence microscope. The expression f mutant Rad50 in the Ad-Rad50-GFP transfected CNE1 cells with optimal MOI was detected by Western Blot after transfection. The biological titer of Ad-Rad50-GFP was 1.26 x 10¹¹ pfu/ml. CNE1 cell growth was not influenced significantly as they were transfected by recombinant adenoviral vector with MOI less than 50. Transfection efficacy of recombinant adenoviral vector was most salient at 24 hours after transfection, with the high expression of mutant Rad50, and the efficiency still remained about 70% after 72 hours. Recombinant adenoviral vector Ad-Rad50-GFP could transfect CNE1 cells as well as result in the expression of mutant Rad50 in CNE1 cells effectively. MOI = 50 was the optimal multiplicity of infection of CNE1 cells transfected by recombinant adenoviral vector Ad-Rad50-GFP.

  10. Effects of sodium lactate Ringer's injection on transfection of human protein kinase C-α antisense oligonucleotide in A549 lung cancer cells.

    Science.gov (United States)

    Wang, Z H; Sun, W W; Han, Y L; Ma, Z

    2016-08-26

    In the present study, we evaluated the effects of four solutions [Dulbecco's modified Eagle's medium (DMEM), sodium lactate Ringer's injection (SLRI), phosphate-buffered saline (PBS), and NaCl] on the transfection of the human protein kinase C-a antisense oligonucleotide (PKC-a ASO) aprinocarsen in human lung carcinoma A549 cells. Specifically, SLRI, DMEM, PBS, or NaCl were used as the growth solutions for A549 cells, and OPTI-MEM was used as the PKC-a ASO diluent for transfection. Additionally, SLRI, DMEM, PBS, or NaCl were used as both the growth solutions and diluents for transfection. The cell viability and transfection efficiency were determined. The results demonstrated that when SLRI was used as either the growth solution or both the growth solution and diluent for aprinocarsen transfection in A549 cells, the effects were close to the best effects observed with DMEM as the growth solution and OPTI-MEM as the diluent, which supported the transfection of aprinocarsen into the cells. Moreover, SLRI resulted in higher transfection efficiency than those of PBS and NaCl. In in vitro experiments, aprinocarsen effectively induced apoptosis in A549 cells. In conclusion, SLRI may replace PBS or NaCl in clinical trials as a transfection solution readily accepted by the human body. To our knowledge, this is the first report demonstrating the use of SLRI as a transfection solution in lung-cancer cell lines.

  11. Recognition and Suppression of Transfected Plasmids by Protein ZNF511-PRAP1, a Potential Molecular Barrier to Transgene Expression

    Science.gov (United States)

    Qiu, Guo-Hua; Leung, Carol Ho-Wing; Yun, Tong; Xie, Xiaojin; Laban, Mirtha; Hooi, Shing Chuan

    2011-01-01

    Nonviral vectors present considerable advantages over viral counterparts in gene transfer. However, the poor expression efficiency of the transfected genes poses a challenge for their use in gene therapy, primarily due to the inability of these vectors to overcome various barriers, including the biological barriers. Here, we report that ZNF511-PRAP1 may be involved in the recognition and inactivation of transfected plasmids. ZNF511-PRAP1 is induced by transfection of plasmid DNA and suppresses the transcription of transfected plasmids. It binds directly to the p21 promoter in transfected plasmids but not the endogenous counterpart. Similarly, ZNF511-PRAP1 suppresses the expression of the green fluorescent protein reporter gene on transiently transfected plasmids but not an integrated red fluorescence reporter gene with the same cytomegalovirus (CMV) promoter. Therefore, ZNF511-PRAP1 is able to differentiate between exogenous/nonintegrated and endogenous/integrated DNA. The suppression by ZNF511-PRAP1 is independent of DNA methylation and can be abolished by trichostatin A (TSA) treatment and knockdown of HDAC2 and/or ZNF511-PRAP1. Furthermore, ZNF511-PRAP1 interacts directly with HDAC2. Our results revealed that transfected plasmids are recognized by ZNF511-PRAP1 and suppressed by a repressor complex comprising ZNF511-PRAP1 and HDAC2 and suggest that ZNF511-PRAP1 could play a role as a potential molecular barrier in nonviral transgene expression. PMID:21540836

  12. Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

    Science.gov (United States)

    Magro, Massimiliano; Martinello, Tiziana; Bonaiuto, Emanuela; Gomiero, Chiara; Baratella, Davide; Zoppellaro, Giorgio; Cozza, Giorgio; Patruno, Marco; Zboril, Radek; Vianello, Fabio

    2017-11-01

    Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Can non-viral technologies knockdown the barriers to siRNA delivery and achieve the next generation of cancer therapeutics?

    Science.gov (United States)

    Guo, Jianfeng; Bourre, Ludovic; Soden, Declan M; O'Sullivan, Gerald C; O'Driscoll, Caitriona

    2011-01-01

    Cancer is one of the most wide-spread diseases of modern times, with an estimated increase in the number of patients diagnosed worldwide, from 11.3 million in 2007 to 15.5 million in 2030 (www.who.int). In many cases, due to the delay in diagnosis and high increase of relapse, survival rates are low. Current therapies, including surgery, radiation and chemotherapy, have made significant progress, but they have many limitations and are far from ideal. Although immunotherapy has recently offered great promise as a new approach in cancer treatment, it is still very much in its infancy and more information on this approach is required before it can be widely applied. For these reasons effective, safe and patient-acceptable cancer therapy is still largely an unmet clinical need. Recent knowledge of the genetic basis of the disease opens up the potential for cancer gene therapeutics based on siRNA. However, the future of such gene-based therapeutics is dependent on achieving successful delivery. Extensive research is ongoing regarding the design and assessment of non-viral delivery technologies for siRNA to treat a wide range of cancers. Preliminary results on the first human Phase I trial for solid tumours, using a targeted non-viral vector, illustrate the enormous therapeutic benefits once the issue of delivery is resolved. In this review the genes regulating cancer will be discussed and potential therapeutic targets will be identified. The physiological and biochemical changes caused by tumours, and the potential to exploit this knowledge to produce bio-responsive 'smart' delivery systems, will be evaluated. This review will also provide a critical and comprehensive overview of the different non-viral formulation strategies under investigation for siRNA delivery, with particular emphasis on those designed to exploit the physiological environment of the disease site. In addition, a section of the review will be dedicated to pre-clinical animal models used to evaluate

  14. A powerful cooperative interaction between a fusogenic peptide and lipofectamine for the enhancement of receptor-targeted, non-viral gene delivery via integrin receptors.

    Science.gov (United States)

    Zhang, X; Collins, L; Fabre, J W

    2001-01-01

    Following receptor-mediated endocytosis, vector/DNA complexes require assistance to exit endocytic vesicles in order to avoid degradation in the lysosomes. Overcoming this barrier is a major challenge for the development of receptor-targeted, non-viral gene delivery. The fusogenic peptide of influenza virus haemagglutinin, lipofectamine and chloroquine were tested singly and in combination in various doses for promoting in vitro gene transfer by an integrin-targeted, non-viral DNA vector (polylysine-molossin). The fusogenic peptide and lipofectamine both individually promoted integrin-targeted gene delivery. However, the combined use of these agents was particularly effective, even at concentrations where neither agent singly had any effect on promoting gene delivery by polylysine-molossin. This optimal combination was effective on several cell lines and primary cell cultures. On the HuH7 cell line, it was approximately five-fold more effective than optimal chloroquine concentrations for integrin-targeted gene delivery and four to five times more effective than commercially available polyethylenimine. With the beta-galactosidase reporter gene, 60-65% of HepG2 cells and 75-80% of HuH7 cells were positive. The surface charge of polylysine-molossin/DNA/lipofectamine/fusogenic peptide complexes was approximately the same as that of polylysine-molossin/DNA complexes. The size distribution of the complexes suggested that competitive binding of polylysine-molossin and lipofectamine to DNA influenced the overall efficacy of this approach. Although the mechanisms are not clear, the combined use of very low doses of two membrane-destabilizing agents results in high levels of receptor-targeted gene delivery.

  15. Optimal Electroporation Condition for Small Interfering RNA Transfection into MDA-MB-468 Cell Line

    Directory of Open Access Journals (Sweden)

    Rita Arabsolghar

    2012-09-01

    Full Text Available Background: Electroporation is a valuable tool for small interfering RNA (siRNA delivery into cells because it efficiently transforms a wide variety of cell types. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA. Methods: To identify the best condition, the cells were firstly electroporated without siRNA and cell viability was determined by trypan blue and MTT assays. Then siRNA transfection in the best condition was performed. Western blot analysis was used for monitoring successful siRNA transfection. Results: The best condition for electroporation of this cell line was 220 volt and 975 µF in exponential decay using the Gene Pulser X cell electroporation system. Our data demonstrated that by using proper electroporation condition, DNA methyl transferase mRNA was silenced by 10 nmol DNMT1 siRNA in MDA-MB 468 cells when compared with negative control siRNA electroporation. Analysis of cell viability demonstrated that optimal electroporation condition resulted in 74% and 78% cell viability by trypan blue staining and MTT assay, respectively. Conclusion: Transfection of the MDA-MB-468 breast cancer cell line with siRNA in the obtained electroporation condition was successful and resulted in effective gene silencing and high cellular viability.

  16. Photoluminescent and biodegradable polycitrate-polyethylene glycol-polyethyleneimine polymers as highly biocompatible and efficient vectors for bioimaging-guided siRNA and miRNA delivery.

    Science.gov (United States)

    Wang, Min; Guo, Yi; Yu, Meng; Ma, Peter X; Mao, Cong; Lei, Bo

    2017-05-01

    Development of biodegradable and biocompatible non-viral vectors with intrinsical multifunctional properties such as bioimaging ability for highly efficient nucleic acids delivery still remains a challenge. Here, a biodegradable poly (1,8-octanedio-citric acid)-co-polyethylene glycol grafted with polyethyleneimine (PEI) (POCG-PEI) polymers with the photoluminescent capacity were synthesized for nucleic acids delivery (siRNA and miRNA). POCG-PEI polymers can efficiently bind various nucleic acids, protect them against enzymatic degradation and release the genes in the presence of polyanionic heparin. POCG-PEI also showed a significantly low cytotoxicity, enhanced cellular uptake and high transfection efficiency of nucleic acids, as compared to commercial transfection agents, lipofectamine 2000 (Lipo) and polyethylenimine (PEI 25K). POCG-PEI polymers demonstrate an excellent photostability, which allows for imaging the cells and real-time tracking the nucleic acids delivery. The photoluminescent property, low cytotoxicity, biodegradation, good gene binding and protection ability and high genes delivery efficiency make POCG-PEI highly competitive as a non-virus vector for genes delivery and real-time bioimaging applications. Our results may be also an important step for designing biodegradable biomaterials with multifunctional properties towards bioimaging-guided genes therapeutic applications. Here, a biodegradable poly (1,8-octanedio-citric acid)-co-polyethylene glycol grafted with polyethyleneimine (PEI) (POCG-PEI) polymers with controlled photoluminescent capacity were synthesized for nucleic acids delivery (siRNA and miRNA). POCG-PEI polymers can efficiently bind various nucleic acids, protect them against enzymatic degradation and release the genes in the presence of polyanionic heparin. POCG-PEI also showed a significantly low cytotoxicity, enhanced cellular uptake and high transfection efficiency of nucleic acids, as compared to commercial transfection agents

  17. SC-17BMP4-SECRETING hAdMSCs ENGINEERED WITH NANOPARTICLES: A NON-VIRAL MSC-BASED THERAPY FOR GLIOBLASTOMA

    Science.gov (United States)

    Mangraviti, Antonella; Tzeng, Stephany; Seng, Michael; Abbadi, Sara; Kozielski, Kristen; Schiapparelli, Paula; Wijesekera, Olindi; Sarabia-Estrada, Rachel; Brem, Henry; Tyler, Betty; Olivi, Alessandro; Green, Jordan; Quinones-Hinojosa, Alfredo

    2014-01-01

    Mesenchymal stem cells (MSCs) have attracted significant attention as effective delivery vehicles for targeting brain disorders. We have shown that MSCs derived from human adipose tissue (hAMSCs) and genetically modified with viruses display high anti-glioma tropism and can deliver bone morphogenetic protein 4 (BMP4), a therapeutic agent that reduces the clonogenic ability of glioma stem cells. Virus-safety concerns have led to the development of biodegradable nanoparticles that can transfect human cells. We identify nanoparticles for engineering hAMCSs to secrete BMP4 both in vitro and in vivo to treat brain cancer. hAMSCs were transfected using poly (beta-amino ester-PBAE)-based nanoparticles. Transfection efficacy and toxicity were evaluated to determine the optimal formulation to transfect hAMSCs with a BMP4-expressing plasmid. In vitro functional assays evaluated the effect of the transfection on hAMSC phenotype. In vivo imaging and histological analyses tracked engineered hAMSCs (NP-AMSCs) in the brain after local and systemic administration to rodents with orthotopic human tumors. Optimized nanoparticles transfected hAMSCs with 75 ± 2% efficacy and 71 ± 7% viability which was significantly superior (p < 0.0001) to LipofectamineTM 2000(16 ± 3% transfection, 54 ± 5% viability) the leading commercial product. PCR and Western blot analysis confirmed BMP4 production from NP-hAMSCs (52.5 ng protein/106 NP-hAMSCs). NP-hAMSCs were significantly better in migration and invasion (p = 0.005) than hAMSCs transduced with lentivirus. Flow cytometry showed that NP-hAMSCs retain multipotency. Intranasal, intravenous, and local delivery of luciferase- and GFP-expressing NP-hAMSCs in a rodent model of human glioma showed transgene expression 5 days after injection and NP-hAMSC migration towards the brain and within the tumor. We demonstrate that PBAE-nanoparticle formulations effectively transfect hAMSCs to produce BMP4. NP-AMSCs migrate towards the brain and penetrate

  18. Plasmid transfection in mammalian cells spatiotemporally tracked by a gold nanoparticle.

    Science.gov (United States)

    Muroski, Megan E; Carnevale, Kate J F; Riskowski, Ryan A; Strouse, Geoffrey F

    2015-01-27

    Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.

