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Sample records for dc gene delivery

  1. Microfluidic electroporation for delivery of small molecules and genes into cells using a common DC power supply.

    Science.gov (United States)

    Wang, Hsiang-Yu; Lu, Chang

    2008-06-15

    Electroporation is an efficient method of introducing foreign impermeant molecules such as drugs and genes into cells. Conventional electroporation has been based on the application of short electrical pulses (electropulsation). Electropulsation requires specialized equipment and cannot be integrated easily with techniques such as electrophoresis which is based on constant voltage. Here we demonstrate the delivery of small molecules and genes into cells, using a microfluidic electroporation technique based on constant direct current (DC) voltage that we developed earlier. We demonstrate the delivery of two molecules into Chinese hamster ovary (CHO-K1) cells: a membrane impermeable nucleic acid dye (SYTOX Green) and a plasmid vector carrying the gene for green fluorescent protein (pEGFP-C1). Our devices can exert field variations to flowing cells that are analogous to the application of single or multiple pulses by having different geometries. We investigate the effects of the electrical parameters and different geometries of the device on the transfection efficiency and cell viability. Our technique provides a simple solution to electroporation-based drug and gene delivery by eliminating the need for a pulse generator. We envision that these simple microscale electroporation devices will have the potential to work in parallel on a microchip platform and such technology will allow high-throughput functional screening of drugs and genes. (c) 2008 Wiley Periodicals, Inc.

  2. Synthetic sustained gene delivery systems.

    Science.gov (United States)

    Agarwal, Ankit; Mallapragada, Surya K

    2008-01-01

    Gene therapy today is hampered by the need of a safe and efficient gene delivery system that can provide a sustained therapeutic effect without cytotoxicity or unwanted immune responses. Bolus gene delivery in solution results in the loss of delivered factors via lymphatic system and may cause undesired effects by the escape of bioactive molecules to distant sites. Controlled gene delivery systems, acting as localized depot of genes, provide an extended sustained release of genes, giving prolonged maintenance of the therapeutic level of encoded proteins. They also limit the DNA degradation in the nuclease rich extra-cellular environment. While attempts have been made to adapt existing controlled drug delivery technologies, more novel approaches are being investigated for controlled gene delivery. DNA encapsulated in nano/micro spheres of polymers have been administered systemically/orally to be taken up by the targeted tissues and provide sustained release once internalized. Alternatively, DNA entrapped in hydrogels or scaffolds have been injected/implanted in tissues/cavities as platforms for gene delivery. The present review examines these different modalities for sustained delivery of viral and non-viral gene-delivery vectors. Design parameters and release mechanisms of different systems made with synthetic or natural polymers are presented along with their prospective applications and opportunities for continuous development.

  3. Cationic Bolaamphiphiles for Gene Delivery

    Science.gov (United States)

    Tan, Amelia Li Min; Lim, Alisa Xue Ling; Zhu, Yiting; Yang, Yi Yan; Khan, Majad

    2014-05-01

    Advances in medical research have shed light on the genetic cause of many human diseases. Gene therapy is a promising approach which can be used to deliver therapeutic genes to treat genetic diseases at its most fundamental level. In general, nonviral vectors are preferred due to reduced risk of immune response, but they are also commonly associated with low transfection efficiency and high cytotoxicity. In contrast to viral vectors, nonviral vectors do not have a natural mechanism to overcome extra- and intracellular barriers when delivering the therapeutic gene into cell. Hence, its design has been increasingly complex to meet challenges faced in targeting of, penetration of and expression in a specific host cell in achieving more satisfactory transfection efficiency. Flexibility in design of the vector is desirable, to enable a careful and controlled manipulation of its properties and functions. This can be met by the use of bolaamphiphile, a special class of lipid. Unlike conventional lipids, bolaamphiphiles can form asymmetric complexes with the therapeutic gene. The advantage of having an asymmetric complex lies in the different purposes served by the interior and exterior of the complex. More effective gene encapsulation within the interior of the complex can be achieved without triggering greater aggregation of serum proteins with the exterior, potentially overcoming one of the great hurdles faced by conventional single-head cationic lipids. In this review, we will look into the physiochemical considerations as well as the biological aspects of a bolaamphiphile-based gene delivery system.