  19. Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

    Science.gov (United States)

    Misra, Santosh K.; Biswas, Joydeep; Kondaiah, Paturu; Bhattacharya, Santanu

    2013-01-01

    Background Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media. Methodology/Principal Findings To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features. Conclusions/Significance -OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies. PMID:23861884

  20. Modulated protonation of side chain aminoethylene repeats in N-substituted polyaspartamides promotes mRNA transfection.

    Science.gov (United States)

    Uchida, Hirokuni; Itaka, Keiji; Nomoto, Takahiro; Ishii, Takehiko; Suma, Tomoya; Ikegami, Masaru; Miyata, Kanjiro; Oba, Makoto; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2014-09-03

    Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

  1. A targeted ultrasound contrast agent carrying gene and cell-penetrating peptide: preparation and gene transfection in vitro.

    Science.gov (United States)

    Ren, Jianli; Zhang, Ping; Tian, Ju; Zhou, Zhiyi; Liu, Xingzhao; Wang, Dong; Wang, Zhigang

    2014-09-01

    Targeted and high efficient gene delivery is a main issue in gene treatment. Taking advantage of ischemic memory target P-selectin and our previous study-synergistic effects of ultrasound-targeted microbubble destruction (UTMD) and TAT peptide on gene transfection, which were characterized by targeted aggregation and high efficient gene transfection, we set up a 'smart' gene delivery system-targeted ultrasound contrast agent (UCA) carrying gene and cell-permeable peptides (CPP). Such UCA had a strong binding force with DNA which was protected from being hydrolysed by nuclease. Moreover, synergistic effects of UTMD and TAT peptide increased gene transfection. Specifically, the UCA were reacted with an ischemic memory target P-selectin overexpressed by ischemic issues (including ischemic heart disease) and loaded with gene and CPP, which enabled targeted localization and gene delivery to ischemic cells overexpressing P-selectin. We demonstrated their targeting affinity for hypoxia human umbilical vein endothelial cell (HUVEC) and gene transfection in vitro. The results of confocal laser scanning microscopy (CLSM) showed that gene and CPP were distributed on the shell of UCA. Red fluorescence was observed on the surface of targeted UCA using immunofluorescent microscopy, which demonstrated that the antibody was successfully connected to the UCA. The targeted UCA was specifically and tightly binded to hypoxia HUVEC, while there were no or little non-targeted UCA binding around hypoxia HUVEC. 24h after transfection, gene transfection efficiency detected by FCM was higher in targeted group than non-targeted group. Overall, the targeted UCA carrying gene and CPP was prepared successfully. It had a strong target binding capacity to hypoxia HUVEC and high efficient gene transfection, which maybe provide a novel strategy for gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Highly Branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate for High Performance Gene Transfection

    Directory of Open Access Journals (Sweden)

    Ming Zeng

    2017-05-01

    Full Text Available The top-performing linear poly(β-amino ester (LPAE, poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate (C32, has demonstrated gene transfection efficiency comparable to viral-mediated gene delivery. Herein, we report the synthesis of a series of highly branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate (HC32 and explore how the branching structure influences the performance of C32 in gene transfection. HC32 were synthesized by an “A2 + B3 + C2” Michal addition strategy. Gaussia luciferase (Gluciferase and green fluorescent protein (GFP coding plasmid DNA were used as reporter genes and the gene transfection efficiency was evaluated in human cervical cancer cell line (HeLa and human recessive dystrophic epidermolysis bullosa keratinocyte (RDEBK cells. We found that the optimal branching structure led to a much higher gene transfection efficiency in comparison to its linear counterpart and commercial reagents, while preserving high cell viability in both cell types. The branching strategy affected DNA binding, proton buffering capacity and degradation of polymers as well as size, zeta potential, stability, and DNA release rate of polyplexes significantly. Polymer degradation and DNA release rate played pivotal parts in achieving the high gene transfection efficiency of HC32-103 polymers, providing new insights for the development of poly(β-amino esters-based gene delivery vectors.

  3. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.

    Science.gov (United States)

    Yin, Hao; Song, Chun-Qing; Dorkin, Joseph R; Zhu, Lihua J; Li, Yingxiang; Wu, Qiongqiong; Park, Angela; Yang, Junghoon; Suresh, Sneha; Bizhanova, Aizhan; Gupta, Ankit; Bolukbasi, Mehmet F; Walsh, Stephen; Bogorad, Roman L; Gao, Guangping; Weng, Zhiping; Dong, Yizhou; Koteliansky, Victor; Wolfe, Scot A; Langer, Robert; Xue, Wen; Anderson, Daniel G

    2016-03-01

    The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

  4. Gene Transfection of Human Turbinate Mesenchymal Stromal Cells Derived from Human Inferior Turbinate Tissues

    Directory of Open Access Journals (Sweden)

    Jin Seon Kwon

    2016-01-01

    Full Text Available Human turbinate mesenchymal stromal cells (hTMSCs are novel stem cells derived from nasal inferior turbinate tissues. They are easy to isolate from the donated tissue after turbinectomy or conchotomy. In this study, we applied hTMSCs to a nonviral gene delivery system using polyethyleneimine (PEI as a gene carrier; furthermore, the cytotoxicity and transfection efficiency of hTMSCs were evaluated to confirm their potential as resources in gene therapy. DNA-PEI nanoparticles (NPs were generated by adding the PEI solution to DNA and were characterized by a gel electrophoresis and by measuring particle size and surface charge of NPs. The hTMSCs were treated with DNA-PEI NPs for 4 h, and toxicity of NPs to hTMSCs and gene transfection efficiency were monitored using MTT assay, fluorescence images, and flow cytometry after 24 h and 48 h. At a high negative-to-positive charge ratio, DNA-PEI NPs treatment led to cytotoxicity of hTMSCs, but the transfection efficiency of DNA was increased due to the electrostatic effect between the NPs and the membranes of hTMSCs. Importantly, the results of this research verified that PEI could deliver DNA into hTMSCs with high efficiency, suggesting that hTMSCs could be considered as untapped resources for applications in gene therapy.

  5. Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

    Science.gov (United States)

    Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

    2016-02-01

    Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

  6. Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound.

    Science.gov (United States)

    Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K Kirk

    2016-02-04

    Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca(2+) and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

  7. Size effect on transfection and cytotoxicity of nanoscale plasmid DNA/polyethyleneimine complexes for aerosol gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Hoon Byeon, Jeong, E-mail: jbyeon@purdue.edu [Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 (United States); Kim, Jang-Woo, E-mail: jwkim@hoseo.edu [Department of Digital Display Engineering, Hoseo University, Asan 336-795 (Korea, Republic of)

    2014-02-03

    Nanoscale plasmid DNA (pDNA)/polyethyleneimine (PEI) complexes were fabricated in the aerosol state using a nebulization system consisting of a collison atomizer and a cool-walled diffusion dryer. The aerosol fabricated nanoscale complexes were collected and employed to determine fundamental properties of the complexes, such as size, structure, surface charge, and in vitro gene transfection efficiency and cytotoxicity. The results showed that mass ratio between pDNA and PEI should be optimized to enhance gene transfection efficiency without a significant loss of cell viability. These findings may support practical advancements in the field of nonviral gene delivery.

  8. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery

    OpenAIRE

    Francesco Cardarelli; Luca Digiacomo; Cristina Marchini; Augusto Amici; Fabrizio Salomone; Giuseppe Fiume; Alessandro Rossetta; Enrico Gratton; Daniela Pozzi; Giulio Caracciolo

    2016-01-01

    Lipofectamine reagents are widely accepted as ?gold-standard? for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find ...

  9. Improvement of spinal non-viral IL-10 gene delivery by D-mannose as a transgene adjuvant to control chronic neuropathic pain.

    Science.gov (United States)

    Dengler, Ellen C; Alberti, Lauren A; Bowman, Brandi N; Kerwin, Audra A; Wilkerson, Jenny L; Moezzi, Daniel R; Limanovich, Eugene; Wallace, James A; Milligan, Erin D

    2014-05-21

    Peri-spinal subarachnoid (intrathecal; i.t.) injection of non-viral naked plasmid DNA encoding the anti-inflammatory cytokine, IL-10 (pDNA-IL-10) suppresses chronic neuropathic pain in animal models. However, two sequential i.t. pDNA injections are required within a discrete 5 to 72-hour period for prolonged efficacy. Previous reports identified phagocytic immune cells present in the peri-spinal milieu surrounding the i.t injection site that may play a role in transgene uptake resulting in subsequent IL-10 transgene expression. In the present study, we aimed to examine whether factors known to induce pro-phagocytic anti-inflammatory properties of immune cells improve i.t. IL-10 transgene uptake using reduced naked pDNA-IL-10 doses previously determined ineffective. Both the synthetic glucocorticoid, dexamethasone, and the hexose sugar, D-mannose, were factors examined that could optimize i.t. pDNA-IL-10 uptake leading to enduring suppression of neuropathic pain as assessed by light touch sensitivity of the rat hindpaw (allodynia). Compared to dexamethasone, i.t. mannose pretreatment significantly and dose-dependently prolonged pDNA-IL-10 pain suppressive effects, reduced spinal IL-1β and enhanced spinal and dorsal root ganglia IL-10 immunoreactivity. Macrophages exposed to D-mannose revealed reduced proinflammatory TNF-α, IL-1β, and nitric oxide, and increased IL-10 protein release, while IL-4 revealed no improvement in transgene uptake. Separately, D-mannose dramatically increased pDNA-derived IL-10 protein release in culture supernatants. Lastly, a single i.t. co-injection of mannose with a 25-fold lower pDNA-IL-10 dose produced prolonged pain suppression in neuropathic rats. Peri-spinal treatment with D-mannose may optimize naked pDNA-IL-10 transgene uptake for suppression of allodynia, and is a novel approach to tune spinal immune cells toward pro-phagocytic phenotype for improved non-viral gene therapy.

  10. [E1A gene transfection of human undifferentiated thyroid cancer cell line HTC/3 by nanoparticles].

    Science.gov (United States)

    He, Xiang-Liang; He, Dong-Hua; Liao, Xiao-Xing; Zhan, Hong; Ma, Zhong-Fu; Wang, Xi-Fu; Li, Qing; Li, Xin; Li, Yu-Jie

    2007-12-01

    To prepare nanoparticles containing E1A gene and observe the efficiency and feasibility of transfecting E1A gene into human undifferentiated thyroid cancer cell line HTC/3. To examine the sensitivity of transgene cells to X-ray and X-ray-induced apoptosis in those cells. Nanoparticle-DNA complex was prepared with PLGA coating adenoviral early expression gene E1A, and the package efficiency, release progress in vitro, and size of the complex were determined. The nanoparticle-DNA was transfected into the HTC/3 cells. Lipofectamine was used to transfect E1A gene as a control. RT-PCR was used to examine E1A gene mRNA expression in the transfected cells. The survival ratio of HTC/3-E1A and control cells, and the growth inhibition ratio induced by different doses of X-ray in HTC/3-E1A cells were examined by MTT assay. The apoptosis in HTC/3-E1A cells induced by 2 Gy X-ray iradiation was examined by flow cytometry and DNA electrophoresis. The package efficiency, release progress in vitro, and size of the nanoparticle-DNA complex were 0.78%, 18 days, and 150-280 nm, respectively when transfected the plasmid at the same level, the nanoparticle group got more positive transgene cell clones than that in lipofectamine group, with a statistically significant difference (P HTC/3-E1A cells grew slowly, and their doubling time was prolongated (1.44 times in comparison with that in parental cells). According to IC50, the sensitivity of HTC/3-E1A cells to X-ray was improved 2.9 and 2.8 times, respectively, in comparison with that in HTC/3-Vect and HTC/3 cells. The ratio of subG0/G1 phase of HTC/3-E1A cells was significantly higher than that in HTC/3-Vect and HTC/3 cells (P HTC/3-E1A cells was significantly lower than that in HTC/3-Vect and HTC/3 cells (P HTC/3-E1A cells was observed by electrophoresis, but not found in HTC/3-Vect and HTC/3 cells. A nanoparticle-DNA complex has been successfully prepared, and it may carry a foreign gene into cells. The sensitivity of HTC/3-E1A cells

  11. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies.

    Science.gov (United States)

    Park, Jae-Seung; Surendran, Sneha; Kamendulis, Lisa M; Morral, Núria

    2011-01-18

    Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA) or microRNA (miRNA). New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells) as well as double stranded RNA (>90% with siRNA or microRNA). In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

  12. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

    Directory of Open Access Journals (Sweden)

    Morral Núria

    2011-01-01

    Full Text Available Abstract Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA or microRNA (miRNA. New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells as well as double stranded RNA (>90% with siRNA or microRNA. In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

  13. Establishing malaria parasite transfection technology in South Africa.

    CSIR Research Space (South Africa)

    Van Brummelen, AC

    2010-01-01

    Full Text Available In order to establish malaria parasite transfection technology in South Africa, firefly luciferase and green fluorescent protein (GFP) reporter constructs were prepared. In attempt to simplify these constructs, a var intron (PFC0005w), previously...

  14. A novel strategy for selective gene delivery by using the inhibitory effect of blue light on jetPRIME-mediated transfection.

    Science.gov (United States)

    Dateki, Minori; Imamura, Osamu; Arai, Masaaki; Shimizu, Hidehisa; Takishima, Kunio

    2016-07-01

    Photodynamic control of gene delivery is a new technology with growing applications in gene therapy and basic cell research. Main approaches of light-selective gene delivery rely on the light-dependent enhancement of transfection efficiency. Studies focused on light-stimulated inhibitory regulation of transfection have rarely been reported. Here, we tried to establish a novel procedure of light-dependent inhibition of transfection. Our experiments, conducted with several types of commercial transfection reagents, revealed that jetPRIME-mediated transfection was strongly inhibited by blue light. Although the uptake of reagent-DNA complex was drastically reduced, preliminary exposure of cells or reagent-DNA complex to blue light had no inhibitory effect on the transfection efficiency. The inhibitory effect was wavelength-dependent and mediated by reactive oxygen species. Partial exposure of a culture vessel to blue light resulted in selective gene delivery into cells grown on the unexposed area of the vessel. By using this approach, different types of plasmid DNA were delivered into different areas in the culture vessel. This novel approach to the inhibitory control of transfection provides practical options for research and therapeutics. Biotechnol. Bioeng. 2016;113: 1560-1567. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  15. New Transfection Agents Based on Liposomes Containing Biosurfactant MEL-A

    Directory of Open Access Journals (Sweden)

    Tadahide Furuno

    2013-08-01

    Full Text Available Nano vectors are useful tools to deliver foreign DNAs, oligonucleotides, and small interfering double-stranded RNAs (siRNAs into mammalian cells with gene transfection and gene regulation. In such experiments we have found the liposomes with a biosurfacant mannosylerythriol lipid (MEL-A are useful because of their high transfer efficiency, and their unique mechanism to transfer genes to target cells with the lowest toxicity. In the present review we will describe our current work, which may contribute to the great advance of gene transfer to target cells and gene regulations. For more than two decades, the liposome technologies have changed dramatically and various methods have been proposed in the fields of biochemistry, cell biology, biotechnology, and so on. In addition, they were towards to pharmaceutics and clinical applications. The liposome technologies were expected to use gene therapy, however, they have not reached a requested goal as of yet. In the present paper we would like to present an approach using a biosurfactant, MEL-A, which is a surface-active compound produced by microorganisms growing on water-insoluble substrates and increases efficiency in gene transfection. The present work shows new transfection agents based on liposomes containing biosurfactant MEL-A.

  16. Gene transfection in primary stem-like cells of giant cell tumor of bone

    Directory of Open Access Journals (Sweden)

    Shalini Singh

    2010-09-01

    Full Text Available Shalini Singh1, Isabella Mak1, Patricia Power1, Melissa Cunnigham2, Robert Turcotte3, Michelle Ghert11Departments of Surgery, 2Biology, McMaster University, Hamilton, Ontario; 3Department of Orthopaedic Surgery, McGill University Medical Centre, Montreal, Quebec, CanadaAbstract: The neoplastic stem-like stromal cell of giant cell tumor of bone (GCT survives for multiple passages in primary culture with a stable phenotype, and exhibits multipotent characteristics. The pathophysiology of this tumor has been studied through the primary culture of these cells. However, successful gene transfer of these cells has not been reported to date. In this short report, we describe the development of the first reported technique that results in efficient gene transfection in primary stem-like cells of GCT.Keywords: gene, transfection, primary cells, TWIST, giant cell tumor

  17. Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

    DEFF Research Database (Denmark)

    Glahder, Jacob; Norrild, Bodil; Persson, Mikael B

    2005-01-01

    Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and puls...

  18. Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection.

    Science.gov (United States)

    Hujaya, Sry D; Marchioli, Giulia; Roelofs, Karin; van Apeldoorn, Aart A; Moroni, Lorenzo; Karperien, Marcel; Paulusse, Jos M J; Engbersen, Johan F J

    2015-05-10

    Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D. Therefore, COS-7 cells were cultured on top of multilayer films formed by layer-by-layer dipcoating of these polymers with GFP-encoded pDNA, and the effect of the number of layers and cell seeding density on the transfection efficiency was evaluated. Multilayer films with pCABOL were found to be superior to pCHIS in facilitating transfection, which was attributed to higher incorporation of pDNA and release of the transfection agent. High amounts of transfected cells were obtained on pCABOL films, correlating proportionally over a wide range with seeding density. Optimal transfection efficiency was obtained with pCABOL films composed of 10 bilayers. Further increase in the number of bilayers only marginally increased transfection efficiency. Using the optimal multilayer and cell seeding conditions, pCABOL multilayers were fabricated on poly(ε-caprolactone) (PCL), heparinized PCL (PCL-HEP), and poly(lactic acid) (PLA) disks as examples of common biomedical supports. The multilayers were found to completely mask the properties of the original substrates, with significant improvement in cell adhesion, which is especially pronounced for PCL and PLA disks. With all these substrates, transfection efficiency was found to be in the range of 25-50% transfected cells. The pCABOL/pDNA multilayer films can also conveniently add transfection capability to 3D scaffolds. Significant improvement in cell adhesion was observed after multilayer coating of 3D-plotted fibers of PCL (with and without an additional covalent heparin layer), especially for the PCL scaffold without heparin layer and transfection was observed on both 3D PCL and PCL-HEP scaffolds. These results show that layer

  19. Laser-induced Stress Wave-assisted Gene Transfection with Cationic Liposome

    Science.gov (United States)

    Terakawa, Mitsuhiro; Otsuka, Risa; Sato, Shunichi; Satoh, Yasushi; Takishima, Kunio; Ashida, Hiroshi; Okano, Hideyuki; Obara, Minoru

    2007-12-01

    We showed that efficiency of gene transfer using laser-induced stress waves (LISWs) can be drastically enhanced by modifying plasmid DNA with cationic liposome. Cultured NIH 3T3 cells were covered with a suspension including plasmid DNA that had been modified with Lipofectamine, and a laser target was placed over the suspension. The cells were exposed to stress waves generated by irradiation of the laser target with 6-ns laser pulses. The cells showed a significantly higher transfection efficiency compared with that of cells treated with LISWs alone or Lipofectamine alone.

  20. Manipulating the membrane penetration mechanism of helical polypeptides via aromatic modification for efficient gene delivery.

    Science.gov (United States)

    Zheng, Nan; Song, Ziyuan; Yang, Jiandong; Liu, Yang; Li, Fangfang; Cheng, Jianjun; Yin, Lichen

    2017-08-01

    The delivery performance of non-viral gene vectors is greatly related to their intracellular kinetics. Cationic helical polypeptides with potent membrane penetration properties and gene transfection efficiencies have been recently developed by us. However, they suffer from severe drawbacks in terms of their membrane penetration mechanisms that mainly include endocytosis and pore formation. The endocytosis mechanism leads to endosomal entrapment of gene cargos, while the charge- and helicity-induced pore formation causes appreciable cytotoxicity at high concentrations. With the attempt to overcome such critical challenges, we incorporated aromatic motifs into the design of helical polypeptides to enhance their membrane activities and more importantly, to manipulate their membrane penetration mechanisms. The aromatically modified polypeptides exhibited higher cellular internalization level than the unmodified analogue by up to 2.5 folds. Such improvement is possibly because aromatic domains promoted the polypeptides to penetrate cell membranes via direct transduction, a non-endocytosis and non-pore formation mechanism. As such, gene cargos were more efficiently delivered into cells by bypassing endocytosis and subsequently avoiding endosomal entrapment, and the material toxicity associated with excessive pore formation was also reduced. The top-performing aromatic polypeptide containing naphthyl side chains at the incorporated content of 20mol% revealed notably higher transfection efficiencies than commercial reagents in melanoma cells in vitro (by 11.7 folds) and in vivo (by 9.1 folds), and thus found potential utilities toward topical gene delivery for cancer therapy. Cationic helical polypeptides, as efficient gene delivery materials, suffer from severe drawbacks in terms of their membrane penetration mechanisms. The main cell penetration mechanisms involved are endocytosis and pore formation. However, the endocytosis mechanism has the limitation of endosomal

  1. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.

    Science.gov (United States)

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.

  2. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    Science.gov (United States)

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. PMID:25834424

  3. Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection

    Science.gov (United States)

    Arsenault, Jason; Cuijpers, Sabine AG; Niranjan, Dhevahi; Davletov, Bazbek

    2014-01-01

    Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30–50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection. J. Cell. Biochem. 115: 2047–2054, 2014. © 2014 Wiley Periodicals, Inc. PMID:25043607

  4. [Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells].

    Science.gov (United States)

    Wang, Wei-rong; Lin, Rong; Yang, Yu-cong; Gan, Wei-jie; Liu, Jun-tian; Lü, She-min

    2005-12-01

    To construct an eukaryotic expression vector containing human CD40 gene for its efficient, continuous and stable expression in human umbilical vein endothelial ECV-304 cells. The recombinant plasmid pUCD40 was digested with endonucleases to obtain human CD40 gene fragment, which was cloned into pCDNA3.1 vector to construct recombinant eukaryotic expression vector pCDNA3.1(+)/CD40. The recombinant vector was identified by enzyme digestion before introduced into ECV-304 cells via liposome, with the positive cell clones selected with G418. The stable transfection and expression of CD40 in ECV-304 cells were identified by reverse transcription (RT)-PCR, Western blotting and flow cytometry, respectively. Enzyme digestion analysis showed that target gene had been cloned into the recombinant vector. The transfected ECV-304 cells successfully expressed human CD40 as determined by RT-PCR and Western-blotting, and 95% of the cells were CD40-positive as shown by flow cytometry. The recombinant eukaryotic expression vector pCDNA3.1(+)/CD40 has been successfully constructed, which is capable of stable transfection and expression of CD40 in ECV-304 cells to facilitate further investigation of the roles of CD40 molecule in antiatherosclerotic drug development.

  5. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina

    Directory of Open Access Journals (Sweden)

    Higgins Dennis

    2001-02-01

    Full Text Available Abstract Background Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli β-galactosidase or enhanced green fluorescence protein (EGFP using PEI. Results Optimal transfection efficiency was observed with 1 μg/ml of plasmid DNA and 5 μg/ml PEI. Expression of β-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with ∼ 9% of the neurons expressing β-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector. Conclusions These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures.

  6. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

    Science.gov (United States)

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes. PMID:28216629

  7. Liver suction-mediated transfection in mice using a pressure-controlled computer system.

    Science.gov (United States)

    Shimizu, Kazunori; Zhang, Guangyuan; Kawakami, Shigeru; Taniguchi, Yota; Hayashi, Kouji; Hashida, Mitsuru; Konishi, Satoshi

    2014-01-01

    We previously developed an in vivo tissue suction-mediated transfection method (denoted as the tissue suction method) for naked nucleic acids, such as plasmid DNA (pDNA) and small interfering RNA (siRNA), in mice. However, it remains unclear whether the suction pressure conditions affect the results of this method. Therefore, in the present study, we assembled a computer system to control the suction pressure and investigate the effects of the suction pressure conditions on the efficiency of the liver suction transfection of naked pDNA that encodes luciferase in mice. Using the developed system, we examined the effects of the minimum magnitude of the suction pressure, suction pressure waveform, and suction times of the luciferase expression level in mice livers. We determined that the liver suction method at 5 kPa was not only effective but also caused the lowest hepatic toxicity in mice. Additionally, the results indicated that the suction pressure waveform affects the luciferase expression levels, and a single period of suction on the targeted portion of the liver is sufficient for transfection. Thus, the developed system is useful for performing the tissue suction method with high accuracy and safety.

  8. Poly(ester-anhydride):poly(beta-amino ester) micro- and nanospheres: DNA encapsulation and cellular transfection.

    Science.gov (United States)

    Pfeifer, Blaine A; Burdick, Jason A; Little, Steve R; Langer, Robert

    2005-11-04

    Poly(ester-anhydride) delivery devices allow flexibility regarding carrier dimensions (micro- versus nanospheres), degradation rate (anhydride versus ester hydrolysis), and surface labeling (through the anhydride functional unit), and were therefore tested for DNA encapsulation and transfection of a macrophage P388D1 cell line. Poly(l-lactic acid-co-sebacic anhydride) and poly(l-lactic acid-co-adipic anhydride) were synthesized through melt condensation, mixed with 25 wt.% poly(beta-amino ester), and formulated with plasmid DNA (encoding firefly luciferase) into micro- and nanospheres using a double emulsion/solvent evaporation technique. The micro- and nanospheres were then characterized (size, morphology, zeta potential, DNA release) and assayed for DNA encapsulation and cellular transfection over a range of poly(ester-anhydride) copolymer ratios. Poly(ester-anhydride):poly(beta-amino ester) composite microspheres (6-12 microm) and nanospheres (449-1031 nm), generated with copolymers containing between 0 and 25% total polyanhydride content, encapsulated plasmid DNA (>or=20% encapsulation efficiency). Within this polyanhydride range, poly(adipic anhydride) copolymers provided DNA encapsulation at an increased anhydride content (10%, microspheres; 10-25%, nanospheres) compared to poly(sebacic anhydride) copolymers (1%, microspheres and nanospheres) with cellular transfection correlating with the observed DNA encapsulation.

  9. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes.

    Science.gov (United States)

    Pappalardo, Juan Sebastián; Langellotti, Cecilia A; Di Giacomo, Sebastián; Olivera, Valeria; Quattrocchi, Valeria; Zamorano, Patricia I; Hartner, William C; Levchenko, Tatyana S; Torchilin, Vladimir P

    2014-01-01

    Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

  10. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Konrad H. Engelhardt

    2017-01-01

    Full Text Available Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc at certain nitrogen-to-phosphorus (N/P ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3. Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS, atomic force microscopy (AFM, and scanning electron microscopy (Cryo-SEM, respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI.