  4. Gene therapy prospects--intranasal delivery of therapeutic genes.

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    Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

    2012-01-01

    Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

  5. Bioreducible polymers for gene silencing and delivery.

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    Son, Sejin; Namgung, Ran; Kim, Jihoon; Singha, Kaushik; Kim, Won Jong

    2012-07-17

    Polymeric gene delivery vectors show great potential for the construction of the ideal gene delivery system. These systems harness their ability to incorporate versatile functional traits to overcome most impediments encountered in gene delivery: from the initial complexation to their target-specific release of the therapeutic nucleic acids at the cytosol. Among the numerous multifunctional polymers that have been designed and evaluated as gene delivery vectors, polymers with redox-sensitive (or bioreducible) functional domains have gained great attention in terms of their structural and functional traits. The redox environment plays a pivotal role in sustaining cellular homeostasis and natural redox potential gradients exist between extra- and intracellular space and between the exterior and interior of subcellular organelles. In some cases, researchers have designed the polymeric delivery vectors to exploit these gradients. For example, researchers have taken advantage of the high redox potential gradient between oxidizing extracellular space and the reducing environment of cytosolic compartments by integrating disulfide bonds into the polymer structure. Such polymers retain their cargo in the extracellular space but selectively release the therapeutic nucleic acids in the reducing space within the cytosol. Furthermore, bioreducible polymers form stable complex with nucleic acids, and researchers can fabricate these structures to impart several important features such as site-, timing-, and duration period-specific gene expression. Additionally, the introduction of disulfide bonds within these polymers promotes their biodegradability and limits their cytotoxicity. Many approaches have demonstrated the versatility of bioreducible gene delivery, but the underlying biological rationale of these systems remains poorly understood. The process of disulfide reduction depends on multiple variables in the cellular redox environment. Therefore, the quest to unravel various

  6. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

    Directory of Open Access Journals (Sweden)

    Shyamal Kumar Kundu

    2014-01-01

    Full Text Available Advancement in the gene delivery system have resulted in clinical successes in gene therapy for patients with several genetic diseases, such as immunodeficiency diseases, X-linked adrenoleukodystrophy (X-ALD blindness, thalassemia, and many more. Among various delivery systems, liposomal mediated gene delivery route is offering great promises for gene therapy. This review is an attempt to depict a portrait about the polymer based liposomal gene delivery systems and their future applications. Herein, we have discussed in detail the characteristics of liposome, importance of polymer for liposome formulation, gene delivery, and future direction of liposome based gene delivery as a whole.

  7. Modified montmorillonite as vector for gene delivery.

    Science.gov (United States)

    Lin, Feng-Huei; Chen, Chia-Hao; Cheng, Winston T K; Kuo, Tzang-Fu

    2006-06-01

    Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system. Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si-O tetrahedrons on the bottom of the layer and Al-O(OH)2 octahedrons on the top. The inter-layer space is about 12 A. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA-HDTMA-MMT is as follows: 1 mg of 1.5CEC HDTMA-MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA-MMT has a great potential as a vector for gene delivery in the future.

  8. Novel Polymeric Nanoparticles for Pulmonary Gene Delivery

    Science.gov (United States)