  11. Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial

    Science.gov (United States)

    Alton, Eric W F W; Armstrong, David K; Ashby, Deborah; Bayfield, Katie J; Bilton, Diana; Bloomfield, Emily V; Boyd, A Christopher; Brand, June; Buchan, Ruaridh; Calcedo, Roberto; Carvelli, Paula; Chan, Mario; Cheng, Seng H; Collie, D David S; Cunningham, Steve; Davidson, Heather E; Davies, Gwyneth; Davies, Jane C; Davies, Lee A; Dewar, Maria H; Doherty, Ann; Donovan, Jackie; Dwyer, Natalie S; Elgmati, Hala I; Featherstone, Rosanna F; Gavino, Jemyr; Gea-Sorli, Sabrina; Geddes, Duncan M; Gibson, James S R; Gill, Deborah R; Greening, Andrew P; Griesenbach, Uta; Hansell, David M; Harman, Katharine; Higgins, Tracy E; Hodges, Samantha L; Hyde, Stephen C; Hyndman, Laura; Innes, J Alastair; Jacob, Joseph; Jones, Nancy; Keogh, Brian F; Limberis, Maria P; Lloyd-Evans, Paul; Maclean, Alan W; Manvell, Michelle C; McCormick, Dominique; McGovern, Michael; McLachlan, Gerry; Meng, Cuixiang; Montero, M Angeles; Milligan, Hazel; Moyce, Laura J; Murray, Gordon D; Nicholson, Andrew G; Osadolor, Tina; Parra-Leiton, Javier; Porteous, David J; Pringle, Ian A; Punch, Emma K; Pytel, Kamila M; Quittner, Alexandra L; Rivellini, Gina; Saunders, Clare J; Scheule, Ronald K; Sheard, Sarah; Simmonds, Nicholas J; Smith, Keith; Smith, Stephen N; Soussi, Najwa; Soussi, Samia; Spearing, Emma J; Stevenson, Barbara J; Sumner-Jones, Stephanie G; Turkkila, Minna; Ureta, Rosa P; Waller, Michael D; Wasowicz, Marguerite Y; Wilson, James M; Wolstenholme-Hogg, Paul

    2015-01-01

    Summary Background Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. Methods We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50–90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene–liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. Findings Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. Interpretation Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of

  12. Lipid-based transfection reagents can interfere with cholesterol biosynthesis.

    Science.gov (United States)

    Danielli, Mauro; Marinelli, Raúl A

    2016-02-15

    Lipid-based transfection reagents are widely used for delivery of small interfering RNA into cells. We examined whether the commonly used commercial transfection reagents DharmaFECT-4 and Lipofectamine 2000 can interfere with lipid metabolism by studying cholesterogenesis. Cholesterol de novo synthesis from [(14)C]acetate was assessed in human hepatocyte-derived Huh-7 cells. The results revealed that DharmaFECT, but not Lipofectamine, markedly inhibited cholesterol biosynthesis by approximately 70%. Cell viability was not significantly altered. These findings suggest that caution is required in the choice of certain lipid-based transfection reagents for gene silencing experiments, particularly when assessing cholesterol metabolism. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Establishment of transient and stable transfection systems for Babesia ovata.

    Science.gov (United States)

    Hakimi, Hassan; Yamagishi, Junya; Kegawa, Yuto; Kaneko, Osamu; Kawazu, Shin-Ichiro; Asada, Masahito

    2016-03-23

    Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study. In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection. After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata. The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.

  14. [Transfection of human umbilical vein endothelial cell line ECV-304 with liposome-oligonucleotide complexes under hypothermic and anoxic conditions].

    Science.gov (United States)

    Feng, Gui-wen; Yu, Li-xin; Zhang, Hong-tao; Zhao, Xian-guo

    2004-05-01

    To investigate the transfection efficiency of nuclear factor (NF)-kappaB decoy oligodeoxynucleotides (ODN) mediated by in vivo liposome in human umbilical vascular endothelial cell line ECV-304 under hypothermic and anoxic conditions. ECV-304 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 degrees Celsius; in the presence of 5% CO2. Liposome-ODN complexes were prepared just before use and added to the cells with a liposome-ODN charge ratio of 2:1. ECV-304 cell monolayers were transfected with liposome-ODN complexes containing 0.50, 0.75, 1.00 and 1.25 micromol/L ODN respectively in Euro-Collins solution (ECs) at 4 degrees celsius; and then stored for 2, 4, 6 and 8 hours respectively under anoxic condition. The ODN without liposome was transfected into ECV-304 cells under identical conditions as the control. The distribution of fluorescein isothiocyanate (FITC)-labeled ODN in ECV-304 cells was observed by fluorescence microscope, and the transfection efficiency and mean fluorescence intensity (MFI) were evaluated by flow cytometry. MFI was enhanced as the storage time extended and ODN concentration increased, reaching the peak level at 6 h (PECV-304 cells by in vivo liposome in ECs under hypothermic and anoxic conditions, which provides an experimental basis for further study of the donor organ preservation at the level of genetic regulation.

  15. Cellular Injury of Cardiomyocytes during Hepatocyte Growth Factor Gene Transfection with Ultrasound-Triggered Bubble Liposome Destruction

    Directory of Open Access Journals (Sweden)

    Kazuo Komamura

    2011-01-01

    Full Text Available We transfected naked HGF plasmid DNA into cultured cardiomyocytes using a sonoporation method consisting of ultrasound-triggered bubble liposome destruction. We examined the effects on transfection efficiency of three concentrations of bubble liposome (1×106, 1×107, 1×108/mL, three concentrations of HGF DNA (60, 120, 180 μg/mL, two insonification times (30, 60 sec, and three incubation times (15, 60, 120 min. We found that low concentrations of bubble liposome and low concentrations of DNA provided the largest amount of the HGF protein expression by the sonoporated cardiomyocytes. Variation of insonification and incubation times did not affect the amount of product. Following insonification, cardiomyocytes showed cellular injury, as determined by a dye exclusion test. The extent of injury was most severe with the highest concentration of bubble liposome. In conclusion, there are some trade-offs between gene transfection efficiency and cellular injury using ultrasound-triggered bubble liposome destruction as a method for gene transfection.

  16. Multi-modal transfection agent based on monodisperse magnetic nanoparticles for stem cell gene delivery and tracking.

    Science.gov (United States)

    Park, Wooram; Yang, Han Na; Ling, Daishun; Yim, Hyeona; Kim, Kyoung Sub; Hyeon, Taeghwan; Na, Kun; Park, Keun-Hong

    2014-08-01

    Directing the controlled differentiation and tracking of stem cells is essential to achieve successful stem cell therapy. In this work, we describe a multi-modal (MR/optical) transfection agent (MTA) for efficient gene delivery and cell tracking of human mesenchymal stem cells (hMSCs). The MTA was synthesized through a facile two-step approach with 1) ligand exchange of a catechol-functionalized polypeptide (CFP) and 2) chemical immobilization of fluorescence labelled cationic polymer via aminolysis reaction. Cationic polymer-immobilized MTAs with size of ~40 nm exhibit greatly enhanced colloidal stability in aqueous solution. In addition, the MTAs were capable of binding DNA molecules for transfection. The MTA/pDNA complex showed relatively good transfection efficiency in hMSCs (compared to the commercial transfection agent, Lipofectamine) and good biocompatibility. MTA-treated hMSCs were successfully visualized after transplantation via MR and optical imaging system over 14 days. These studies highlight the challenges associated with the potential advantages of designing multi-modal nanostructured materials as tools for genetic materials delivery and cell-tracking in stem cell therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Transfection of rat brain endothelium in a primary culture model of the blood-brain barrier at different states of barrier maturity

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Lichota, Jacek

    approaches have been developed. Gene therapy could be a promising and novel approach to overcome the restricting properties of the BBB to polypeptides and proteins. Gene therapy is based on the delivery of genetic material into brain capillary endothelial cells (BCECs), which, theoretically, will result...... in expression and secretion of the recombinant protein from the BCECs and into the brain, thus turning BCECs into small recombinant protein factories. In this study, the possibility of using BCECs as small factories for recombinant protein production was investigated. To mimic the in-vivo situation as closely...... transfection is positively correlated with cell division, with dividing cells being more sensitive to gene transfection. Therefore, by using this model, the transfection efficiency of the rat endothelium was investigated in an immature state and mature state of the BBB. During the transfection period...

  18. Transfection of RNA from organ samples of infected animals represents a highly sensitive method for virus detection and recovery of classical swine fever virus.

    Science.gov (United States)

    Meyer, Denise; Schmeiser, Stefanie; Postel, Alexander; Becher, Paul

    2015-01-01

    Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.

  19. Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes.

    Science.gov (United States)

    Teagle, Alexandra R; Birchall, James C; Hargest, Rachel

    2016-01-01

    Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy. Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab). The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab. Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent. © 2016 The Author(s) Published by S. Karger AG, Basel.

  20. Influence of nanoparticle-mediated transfection on proliferation of primary immune cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Susanne Przybylski

    Full Text Available One of the main obstacles in the widespread application of gene therapeutic approaches is the necessity for efficient and safe transfection methods. For the introduction of small oligonucleotide gene therapeutics into a target cell, nanoparticle-based methods have been shown to be highly effective and safe. While immune cells are a most interesting target for gene therapy, transfection might influence basic immune functions such as cytokine expression and proliferation, and thus positively or negatively affect therapeutic intervention. Therefore, we investigated the effects of nanoparticle-mediated transfection such as polyethylenimine (PEI or magnetic beads on immune cell proliferation.Human adherent and non-adherent PBMCs were transfected by various methods (e.g. PEI, Lipofectamine® 2000, magnetofection and stimulated. Proliferation was measured by lymphocyte transformation test (LTT. Cell cycle stages as well as expression of proliferation relevant genes were analyzed. Additionally, the impact of nanoparticles was investigated in vivo in a murine model of the severe systemic immune disease GvHD (graft versus host disease.The proliferation of primary immune cells was influenced by nanoparticle-mediated transfection. In particular in the case of magnetic beads, proliferation inhibition coincided with short-term cell cycle arrest and reduced expression of genes relevant for immune cell proliferation. Notably, proliferation inhibition translated into beneficial effects in a murine GvHD model with animals treated with PEI-nanoparticles showing increased survival (pPEI = 0.002 most likely due to reduced inflammation.This study shows for the first time that nanoparticles utilized for gene therapeutic transfection are able to alter proliferation of immune cells and that this effect depends on the type of nanoparticle. For magnetic beads, this was accompanied by temporary cell cycle arrest. Notably, in GvHD this nonspecific anti-proliferative effect

  1. Influence of nanoparticle-mediated transfection on proliferation of primary immune cells in vitro and in vivo.

    Science.gov (United States)

    Przybylski, Susanne; Gasch, Michaela; Marschner, Anne; Ebert, Marcus; Ewe, Alexander; Helmig, Gisa; Hilger, Nadja; Fricke, Stephan; Rudzok, Susanne; Aigner, Achim; Burkhardt, Jana

    2017-01-01

    One of the main obstacles in the widespread application of gene therapeutic approaches is the necessity for efficient and safe transfection methods. For the introduction of small oligonucleotide gene therapeutics into a target cell, nanoparticle-based methods have been shown to be highly effective and safe. While immune cells are a most interesting target for gene therapy, transfection might influence basic immune functions such as cytokine expression and proliferation, and thus positively or negatively affect therapeutic intervention. Therefore, we investigated the effects of nanoparticle-mediated transfection such as polyethylenimine (PEI) or magnetic beads on immune cell proliferation. Human adherent and non-adherent PBMCs were transfected by various methods (e.g. PEI, Lipofectamine® 2000, magnetofection) and stimulated. Proliferation was measured by lymphocyte transformation test (LTT). Cell cycle stages as well as expression of proliferation relevant genes were analyzed. Additionally, the impact of nanoparticles was investigated in vivo in a murine model of the severe systemic immune disease GvHD (graft versus host disease). The proliferation of primary immune cells was influenced by nanoparticle-mediated transfection. In particular in the case of magnetic beads, proliferation inhibition coincided with short-term cell cycle arrest and reduced expression of genes relevant for immune cell proliferation. Notably, proliferation inhibition translated into beneficial effects in a murine GvHD model with animals treated with PEI-nanoparticles showing increased survival (pPEI = 0.002) most likely due to reduced inflammation. This study shows for the first time that nanoparticles utilized for gene therapeutic transfection are able to alter proliferation of immune cells and that this effect depends on the type of nanoparticle. For magnetic beads, this was accompanied by temporary cell cycle arrest. Notably, in GvHD this nonspecific anti-proliferative effect might

  2. A cost-effective approach to microporate mammalian cells with the Neon Transfection System.

    Science.gov (United States)

    Brees, Chantal; Fransen, Marc

    2014-12-01

    Electroporation is one of the most efficient nonviral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name Neon Transfection System. This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon kit accessories, thereby reducing the cost of electroporation at least 10-fold. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Improved transfection of HUVEC and MEF cells using DNA ...

    Indian Academy of Sciences (India)

    [Lim J. and Dobson J. 2012 Improved transfection of HUVEC and MEF cells using DNA complexes with magnetic nanoparticles in an oscillating field. J. Genet. ... in stem cell research and endothelial cell physiology and pathology studies are ... It also has potential therapeutic applications (Muthana et al. 2008). Here, we ...

  4. Nanothermite-Based Microsystem for Drug Delivery and Cell Transfection

    Science.gov (United States)

    2008-12-01

    counting individual cells inside the tissues. Fig. 14 shows fluorescence microscope images of the tissues (spinal cord and artery) from the chicken...Fig. 14 Fluorescence microscope images of transfected Spinal cord (Left) and artery (Right). CONCLUSION A new system has been developed

  5. Cell transfection as a tool to study growth hormone action

    DEFF Research Database (Denmark)

    Norstedt, G; Enberg, B; Francis, S

    1994-01-01

    The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration...

  6. A novel transfection method for eukaryotic cells using polyethylenimine coated albumin microbubbles.

    Science.gov (United States)

    Dang, Shi-peng; Wang, Ru-xing; Qin, Ming-de; Zhang, Yan; Gu, Yan-zheng; Wang, Ming-yuan; Yang, Qiao-lin; Li, Xiao-rong; Zhang, Xue-guang

    2011-10-01

    Albumin microbubbles have been intensively studied for their application in gene delivery. However, with negative surface potential, albumin microbubbles hardly bind plasmid DNA, which might contribute to their low transgene efficiency. In this study, we developed polyethylenimine (PEI) coated albumin microbubbles (PAMB) which were prepared by sonicating the mixture of human albumin, PEI, polyethylene glycol and glucose. CHO cells, COS cells and 293T cells were transfected with PEI, PEI+albumin, PAMB and Lipofectamine 2000, respectively. Our results showed that the surface potential was elevated and PAMB could bind plasmid DNA. The transgene efficiency of PAMB was higher than PEI and PEI+albumin (PLipofectamine 2000 did but with lower cytotoxicity than Lipofectamine 2000. Albumin microbubbles modified by PEI has high transgene efficiency and low cytotoxicity even without ultrasound medication, making it a useful non-virus gene delivery method in vitro. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. [RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO].

    Science.gov (United States)

    Zhang, Guangde; Su, Chengshuai; Jin, Xia; Yang, Shimao; Fang, Dianji; Guo, Yanwei

    2016-03-01

    To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the

  8. Improving the Gene Transfection in Human Embryonic Stem Cells: Balancing with Cytotoxicity and Pluripotent Maintenance.

    Science.gov (United States)

    Luo, Chunhua; Lü, Dongyuan; Pan, Jun; Long, Mian

    2016-04-06

    Manipulation of genes in human embryonic stem cells (hESCs) is imperative for their highly potential applications; however, the transduction efficiency remains very low. Although existing evidence revealed the type, size, and zeta potential of vector affect gene transfection efficiency in cells, the systematic study in hESCs is scarce. In this study, using poly(amidoamine) (PAMAM) dendrimers ended with amine, hydroxyl, or carboxyl as model, we tested the influences of size and surface group as well as cytotoxicity and endocytosis on hESC gene transfection. We found that in culture medium of mTeSR the particle sizes of G5, G7, G4.5COOH, and G5OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential, respectively, whereas G1 was slightly negative, and G4.5COOH and G5OH were significantly negative. We demonstrated that only amine-terminated dendrimers accomplished gene transfection in hESCs, which is greater than that from Lipofectamine 2000 transfection. Ten micromolar G5 had the greatest efficiency and was better than 1000 μM G1. Only a low concentration (0.5 and 1 μM) of G7 realized gene delivery. Amine-ended dendrimers, especially with higher generations, were detrimental to the growth and pluripotent maintenance of hESCs. In contrast, similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity, in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. We also confirmed the endocytosis into and significant exocytosis from hESCs using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size, concentration, and zeta potential, particularly the identity and position of groups, as well as minimized exocytosis in the design of a vector for hESC gene delivery are necessary, which helps to better design an effective vector in hESC gene transduction.

  9. Chitosan-pDNA nanoparticle characteristics determine the transfection efficacy of gene delivery to human mesenchymal stem cells.

    Science.gov (United States)

    Malakooty Poor, Elham; Baghaban Eslaminejad, Mohamadreza; Gheibi, Nematollah; Bagheri, Fatemeh; Atyabi, Fatemeh

    2014-12-01

    This study evaluated the potential for prepared chitosan-plasmid DNA (pDNA) nanoparticles to transfer an exogenous gene into human bone marrow-derived mesenchymal stem cells (MSCs). Chitosan-pDNA nanoparticles were synthesized by the complex coacervation method. We used 18, 50 and 136 KD chitosan at concentrations of 0.05%, 0.1%, 0.5% and 1%, in addition to a pTracer-CMV2 plasmid that contained the green fluorescent protein (GFP) gene. To examine the complexation, samples were run through an agarose gel. The sizes and zeta potential of the nanoparticles were measured by a nanosizer. Scanning electron microscopy (SEM) imaging was used to observe the nanoparticle morphology. MSCs were prepared from human bone marrow and transfected with chitosan-pDNA nanoparticles. The cultures transfected by lipofectamine(2000) were taken as the control. Cell viability was determined by MTT assay and transfection efficiency by flow cytometry. The smallest size of complexes was obtained with 18 KD chitosan (211 nm) and the highest zeta potential was observed with 136 KD chitosan (29.61 mV). The best transfection rate (18.43%) was achieved with the 0.1% concentration of 18 KD chitosan nanoparticles versus 40.57% for commercial lipofectamine (p Lipofectamine(2000). Nanoparticles produced by 18 KD chitosan at the 0.1% concentration and pDNA may be a promising gene delivery system for human marrow-derived MSCs. Although transfection efficiency of such nanoparticles is lower than that of Lipofectamine(2000), however comparatively they possess less cytotoxic effects.

  10. Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts.

    Science.gov (United States)

    Kraja, Iv; Bing, Renjie; Hiwatashi, Nao; Rousseau, Bernard; Nalband, Danielle; Kirshenbaum, Kent; Branski, Ryan C

    2017-07-01

    An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent. In vitro. We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-β cascade implicated in fibroplasia in the vocal folds and other tissues. The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility. NA. Laryngoscope, 127:E231-E237, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  11. Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

    Science.gov (United States)

    2011-01-01

    Background During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. Results We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. Conclusions Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells. PMID:21663599

  12. Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity.

    Science.gov (United States)

    Yang, Jing-Yue; Li, Xiao; Gao, Li; Teng, Zeng-Hui; Liu, Wen-Chao

    2012-10-01

    Dendritic cells (DCs) are highly efficient, specialized antigen-presenting cells and DCs transfected with tumor-related antigens are regarded as promising vaccines in cancer immunotherapy. The aim of the present study was to investigate whether DCs co-transfected with the α-fetoprotein (AFP) and human interleukin-2 (IL-2) genes were able to induce stronger therapeutic antitumor immunity in transfected DCs. In this study, DCs from hepatocellular carcinoma (HCC) patients were co-transfected with the IL-2 gene and/or the AFP gene. The reverse transcription-PCR (RT-PCR) data revealed that the DCs transfected with the adenovirus AdAFP/IL-2 expressed AFP and IL-2. The DCs co-transfected with IL-2 and AFP (AFP/IL-2-DCs) enhanced the cytotoxicities of cytotoxic T lymphocytes (CTLs) and increased the production of IL-2 and interferon-γ significantly compared with their AFP-DC, green fluorescent protein (GFP)-DC, DC or phosphate-buffered saline (PBS) counterparts. In vivo data suggested that immunization with AFP-DCs enhances antigen-specific antitumor efficacy more potently than immunization with IL-2-DCs or AFP-DCs. These findings provide a potential strategy to improve the efficacy of DC-based tumor vaccines.

  13. Specific Transfection of Inflamed Brain by Macrophages: A New Therapeutic Strategy for Neurodegenerative Diseases

    Science.gov (United States)

    Haney, Matthew J.; Zhao, Yuling; Harrison, Emily B.; Mahajan, Vivek; Ahmed, Shaheen; He, Zhijian; Suresh, Poornima; Hingtgen, Shawn D.; Klyachko, Natalia L.; Mosley, R. Lee; Gendelman, Howard E.; Kabanov, Alexander V.; Batrakova, Elena V.

    2013-01-01

    The ability to precisely upregulate genes in inflamed brain holds great therapeutic promise. Here we report a novel class of vectors, genetically modified macrophages that carry reporter and therapeutic genes to neural cells. Systemic administration of macrophages transfected ex vivo with a plasmid DNA (pDNA) encoding a potent antioxidant enzyme, catalase, produced month-long expression levels of catalase in the brain resulting in three-fold reductions in inflammation and complete neuroprotection in mouse models of Parkinson's disease (PD). This resulted in significant improvements in motor functions in PD mice. Mechanistic studies revealed that transfected macrophages secreted extracellular vesicles, exosomes, packed with catalase genetic material, pDNA and mRNA, active catalase, and NF-κb, a transcription factor involved in the encoded gene expression. Exosomes efficiently transfer their contents to contiguous neurons resulting in de novo protein synthesis in target cells. Thus, genetically modified macrophages serve as a highly efficient system for reproduction, packaging, and targeted gene and drug delivery to treat inflammatory and neurodegenerative disorders. PMID:23620794

  14. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    Energy Technology Data Exchange (ETDEWEB)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad, E-mail: amtamadon@gmail.com [Shiraz University of Medical Sciences, Department of Pharmaceutics, School of Pharmacy (Iran, Islamic Republic of)

    2016-05-15

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and {sup 1}H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol{sub 40 %}) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  15. Synthesis, characterization and transfection activity of new saturated and unsaturated cationic lipids.

    Science.gov (United States)

    Arpicco, Silvia; Canevari, Silvana; Ceruti, Maurizio; Galmozzi, Enrico; Rocco, Flavio; Cattel, Luigi

    2004-11-01

    We synthesized new cationic lipids, analogue to N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), in order to compare those containing a dodecyl chain with those having a relatively long chain with two or five double bonds, such as squalenyl and dihydrofarnesyl derivatives, or complex saturated structures, such as squalane derivatives. The fusogenic helper lipid dioleoylphosphatidylethanolamine (DOPE) was added to cationic lipids to form a stable complex. Liposomes composed of 50:50 w/w cationic lipid/DOPE were prepared and incubated with plasmidic DNA at various charge ratios and the diameter and zeta potential of the complexes were measured. The surface charge of the DNA/lipid complexes can be controlled by adjusting the cationic lipid/DNA ratio. Finally, we tested the in vitro transfection efficiency of the cationic lipid/DNA complexes using different cell lines. The transfection efficiency was highest for the dodecyloxy derivative containing a single hydroxyethyl group in the head, followed by the dodecyloxy and the farnesyloxy trimethylammonium derivatives. Instead the C27 squalenyl and C27 squalanyl derivatives resulted inactive.

  16. Specific transfection of inflamed brain by macrophages: a new therapeutic strategy for neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Matthew J Haney

    Full Text Available The ability to precisely upregulate genes in inflamed brain holds great therapeutic promise. Here we report a novel class of vectors, genetically modified macrophages that carry reporter and therapeutic genes to neural cells. Systemic administration of macrophages transfected ex vivo with a plasmid DNA (pDNA encoding a potent antioxidant enzyme, catalase, produced month-long expression levels of catalase in the brain resulting in three-fold reductions in inflammation and complete neuroprotection in mouse models of Parkinson's disease (PD. This resulted in significant improvements in motor functions in PD mice. Mechanistic studies revealed that transfected macrophages secreted extracellular vesicles, exosomes, packed with catalase genetic material, pDNA and mRNA, active catalase, and NF-κb, a transcription factor involved in the encoded gene expression. Exosomes efficiently transfer their contents to contiguous neurons resulting in de novo protein synthesis in target cells. Thus, genetically modified macrophages serve as a highly efficient system for reproduction, packaging, and targeted gene and drug delivery to treat inflammatory and neurodegenerative disorders.

  17. Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection.

    Science.gov (United States)

    Namvar, Ali; Bolhassani, Azam; Khairkhah, Niloofardokht; Motevalli, Fatemeh

    2015-07-01

    Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer-based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra- and extracellular barriers. The recent progress has shown that stimuli-responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA-polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared. © 2015 Wiley Periodicals, Inc.

  18. Uptake of DNA by cancer cells without a transfection reagent.

    Science.gov (United States)

    Kong, Yanping; Zhang, Xianbo; Zhao, Yongliang; Xue, Yanfang; Zhang, Ye

    2017-01-21

    Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting

  19. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    Energy Technology Data Exchange (ETDEWEB)

    Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  20. Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

    DEFF Research Database (Denmark)

    Yang, Hsiao Yin; Vonk, Lucienne A.; Licht, Ruud

    2014-01-01

    % amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day...... 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA...... delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were...

  1. Characterization of cell lines stably transfected with rubella virus replicons

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  2. Paclitaxel tumor priming promotes delivery and transfection of intravenous lipid-siRNA in pancreatic tumors.

    Science.gov (United States)

    Wang, Jie; Lu, Ze; Wang, Junfeng; Cui, Minjian; Yeung, Bertrand Z; Cole, David J; Wientjes, M Guillaume; Au, Jessie L-S

    2015-10-28

    The major barrier for using small interfering RNA (siRNA) as cancer therapeutics is the inadequate delivery and transfection in solid tumors. We have previously shown that paclitaxel tumor priming, by inducing apoptosis, expands the tumor interstitial space, improves the penetration and dispersion of nanoparticles and siRNA-lipoplexes in 3-dimensional tumor histocultures, and promotes the delivery and transfection efficiency of siRNA-lipoplexes under the locoregional setting in vivo (i.e., intraperitoneal treatment of intraperitoneal tumors). The current study evaluated whether tumor priming is functional for systemically delivered siRNA via intravenous injection, which would subject siRNA to several additional delivery barriers and elimination processes. We used the same pegylated cationic (PCat)-siRNA lipoplexes as in the intraperitoneal study to treat mice bearing subcutaneous human pancreatic Hs766T xenograft tumors. The target gene was survivin, an inducible chemoresistance gene. The results show single agent paclitaxel delayed tumor growth but also significantly induced the survivin protein level in residual tumors, whereas addition of PCat-siSurvivin completely reversed the paclitaxel-induced survivin and enhanced the paclitaxel activity (ppriming, by promoting the interstitial transport and cytoplasmic release, is critical to promote the delivery and transfection of siRNA in vivo. In addition, because paclitaxel has broad spectrum activity and is used to treat multiple types of solid tumors including the hard-to-treat pancreatic cancer, the synergistic paclitaxel+siSurvivin combination represents a potentially useful chemo-gene therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

    Science.gov (United States)

    Lang, Annemarie; Neuhaus, Johannes; Pfeiffenberger, Moritz; Schröder, Erik; Ponomarev, Igor; Weber, Yvonne; Gaber, Timo; Schmidt, Michael F G

    2014-01-01

    Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy. Copyright © 2014 John Wiley & Sons, Ltd.