    Fields, Rachel Jennifer

    The lung is an important target for gene and drug therapy of many diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), tubuerculosis (TB) and lung cancer. In fact, the pulmonary route has been employed as a means of delivering drugs for centuries, dating back 4000 years to India where inhaled vapors were used for medicinal purpose. Currently, pulmonary administration of small, hydrophobic drugs leads to rapid local and systemic absorption. However, delivery of large biomacromolecules, such as therapeutic genes, has not yet been accomplished. Here, I test the hypothesis that a rationally engineered nanoparticle (NP) vector can improve delivery of large biomacromolecules. . In this dissertation I tested this hypothesis using a hybrid NP delivery system consisting of a blend of poly(lactic-co-glycolic acid) (PLGA) and a poly(beta-amino ester) (PBAE), a cationic polymer that is particularly useful for delivery of nucleic acids.. PBAE/PLGA nanoparticles (15% PBAE) loaded with plasmid DNA were surface modified with cell-penetrating peptides (CPPs) via a PEGylated phospholipid linker. This optimized NP formulation was able to induce substantial intracellular uptake and transfect lung epithelial cells in vitro while imparting minimal cellular toxicity. In order to determine the most effective method to deliver these NPs to the lung I used fluorescently labeled particles to study the biodistribution of particles after administration to the lung of mice via various administration routes. I determined that the intranasal route was most effective. I further investigated this route and determined that an average of 37.1 +/- 15.1 % of lung cells had NP association after 4hrs. I also investigated the association of particles with different lung cell types like macrophages and alveolar epithelial cells and determined that our best particle formulations associated with approximately 80% of both of these cell types. To demonstrate the ability of the

  9. Genetic nanomedicine: gene delivery by targeted lipoplexes.

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    Düzgüneş, Nejat; de Ilarduya, Conchita Tros

    2012-01-01

    Cationic liposome-DNA complexes (lipoplexes) are used for the delivery of plasmid DNA to cultured cells and various tissues in vivo. In this chapter, we describe the preparation and evaluation of plain and targeted lipoplexes, using targeting ligands, including epidermal growth factor and transferrin. Ligand-associated lipoplexes may be used to target DNA or other nucleic acid drugs to specific cells, particularly cancer cells that overexpress the receptors for the ligands. We provide examples of the enhancement of gene expression mediated by epidermal growth factor in murine and human oral squamous cell carcinoma cells, and human hepatoblastoma and rat colon adenocarcinoma cells. We also summarize the studies on the use of transferrin-lipoplexes for enhancing gene delivery to cervical carcinoma, murine colon carcinoma, and African green monkey kidney cells. We outline two animal models in which transferrin-lipoplexes have been used for antitumor therapy by delivering either the gene encoding interleukin-12 or a suicide gene: a CT26 murine colon carcinoma, and a syngeneic, orthotopic murine oral squamous cell carcinoma. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Synthetic virology: engineering viruses for gene delivery.

    Science.gov (United States)

    Guenther, Caitlin M; Kuypers, Brianna E; Lam, Michael T; Robinson, Tawana M; Zhao, Julia; Suh, Junghae

    2014-01-01

    The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. © 2014 Wiley Periodicals, Inc.

  11. Liposomes for Use in Gene Delivery

    Directory of Open Access Journals (Sweden)

    Daniel A. Balazs

    2011-01-01

    Full Text Available Liposomes have a wide array of uses that have been continuously expanded and improved upon since first being observed to self-assemble into vesicular structures. These arrangements can be found in many shapes and sizes depending on lipid composition. Liposomes are often used to deliver a molecular cargo such as DNA for therapeutic benefit. The lipids used to form such lipoplexes can be cationic, anionic, neutral, or a mixture thereof. Herein physical packing parameters and specific lipids used for gene delivery will be discussed, with lipids classified according to overall charge.

  12. Polyelectrolyte nanoparticles mediate vascular gene delivery.

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    Zaitsev, Sergey; Cartier, Régis; Vyborov, Oleg; Sukhorukov, Gleb; Paulke, Bernd-Reiner; Haberland, Annekathrin; Parfyonova, Yelena; Tkachuk, Vsevolod; Böttger, Michael

    2004-09-01

    The purpose is to develop a non-viral gene delivery system that meets the requirements of colloidal stability of DNA complexes expressed in terms of no particle aggregation under physiologic conditions. The system should be used to transfect cardiovascular tissues. We used a strategy based on the formation of polyelectrolyte nanoparticles by deposition of alternatively charged polyelectrolytes onto a DNA core. Polyelectrolytes were transfer RNA as well as the synthetic polyanion, polyvinyl sulfate (PVS), and the polycation polyethylenimine (PEI). The PEI/DNA complex formed the DNA core. We observed that the DNA is condensed by polycations and further packaged by association with a polyanion. These nanoparticles exhibited negative surface charge and low aggregation tendency. In vivo rat carotid artery experiments revealed high transfection efficiency, not only with the reporter gene but also with the gene encoding human urokinase plasminogen activator (Hu-uPA). Hu-uPA is one of the proteins involved in the recovery of the blood vessels after balloon catheter injury and therefore clinically relevant. A strategy for in vivo gene transfer is proposed that uses the incorporation of polyanions as RNA or PVS into PEI/DNA complexes in order to overcome colloidal instability and to generate a negative surface charge. The particles proved to be transfectionally active in vascular gene transfer.