  4. Mechanism of Introduction of Exogenous Genes into Cultured Cells Using DEAE-Dextran-MMA Graft Copolymer as Non-Viral Gene Carrier

    OpenAIRE

    Yasuhiko Onishi; Tomohiko Takasaki; Naoji Kubota; Jun Yoshida; Masaaki Mizuno; Junko Higashihara; Masayasu Onishi; Yuki Eshita

    2009-01-01

    Comparative investigations were carried out regarding the efficiency of introduction of exogenous genes into cultured cells using a cationic polysaccharide DEAE-dextran-MMA (methyl methacrylate ester) graft copolymer (2-diethylaminoethyl-dextran-methyl methacrylate graft copolymer; DDMC) as a nonviral carrier for gene introduction. The results confirmed that the gene introduction efficiency was improved with DDMC relative to DEAE-dextran. Comparative investigations were carried out using vari...

  5. Persistent human cardiac Na+ currents in stably transfected mammalian cells

    Science.gov (United States)

    Wang, Ging Kuo; Russell, Gabriella; Wang, Sho-Ya

    2013-01-01

    Miniature persistent late Na+ currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na+ currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na+ channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na+ currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na+ currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na+ currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na+ channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na+ currents were blocked by tetrodotoxin with an IC50 value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na+ currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na+ currents are suitable for studying as well as screening potent open-channel blockers. PMID:23695971

  6. Tissue Engineering Using Transfected Growth-Factor Genes

    Science.gov (United States)

    Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

  7. A Model to Study the Phenotypic Changes of Insect Cell Transfection by Copepod Super Green Fluorescent Protein (cop-GFP) in Baculovirus Expression System.

    Science.gov (United States)

    Shokrollahi, Narjes; Shahbazzadeh, Delavar; Pooshang-Bagheri, Kamran; Habibi-Anbouhi, Mahdi; Jahanian-Najafabadi, Ali; Behdani, Mahdi

    2016-07-01

    Baculovirus expression system is one of the most attractive and powerful eukaryotic expression systems for the production of recombinant proteins. The presence of a biomarker is required to monitor transfection efficiency or protein expression levels in insect cells. The aim of this study was to construct a baculovirus expression vector encoding a copepod super green fluorescent protein (copGFP). In this light, the resultant vector was constructed and used for transfection of Spodoptera frugiperda cells. Expression of the copGFP protein in insect cells was confirmed by fluorescent microscopy and Western-blot analysis. The application of copGFP control bacmid can be considered as an appropriate control for insect cell transfection.

  8. Cell Internalization of Magnetic Nanoparticles Using Transfection Agents

    Directory of Open Access Journals (Sweden)

    Karin Montet-Abou

    2007-01-01

    Full Text Available Transfection agent (TFA-induced magnetic cell labeling with Feridex IV is an attractive method of loading cells because it employs a pharmaceutical source of iron oxide. Although attractive, the method has two significant drawbacks. First, it requires mixing positively charged transfection agents and negatively charged magnetic nanoparticles, and the resulting loss of nanoparticle surface charge causes nanoparticle precipitation. Second, it can result in nanoparticle adsorption to the cell surface rather than internalization. Internalization of Feridex (and associated dextran is important since dextran cell exterior can react with the antidextran antibodies, commonly present in human populations, and trigger an antibody-mediated cytotoxicity. Here we employed three assays for selecting Feridex/TFA mixtures to minimize nanoparticle precipitation and surface adsorption: (1 an assay for precipitation or stability (light scattering, (2 an assay for labeled cells (percentage of cells retained by a magnetic filter, and (3 an antidextran-based assay for nanoparticle internalization. Cells loaded with Feridex/protamine had internalized iron, whereas cells loaded with Feridex/Lipofectamine had surface-adsorbed iron. Optimal conditions for loading cells were 10 mg/Feridex and 3 mg/mL protamine sulfate. Conditions for loading cells with Feridex and a TFA need to be carefully selected to minimize nanoparticle precipitation and dextran adsorption to the cell surface.

  9. Enhanced transfection of tumor cells in vivo using “Smart” pH-sensitive TAT-modified pegylated liposomes

    Science.gov (United States)

    Kale, Amit A.; Torchilin, Vladimir P.

    2012-01-01

    Liposomes have been prepared loaded with DNA (plasmid encoding for the green fluorescent protein, GFP) and additionally modified with TATp and PEG, with PEG being attached to the liposome surface via both pH-sensitive hydrazone and non-pH-sensitive bonds. The pGFP-loaded liposomal preparations have been administered intratumorarly in tumor-bearing mice and the efficacy of tumor cell transfection was followed after 72 h. The administration of pGFP–TATp–liposomes with non-pH-sensitive PEG coating has resulted in only minimal transfection of tumor cells because of steric hindrances for the liposome-to-cell interaction created by the PEG coat, which shielded the surface-attached TATp. At the same time, the administration of pGFP–TATp–liposomes with the low pH-detachable PEG resulted in at least three times more efficient transfection since the removal of PEG under the action of the decreased intratumoral pH leads to the exposure of the liposome-attached TATp residues, enhanced penetration of the liposomes inside tumor cells and more effective intracellular delivery of the pGFP. This result can be considered as an important step in the development of tumor-specific stimuli-sensitive drug and gene delivery systems. PMID:17671900

  10. Mechanism of introduction of exogenous genes into cultured cells using DEAE-dextran-MMA graft copolymer as non-viral gene carrier.

    Science.gov (United States)

    Eshita, Yuki; Higashihara, Junko; Onishi, Masayasu; Mizuno, Masaaki; Yoshida, Jun; Takasaki, Tomohiko; Kubota, Naoji; Onishi, Yasuhiko

    2009-07-23

    Comparative investigations were carried out regarding the efficiency of introduction of exogenous genes into cultured cells using a cationic polysaccharide DEAE-dextran-MMA (methyl methacrylate ester) graft copolymer (2-diethylaminoethyl-dextran-methyl methacrylate graft copolymer; DDMC) as a nonviral carrier for gene introduction. The results confirmed that the gene introduction efficiency was improved with DDMC relative to DEAE-dextran. Comparative investigations were carried out using various concentrations of DDMC and DNA in the introduction of DNA encoding luciferase (pGL3 control vector; Promega) into COS-7 cells derived from African green monkey kidney cells. The complex formation reaction is thought to be directly proportional to the transformation rate, but the complex formation reaction between DDMC and DNA is significantly influenced by hydrophobic bonding strength along with hydrogen bonding strength and Coulomb forces due to the hydrophobicity of the grafted MMA sections. It is thought that the reaction is a Michaelis-Menten type complex formation reaction described by the following equation: Complex amount = K1 (DNA concentration)(DDMC concentration). In support of this equation, it was confirmed that the amount of formed complex was proportional to the RLU value.

  11. Mechanism of Introduction of Exogenous Genes into Cultured Cells Using DEAE-Dextran-MMA Graft Copolymer as Non-Viral Gene Carrier

    Directory of Open Access Journals (Sweden)

    Yasuhiko Onishi

    2009-07-01

    Full Text Available Comparative investigations were carried out regarding the efficiency of introduction of exogenous genes into cultured cells using a cationic polysaccharide DEAE-dextran-MMA (methyl methacrylate ester graft copolymer (2-diethylaminoethyl-dextran-methyl methacrylate graft copolymer; DDMC as a nonviral carrier for gene introduction. The results confirmed that the gene introduction efficiency was improved with DDMC relative to DEAE-dextran. Comparative investigations were carried out using various concentrations of DDMC and DNA in the introduction of DNA encoding luciferase (pGL3 control vector; Promega into COS-7 cells derived from African green monkey kidney cells. The complex formation reaction is thought to be directly proportional to the transformation rate, but the complex formation reaction between DDMC and DNA is significantly influenced by hydrophobic bonding strength along with hydrogen bonding strength and Coulomb forces due to the hydrophobicity of the grafted MMA sections. It is thought that the reaction is a Michaelis-Menten type complex formation reaction described by the following equation: Complex amount = K1 (DNA concentration(DDMC concentration. In support of this equation, it was confirmed that the amount of formed complex was proportional to the RLU value.

  12. Immune monitoring using mRNA-transfected dendritic cells

    DEFF Research Database (Denmark)

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by m......RNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate...... and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA....

  13. Co-liposomes having anisamide tagged lipid and cholesteryl tryptophan trigger enhanced gene transfection in sigma receptor positive cells.

    Science.gov (United States)

    Misra, Santosh K; Moitra, Parikshit; Kondaiah, Paturu; Bhattacharya, Santanu

    2016-06-01

    Selective gene transfection could be strategy of interest for reducing off-target gene expression and toxicity. In this respect, sigma receptors are found to be over-expressed in many human tumors and liposomal formulations with ability to target these sigma receptors may improve the transfection efficiency to a significant level. To this direction, six novel lipids have been synthesized with different hydrophobic segments such as a long hydrophobic chain or a cholesteryl group and L-tryptophan as the head group. Three of them, Lipid 1, 3 and 5 possessed cationic Me3N(+) moiety at the distal end. In contrast each of the other three Lipid 2, 4 and 6 possessed sigma receptor targeting anisamide group with no cationic charge. Mixing of cationic and anisamide counterparts of the same lipid in a molar ratio of 1:1 produced co-liposomes L-M-1 (Lipid 1+2), L-M-2 (Lipid 3+4) and L-M-3 (Lipid 5+6). These co-liposomes, while keeping the sigma targeting anisamide tag intact, showed good DNA binding and release which were optimized from EB intercalation and gel electrophoresis assays. Inclusion of a zwitterionic, fusogenic natural lipid, DOPE, into the co-liposomes further improved the binding efficiencies of the lipid mixtures with DNA. These co-liposomes having cationic and anisamide lipids and DOPE were highly selective toward sigma positive HEK293 and HEK293T cells compared to the sigma negative HeLa cells. As evidenced from both FACS and luciferase assay, a lipid mixture comprising Lipid 3, 4 and DOPE in a molar ratio of 1:1:1 (L-M-2D1) was the best for transfection of reporter pEGFP-C3 and functional pCEP4-p53 gene plasmids. Anisamide mediated sigma receptor selectivity was further probed by pre-incubating the transfecting cells with lipids possessing anisamide and by quantification of the un-transfected plasmid DNA. Also each formulation was highly non-toxic in the cell lines examined. Copyright © 2016. Published by Elsevier B.V.

  14. Multifunctional core/shell nanoparticles cross-linked polyetherimide-folic acid as efficient Notch-1 siRNA carrier for targeted killing of breast cancer.

    Science.gov (United States)

    Yang, Hong; Li, Ying; Li, Tingting; Xu, Min; Chen, Yin; Wu, Chunhui; Dang, Xitong; Liu, Yiyao

    2014-11-17

    In gene therapy, how genetic therapeutics can be efficiently and safely delivered into target tissues/cells remains a major obstacle to overcome. To address this issue, nanoparticles consisting of non-covalently coupled polyethyleneimine (PEI) and folic acid (FA) to the magnetic and fluorescent core/shell of Fe3O4@SiO2(FITC) was tested for their ability to deliver Notch-1 shRNA. Our results showed that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles are 64 nm in diameter with well dispersed and superparamagnetic. These nanoparticles with on significant cytotoxicity are capable of delivering Notch-1 shRNA into human breast cancer MDA-MB-231 cells with high efficiency while effectively protected shRNA from degradation by exogenous DNaseI and nucleases. Magnetic resonance (MR) imaging and fluorescence microscopy showed significant preferential uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by MDA-MB-231 cells. Transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis, leading to the killing of MDA-MB-231 cells. In light of the magnetic targeting capabilities of Fe3O4@SiO2(FITC)/PEI-FA, our results show that by complexing with a second molecular targeting therapeutic, such as Notch-1 shRNA in this report, Fe3O4@SiO2(FITC)/PEI-FA can be exploited as a novel, non-viral, and concurrent targeting delivery system for targeted gene therapy as well as for MR imaging in cancer diagnosis.

  15. A simple protocol for preparation of a liposomal vesicle with encapsulated plasmid DNA that mediate high accumulation and reporter gene activity in tumor tissue

    DEFF Research Database (Denmark)

    Gjetting, Torben; Andresen, Thomas Lars; Christensen, Camilla Laulund

    2011-01-01

    The systemic delivery of gene therapeutics by non-viral methods has proven difficult. Transfection systems that are performing well in vitro have been reported to have disadvantageous properties such as rapid clearance and short circulation time often resulting in poor transfection efficiency when...

  16. Insulin receptor activation and down-regulation by cationic lipid transfection reagents

    OpenAIRE

    Renström Ing-Marie; Kaiser Christina; Danielsson Eva; Andersson Monica; Lanner Johanna; Pramfalk Camilla; Warolén Malin; James Stephen R

    2004-01-01

    Abstract Background Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000™ and FuGENE-6™. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphoryl...

  17. Important role of phosphoramido linkage in imidazole-based dioleyl helper lipids for liposome stability and primary cell transfection.

    Science.gov (United States)

    Mével, Mathieu; Haudebourg, Thomas; Colombani, Thibault; Peuziat, Pauline; Dallet, Laurence; Chatin, Benoît; Lambert, Olivier; Berchel, Mathieu; Montier, Tristan; Jaffrès, Paul-Alain; Lehn, Pierre; Pitard, Bruno

    2016-01-01

    To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain 'helper' neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA molecules via the pH-buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes. We evaluated the influence of the linker structure between the two oleyl chains in the helper lipid on transfection efficiency in cell lines, as well as in primary cells (hepatocytes/cardiomyocytes). We reported the synthesis of two new pH-buffering imidazole helper lipids characterized by a polar headgroup containing one (compound 6) or two (compound 5) imidazole groups and two oleyl chains linked by an amide group. We studied their association with the aminoglycoside lipidic derivative dioleylsuccinylparomomycin (DOSP), which contains two oleyl chains linked to the aminoglycoside polar headgroup via an amide function. We compared the morphology and transfection properties of such binary liposomes of DOSP/5 and DOSP/6 with those of liposomes combining DOSP with another imidazole-based dioleyl helper lipid (MM27) in which a phosphoramido group acts as a linker between the two oleyl chains and imidazole function. The phosphoramido linker in the helper lipid induces a major difference in terms of morphology and resistance to decomplexation at physical pH for DOSP/helper lipid complexes. This hybrid dioleyl linker composition of DOSP/MM27 led to higher transfection efficiency in cell lines and in primary cells compared to complexes with homogeneous dioleyl linker. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Influenza A virus transfectants with chimeric hemagglutinins containing epitopes from different subtypes.

    OpenAIRE

    Li, S Q; Schulman, J L; Moran, T; Bona, C; Palese, P

    1992-01-01

    Influenza virus transfectants with chimeric hemagglutinins were constructed by using a ribonucleoprotein transfection method. Transfectants W(H1)-H2 and W(H1)-H3 contained A/WSN/33(H1N1) (WSN) hemagglutinins in which the six-amino-acid loop (contained in antigenic site B) was replaced by the corresponding structures of influenza viruses A/Japan/57(H2N2) and A/Hong Kong/8/68(H3N2) (HK), respectively. Serological analysis indicated that the W(H1)-H3 transfectant virus reacted with antibodies ag...

  19. Effect of NCAM-transfection on growth and invasion of a human cancer cell line

    DEFF Research Database (Denmark)

    Edvardsen, K; Bock, E; Jirus, S

    1997-01-01

    A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial...... regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM...

  20. Non-viral VEGF165 gene therapy – magnetofection of acoustically active magnetic lipospheres (‘magnetobubbles’) increases tissue survival in an oversized skin flap model

    Science.gov (United States)

    Holzbach, Thomas; Vlaskou, Dialekti; Neshkova, Iva; Konerding, Moritz A; Wörtler, Klaus; Mykhaylyk, Olga; Gänsbacher, Bernd; Machens, H-G; Plank, Christian; Giunta, Riccardo E

    2010-01-01

    Abstract Adenoviral transduction of the VEGF gene in an oversized skin flap increases flap survival and perfusion. In this study, we investigated the potential of magnetofection of magnetic lipospheres containing VEGF165-cDNA on survival and perfusion of ischemic skin flaps and evaluated the method with respect to the significance of applied magnetic field and ultrasound. We prepared perfluoropropane-filled magnetic lipospheres (‘magnetobubbles’) from Tween60-coated magnetic nanoparticles, Metafectene, soybean-oil and cDNA and studied the effect in an oversized random-pattern-flap model in the rats (n= 46). VEGF-cDNA-magnetobubbles were administered under a magnetic field with simultaneously applied ultrasound, under magnetic field alone and with applied ultrasound alone. Therapy was conducted 7 days pre-operative. Flap survival and necrosis were measured 7 days post-operatively. Flap perfusion, VEGF-protein concentration in target and surrounding tissue, formation and appearance of new vessels were analysed additionally. Magnetofection with VEGF-cDNA-magnetobubbles presented an increased flap survival of 50% and increased flap perfusion (P < 0.05). Without ultrasound and without magnetic field, the effect is weakened. VEGF concentration in target tissue was elevated (P < 0.05), while underlying muscle was not affected. Our results demonstrate the successful VEGF gene therapy by means of magnetobubble magnetofection. Here, the method of magnetofection of magnetic lipospheres is equally efficient as adenoviral transduction, but has a presumable superior safety profile. PMID:19040418

  1. Non-viral VEGF(165) gene therapy--magnetofection of acoustically active magnetic lipospheres ('magnetobubbles') increases tissue survival in an oversized skin flap model.

    Science.gov (United States)

    Holzbach, Thomas; Vlaskou, Dialekti; Neshkova, Iva; Konerding, Moritz A; Wörtler, Klaus; Mykhaylyk, Olga; Gänsbacher, Bernd; Machens, H-G; Plank, Christian; Giunta, Riccardo E

    2010-03-01

    Adenoviral transduction of the VEGF gene in an oversized skin flap increases flap survival and perfusion. In this study, we investigated the potential of magnetofection of magnetic lipospheres containing VEGF(165)-cDNA on survival and perfusion of ischemic skin flaps and evaluated the method with respect to the significance of applied magnetic field and ultrasound. We prepared perfluoropropane-filled magnetic lipospheres ('magnetobubbles') from Tween60-coated magnetic nanoparticles, Metafectene, soybean-oil and cDNA and studied the effect in an oversized random-pattern-flap model in the rats (n= 46). VEGF-cDNA-magnetobubbles were administered under a magnetic field with simultaneously applied ultrasound, under magnetic field alone and with applied ultrasound alone. Therapy was conducted 7 days pre-operative. Flap survival and necrosis were measured 7 days post-operatively. Flap perfusion, VEGF-protein concentration in target and surrounding tissue, formation and appearance of new vessels were analysed additionally. Magnetofection with VEGF-cDNA-magnetobubbles presented an increased flap survival of 50% and increased flap perfusion (P < 0.05). Without ultrasound and without magnetic field, the effect is weakened. VEGF concentration in target tissue was elevated (P < 0.05), while underlying muscle was not affected. Our results demonstrate the successful VEGF gene therapy by means of magnetobubble magnetofection. Here, the method of magnetofection of magnetic lipospheres is equally efficient as adenoviral transduction, but has a presumable superior safety profile.

  2. Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

    Science.gov (United States)

    Arias, María Elena; Sánchez-Villalba, Esther; Delgado, Andrea; Felmer, Ricardo

    2017-02-01

    Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

  3. Characterization of PEI-coated superparamagnetic iron oxide nanoparticles for transfection: Size distribution, colloidal properties and DNA interaction

    Energy Technology Data Exchange (ETDEWEB)

    Steitz, Benedikt [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland); Hofmann, Heinrich [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland); Kamau, Sarah W. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zuerich, Zurich (Switzerland); Hassa, Paul O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zuerich, Zurich (Switzerland); Hottiger, Michael O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zuerich, Zurich (Switzerland); Rechenberg, Brigitte von [Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich (Switzerland); Hofmann-Amtenbrink, Magarethe [MatSearch, Chemin Jean Pavillard 14, 1009 Pully (Switzerland); Petri-Fink, Alke [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland)]. E-mail: alke.fink@epfl.ch

    2007-04-15

    Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyethylenimine. Here, we briefly describe the synthesis as well as DNA:PEI:SPION complexes and the characterization of the compounds according to their particle size, {zeta}-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability. PEI coating of SPIONs led to colloidally stable beads even in high salt concentrations over a wide pH range. DNA plasmids and PCR products encoding for green fluorescent protein were associated with the described beads. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency.

  4. Revisit complexation between DNA and polyethylenimine — Effect of length of free polycationic chains on gene transfection

    DEFF Research Database (Denmark)

    Yue, Yanan; Jin, Fan; Deng, Rui

    2011-01-01

    Our revisit of the complexation between DNA and polyethylenimine (PEI) by using a combination of laser light scattering and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) r...... the endosomes. Our result shows that the “proton sponge” effect is not dominant because the shut-down of the proton pump only partially attenuates the transfection efficiency. A possible mechanism is speculated and presented....

  5. Bioreducible PEI-siRNA Nanocomplex for Liver Cancer Therapy: Transfection, Biodistribution, and Tumor Growth Inhibition In Vivo

    Directory of Open Access Journals (Sweden)

    Wei Xia

    2013-01-01

    Full Text Available A bioreducible polyethylenimine (SS-PEI was successfully applied as a nonviral carrier for the delivery of plasmid DNA and VEGF-siRNA in vitro and in vivo. The SS-PEI could strongly condense DNA or siRNA into nanosized complexes (below 200 nm with positive surface charges. In vitro transfection experiments using GFP plasmid as gene reporter showed that the complexes of SS-PEI/DNA were able to efficiently transfect HepG2 cells, with efficiency comparable to that of polyethylenimine, a gold standard for nonviral gene delivery. Moreover, the complexes of SS-PEI/VEGF-siRNA could lead to reduced levels of VEGF protein in HepG2 cells in vitro. Treatment with the complexes of SS-PEI/VEGF-siRNA efficiently inhibited HepG2 tumor growth in an xenograft mouse model. The data of this study imply that the SS-PEI is a potent nucleic acid carrier applicable for liver cancer gene therapy.

  6. Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery.

    Science.gov (United States)

    Chahal, Parminder Singh; Schulze, Erica; Tran, Rosa; Montes, Johnny; Kamen, Amine A

    2014-02-01

    Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10(13)Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10(13)Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10(13)Vg/L cell culture (6.8×10(11)IVP/L) were measured between 48 and 64h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800Vg per cell and 460IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  7. Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.

    Science.gov (United States)

    Fietz, D; Markmann, M; Lang, D; Konrad, L; Geyer, J; Kliesch, S; Chakraborty, T; Hossain, H; Bergmann, M

    2015-12-29

    Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR. Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes. We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.

  8. [Effect of different transfection reagents and injection methods in mice testicular injection on the expression of exogenous gene].

    Science.gov (United States)

    Dai, Jianjun; Li, Xiang; Wu, Caifeng; Zhang, Shushan; Zhang, Tingyu; Zhang, Defu

    2014-10-01

    The purpose of this study was to study the effect of three different transfection reagents (Lipofectamine™ LTX & PLUS™, Lipofectamine 2000 and Nano-PAMAM-D) and three different testicular injection methods (rete testicular injection, seminiferous tubules injection and testicular interstitial injection) on the efficiency of production transgenic mice. After the mixtures of plasmid DNA (pEFP-C1) and transfection reagent were injected with different testicular injection methods, the sperm density, vitality, positive sperm rates and PCR positive transgenic mice rate were examined 30 days after injection. The results showed that the damage degree from slight to serious of three transfection reagents was Lipofectamine™ LTX & PLUS™, Lipofectamine 2000, and PAMAM-D. The sperm positive rates with green fluorescence of these three groups were 35.65%±0.69%, 12.86%±0.35% and 10.04%±0.20%, respectively. The PCR positive rates of transgenic newborn mice were 29.17%, 13.70% and 5.88%, respectively. Among the groups of different testicular injection methods, the damage degree from slight to serious was rete testicular injection, seminiferous tubules injection, and testicular interstitial injection, whereas the sperm positive rates with green fluorescence were 35.13%, 15.13%, and 0%, respectively. The PCR positive rates of transgenic newborn mice among different testicular injection groups were 33.3%, 12.5%, and 0.0%. The combination of rete testicular injection and Lipofectamine™ LTX & PLUS™ had the lowest toxicity and highest transgenic efficiency in the production of transgenic mice.

  9. Transfection of glioma cells with the neural-cell adhesion molecule NCAM

    DEFF Research Database (Denmark)

    Edvardsen, K; Pedersen, P H; Bjerkvig, R

    1994-01-01

    The tumor growth and the invasive capacity of a rat glioma cell line (BT4Cn) were studied after transfection with the human transmembrane 140-kDa isoform of the neural-cell adhesion molecule, NCAM. After s.c. injection, the NCAM-transfected cells showed a slower growth rate than the parent cell...

  10. Co-self-assembly of cationic microparticles to deliver pEGFP-ZNF580 for promoting the transfection and migration of endothelial cells.

    Science.gov (United States)

    Feng, Yakai; Guo, Mengyang; Liu, Wen; Hao, Xuefang; Lu, Wei; Ren, Xiangkui; Shi, Changcan; Zhang, Wencheng

    The gene transfection efficiency of polyethylenimine (PEI) varies with its molecular weight. Usually, high molecular weight of PEI means high gene transfection, as well as high cytotoxicity in gene delivery in vivo. In order to enhance the transfection efficiency and reduce the cytotoxicity of PEI-based gene carriers, a novel cationic gene carrier was developed by co-self-assembly of cationic copolymers. First, a star-shaped copolymer poly(3(S)-methyl-morpholine-2,5-dione-co-lactide) (P(MMD-co-LA)) was synthesized using D-sorbitol as an initiator, and the cationic copolymer (P(MMD-co-LA)-g-PEI) was obtained after grafting low-molecular weight PEI. Then, by co-self-assembly of this cationic copolymer and a diblock copolymer methoxy-poly(ethylene glycol) (mPEG)-b-P(MMD-co-LA), microparticles (MPs) were formed. The core of MPs consisted of a biodegradable block of P(MMD-co-LA), and the shell was formed by mPEG and PEI blocks. Finally, after condensation of pEGFP-ZNF580 by these MPs, the plasmids were protected from enzymatic hydrolysis effectively. The result indicated that pEGFP-ZNF580-loaded MP complexes were suitable for cellular uptake and gene transfection. When the mass ratio of mPEG-b-P(MMD-co-LA) to P(MMD-co-LA)-g-PEI reached 3/1, the cytotoxicity of the complexes was very low at low concentration (20 μg mL(-1)). Additionally, pEGFP-ZNF580 could be transported into endothelial cells (ECs) effectively via the complexes of MPs/pEGFP-ZNF580. Wound-healing assay showed that the transfected ECs recovered in 24 h. Cationic MPs designed in the present study could be used as an applicable gene carrier for the endothelialization of artificial blood vessels.

  11. Aspects of nonviral gene therapy: correlation of molecular parameters with lipoplex structure and transfection efficacy in pyridinium-based cationic lipids.

    Science.gov (United States)

    Parvizi, Paria; Jubeli, Emile; Raju, Liji; Khalique, Nada Abdul; Almeer, Ahmed; Allam, Hebatalla; Manaa, Maryem Al; Larsen, Helge; Nicholson, David; Pungente, Michael D; Fyles, Thomas M

    2014-01-30

    This study seeks correlations between the molecular structures of cationic and neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All lipoplex formulations exhibited plasmid DNA binding and a level of protection from DNase I degradation. Composition-dependent transfection (beta-galactosidase and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The most active formulations containing the pyridinium lipids were less cytotoxic but of comparable activity to a Lipofectamine 2000™ control. Molecular structure parameters and partition coefficients were calculated for all lipids using fragment additive methods. The derived shape parameter values correctly correlated with observed hexagonal lipid phase behavior of lipoplexes as derived from small-angle X-ray scattering experiments. A transfection index applicable to hexagonal phase lipoplexes derived from calculated parameters of the lipid mixture (partition coefficient, shape parameter, lipoplex packing) produced a direct correlation with transfection efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Water-soluble cationic poly(ferrocenylsilane): an efficient DNA condensation and transfection agent

    NARCIS (Netherlands)

    Zhong, Zhiyuan; Lin, C.; Ma, Y.; Hempenius, Mark A.; Lok, M.C.; Fretz, M.M.; Engbersen, Johannes F.J.; Vancso, Gyula J.; Hennink, W.E.; Feijen, Jan; Hennink, W.E.; Feijen, J.; Sam, T

    2006-01-01

    The DNA condensation and in vitro gene transfer properties of an organo-iron polymer, cationic poly(ferrocenylsilane), were investigated. Gel retardation assay, dynamic light scattering (DLS), atomic force microscopy (AFM) and zeta-potential measurements showed that cationic poly(ferrocenylsilane)

  13. Inclusion of the helper lipid dioleoyl-phosphatidylethanolamine in solid lipid nanoparticles inhibits their transfection efficiency

    NARCIS (Netherlands)

    de Jesus, Marcelo B.; Radaic, Allan; Hinrichs, Wouter L J; Ferreira, Carmen V; de Paula, Eneida; Hoekstra, Dirk; Zuhorn, Inge S

    Solid lipid nanoparticles (SLNs) are a promising system for the delivery of lipophilic and hydrophilic drugs. They consist of a solid lipid core that is stabilized by a layer of surfactants. By the incorporation of cationic lipids in the formulation, positively charged SLNs can be generated, that

  14. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    CSIR Research Space (South Africa)

    Mthunzi, P

    2014-02-01

    Full Text Available Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including...

  15. Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA.

    Science.gov (United States)

    Miller, Jason B; Zhang, Shuyuan; Kos, Petra; Xiong, Hu; Zhou, Kejin; Perelman, Sofya S; Zhu, Hao; Siegwart, Daniel J

    2017-01-19

    CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg(-1) ). Intravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Development of Polypeptide-based Nanoparticles for Non-viral Delivery of CD22 RNA Trans-splicing Molecule as a New Precision Medicine Candidate Against B-lineage ALL

    Directory of Open Access Journals (Sweden)

    Fatih M. Uckun

    2015-07-01

    Full Text Available CD22ΔE12 has emerged as a driver lesion in the pathogenesis of pediatric B-lineage acute lymphoblastic leukemia (ALL and a new molecular target for RNA therapeutics. Here we report a 43-gene CD22ΔE12 signature transcriptome that shows a striking representation in primary human leukemia cells from patients with relapsed BPL. Our data uniquely indicate that CD22ΔE12 is a candidate driver lesion responsible for the activation of MAPK and PI3-K pathways in aggressive forms of B-lineage ALL. We also show that the forced expression of a CD22 RNA trans-splicing molecule (RTM markedly reduces the capacity of the leukemic stem cell fraction of CD22ΔE12+ B-lineage ALL cells to engraft and cause overt leukemia in NOD/SCID mice. We have successfully complexed our rationally designed lead CD22-RTM with PVBLG-8 to prepare a non-viral nanoscale formulation of CD22ΔE12-RTM with potent anti-cancer activity against CD22ΔE12+ B-lineage leukemia and lymphoma cells. CD22-RTM nanoparticles effectively delivered the CD22-RTM cargo into B-lineage ALL cells and exhibited significant anti-leukemic activity in vitro.

  17. Biodegradable gadolinium-chelated cationic poly(urethane amide) copolymers for gene transfection and magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiaolong [Department of Radiology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065 (China); Wang, Gangmin [Department of Urology, Huashan Hospital, Fudan University, Shanghai 200040 (China); Shi, Ting [The Institute for Translational Nanomedicine, Shanghai East Hospital, Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Shanghai 200092 (China); Shao, Zhihong [Department of Radiology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065 (China); Zhao, Peng; Shi, Donglu [The Institute for Translational Nanomedicine, Shanghai East Hospital, Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Shanghai 200092 (China); Ren, Jie [Institute of Nano and Biopolymeric Materials, School of Materials Science and Engineering, Tongji University, 4800 Caoan Road, Shanghai 201804 (China); Lin, Chao, E-mail: chaolin@tongji.edu.cn [The Institute for Translational Nanomedicine, Shanghai East Hospital, Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Shanghai 200092 (China); Wang, Peijun, E-mail: tjpjwang@sina.com [Department of Radiology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065 (China)

    2016-08-01

    Theranostic nano-polyplexes containing gene and imaging agents hold a great promise for tumor diagnosis and therapy. In this work, we develop a group of new gadolinium (Gd)-chelated cationic poly(urethane amide)s for gene delivery and T{sub 1}-weighted magnetic resonance (MR) imaging. Cationic poly(urethane amide)s (denoted as CPUAs) having multiple disulfide bonds, urethane and amide linkages were synthesized by stepwise polycondensation reaction between 1,4-bis(3-aminopropyl)piperazine and a mixture of di(4-nitrophenyl)-2, 2′-dithiodiethanocarbonate (DTDE-PNC) and diethylenetriaminepentaacetic acid (DTPA) dianhydride at varied molar ratios. Then, Gd-chelated CPUAs (denoted as GdCPUAs) were produced by chelating Gd(III) ions with DTPA residues of CPUAs. These GdCPUAs could condense gene into nanosized and positively-charged polyplexes in a physiological condition and, however, liberated gene in an intracellular reductive environment. In vitro transfection experiments revealed that the GdCPUA at a DTDE-PNC/DTPA residue molar ratio of 85/15 induced the highest transfection efficiency in different cancer cells. This efficiency was higher than that yielded with 25 kDa branched polyethylenimine as a positive control. GdCPUAs and their polyplexes exhibited low cytotoxicity when an optimal transfection activity was detected. Moreover, GdCPUAs may serve as contrast agents for T{sub 1}-weighted magnetic resonance imaging. The results of this work indicate that biodegradable Gd-chelated cationic poly(urethane amide) copolymers have high potential for tumor theranostics. - Highlights: • Novel cationic gadolinium-chelated poly(urethane amide)s (GdCPUAs) are prepared. • GdCPUAs can induce a high transfection efficacy in different cancer cells. • GdCPUAs reveal good cyto-compatibility against cancer cells. • GdCPUAs may be applied as T{sub 1}-contrast agents for magnetic resonance imaging. • GdCPUAs hold high potential for cancer theranostics.

  18. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.

    Science.gov (United States)

    Li, Pu; Zhang, Lei

    2015-08-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial

  19. Nucleobase-modified dendrimers as nonviral vectors for efficient and low cytotoxic gene delivery.

    Science.gov (United States)

    Wang, Hui; Wei, Haifeng; Huang, Quan; Liu, Hongmei; Hu, Jingjing; Cheng, Yiyun; Xiao, Jianru

    2015-12-01

    Cationic dendrimers are associated with relatively low transfection efficacy and high toxicity in gene delivery. Surface modification of these dendrimers with functional ligands may resolve these issues. Here, we proposed a novel strategy to prepare surface-engineered dendrimers with high transfection efficacy and low toxicity on transfected cells. Several nucleobase analogues were modified on cationic dendrimers by a facile method. These nucleobase-modified dendrimers show improved transfection efficacy and reduced cytotoxicity compared to unmodified dendrimers on HEK293 and HeLa cells. Efficacy of the most efficient polymer is comparable to that of commercial transfection reagents such as SuperFect, PolyFect, and Lipofectamine 2000. The improved transfection efficacy of dendrimers after nucleobase modification is probably attributed to easier intracellular DNA unpacking and lower cytotoxicity. The results provide a valuable insight to guide the design of efficient and low cytotoxic gene vectors. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

    Science.gov (United States)

    Cooper, Caitlin A; Challagulla, Arjun; Jenkins, Kristie A; Wise, Terry G; O'Neil, Terri E; Morris, Kirsten R; Tizard, Mark L; Doran, Timothy J

    2017-06-01

    Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

  1. Miniaturization of Gene Transfection Assays in 384 and 1536-Well Microplates

    Science.gov (United States)

    Li, Jing; Crowley, Samuel T.; Duskey, Jason; Khargharia, Sanjib; Wu, Meng; Rice, Kevin G.

    2015-01-01

    The miniaturization of gene transfer assays to either 384 or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared to more conventional 96-well assays. To validate the approach, luciferase and GFP reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO and 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384 and 1536 well plates. The results established optimal transfection parameters in 384-well plates in an total assay volume of 35 μl and in 1536-well plates in a total volume of 8 μl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI-DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250 cells per well in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI. PMID:25448623

  2. Miniaturization of gene transfection assays in 384- and 1536-well microplates.

    Science.gov (United States)

    Li, Jing; Crowley, Samuel T; Duskey, Jason; Khargharia, Sanjib; Wu, Meng; Rice, Kevin G

    2015-02-01

    The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35μl and in 1536-well plates in a total assay volume of 8μl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. The Induction of HMGB1 Release from RAW 264.7 Cells by Transfected DNA

    Science.gov (United States)

    Jiang, Weiwen; Pisetsky, David S.

    2013-01-01

    High mobility group protein 1 (HMGB1) is a non-histone nuclear protein that can activate innate immunity when in an extracellular location. As shown in in vitro studies, while polyinosinic-polycytidylic acid [poly (I:C)] and LPS, TLR3 and TLR4 ligands respectively, can induce HMGB1 release from macrophages, CpG DNA, a TLR 9 ligand, does not. Since DNA displays distinct immunostimulatory activity when transfected into cells, we investigated whether transfected DNA can induce HMGB1 release from macrophages. In these experiments, using RAW 264.7 cells as model, we show that DNA, either natural DNA or synthetic oligonucleotides, can induce HMGB1 release when used to stimulate cells with the transfection reagent Lipofectamine 2000®; release occurred irrespective of the intrinsic activity of the DNA. The induction of HMGB1 release by transfected DNA was dependent on IFN-β as shown by the inhibitory effects of an antibody. In addition, JNK activation mediated HMGB1 release induced by a transfected phosphorothioate oligonucleotide but not by transfected natural DNA. Together, these findings indicate that transfected DNA can stimulate macrophages to release HMGB1 under conditions in which free DNA is inactive and suggest a role of DNA in inducing inflammation when bound to molecules that influence its entry into cells. PMID:18031817

  4. Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells.

    Science.gov (United States)

    Kaufman, R J; Murtha, P

    1987-01-01

    The translational efficiency of mRNA molecules transcribed from plasmid DNA transfected into COS-1 monkey cells can be increased 10- to 20-fold by the coexpression of the adenovirus virus-associated RNAs I and II. Experiments described here demonstrate a similar increase in translational efficiency by the addition of 2-aminopurine, an inhibitor of double-stranded RNA-activated protein kinase, to the culture medium. Both virus-associated RNA and 2-aminopurine presumably exert their effect by alteration of the functional level of eucaryotic initiation factor-2. The translational stimulation mediated by both means is shown to be restricted to the plasmid-derived mRNAs because there is no qualitative or quantitative alteration in host protein synthesis. The results are consistent with models invoking a localized activation of double-stranded RNA-activated kinase leading to a translational block. Images PMID:3600637

  5. Poly-L-Lysine-Poly[HPMA] Block Copolymers Obtained by RAFT Polymerization as Polyplex-Transfection Reagents with Minimal Toxicity.

    Science.gov (United States)

    Tappertzhofen, Kristof; Weiser, Franziska; Montermann, Evelyn; Reske-Kunz, Angelika; Bros, Matthias; Zentel, Rudolf

    2015-08-01

    Herein we describe the synthesis of poly-L-lysine-b-poly[N-(2-hydroxypropyl)-metha-crylamide)] (poly[HPMA]) block copolymers by combination of solid phase peptide synthesis or polymerization of α-amino acid-N-carboxy-anhydrides (NCA-polymerization) with the reversible addition-fragmentation chain transfer polymerization (RAFT). In the presence of p-DNA, these polymers form polyplex micelles with a size of 100-200 nm in diameter (monitored by SDS-PAGE and FCS). Primary in vitro studies with HEK-293T cells reveal their cellular uptake (FACS studies and CLSM) and proof successful transfection with efficiencies depending on the length of polylysine. Moreover, these polyplexes display minimal toxicity (MTT-assay and FACS-measurements) featuring a p[HPMA] corona for efficient extracellular shielding and the potential ligation with antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

    Directory of Open Access Journals (Sweden)

    Min Koo Seo

    2011-02-01

    Full Text Available BackgroundPreviously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF gene in an Epstein-Barr virus (EBV-based plasmid (pEBVHGF showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model.MethodsNeonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry.ResultsRe-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF.ConclusionFor clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

  7. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-09-21

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

  8. Photo-transfection of mammalian cells via femtosecond laser pulses

    CSIR Research Space (South Africa)

    Mthunzi, P

    2009-06-01

    Full Text Available Various methods including chemical, viral or physical approaches have been developed to trigger transient membrane permeabilisation with limited cytotocixity in cells. Such developments permit efficient cytoplasmic delivery of genes, fluorochromes...

  9. [Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells].

    Science.gov (United States)

    Yin, Kai; Ma, Li; Shen, Chuan'an; Shang, Yuru; Li, Dawei; Li, Longzhu; Zhao, Dongxu; Cheng, Wenfeng

    2016-05-01

    To investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs. hECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured wit