  13. Nonviral Delivery Systems For Cancer Gene Therapy: Strategies And Challenges.

    Science.gov (United States)

    Shim, Gayong; Kim, Dongyoon; Le, Quoc-Viet; Park, Gyu Thae; Kwon, Taekhyun; Oh, Yu-Kyoung

    2018-01-19

    Gene therapy has been receiving widespread attention due to its unique advantage in regulating the expression of specific target genes. In the field of cancer gene therapy, modulation of gene expression has been shown to decrease oncogenic factors in cancer cells or increase immune responses against cancer. Due to the macromolecular size and highly negative physicochemical features of plasmid DNA, efficient delivery systems are an essential ingredient for successful gene therapy. To date, a variety of nanostructures and materials have been studied as nonviral gene delivery systems. In this review, we will cover nonviral delivery strategies for cancer gene therapy, with a focus on target cancer genes and delivery materials. Moreover, we will address current challenges and perspectives for nonviral delivery-based cancer gene therapeutics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Gene delivery in tissue engineering and regenerative medicine.

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    Fang, Y L; Chen, X G; W T, Godbey

    2015-11-01

    As a promising strategy to aid or replace tissue/organ transplantation, gene delivery has been used for regenerative medicine applications to create or restore normal function at the cell and tissue levels. Gene delivery has been successfully performed ex vivo and in vivo in these applications. Excellent proliferation capabilities and differentiation potentials render certain cells as excellent candidates for ex vivo gene delivery for regenerative medicine applications, which is why multipotent and pluripotent cells have been intensely studied in this vein. In this review, gene delivery is discussed in detail, along with its applications to tissue engineering and regenerative medicine. A definition of a stem cell is compared to a definition of a stem property, and both provide the foundation for an in-depth look at gene delivery investigations from a germ lineage angle. © 2014 Wiley Periodicals, Inc.

  15. Polymeric Nanoparticle-Mediated Gene Delivery for Lung Cancer Treatment.

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    Amreddy, Narsireddy; Babu, Anish; Muralidharan, Ranganayaki; Munshi, Anupama; Ramesh, Rajagopal

    2017-04-01

    In recent years, researchers have focused on targeted gene therapy for lung cancer, using nanoparticle carriers to overcome the limitations of conventional treatment methods. The main goal of targeted gene therapy is to develop more efficient therapeutic strategies by improving the bioavailability, stability, and target specificity of gene therapeutics and to reduce off-target effects. Polymer-based nanoparticles, an alternative to lipid and inorganic nanoparticles, efficiently carry nucleic acid therapeutics and are stable in vivo. Receptor-targeted delivery is a promising approach that can limit non-specific gene delivery and can be achieved by modifying the polymer nanoparticle surface with specific receptor ligands or antibodies. This review highlights the recent developments in gene delivery using synthetic and natural polymer-based nucleic acid carriers for lung cancer treatment. Various nanoparticle systems based on polymers and polymer combinations are discussed. Further, examples of targeting ligands or moieties used in targeted, polymer-based gene delivery to lung cancer are reviewed.

  16. Will Nanotechnology Bring New Hope for Gene Delivery?

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    Wong, Joanna K L; Mohseni, Rashin; Hamidieh, Amir Ali; MacLaren, Robert E; Habib, Nagy; Seifalian, Alexander M

    2017-05-01

    The high mortality rates of cancer patients receiving standard treatments emphasize the crucial need for alternative treatments. One promising strategy is to use organic nanoparticles (NPs) for gene delivery. Although multifunctional NPs theoretically have many desirable properties as gene vectors, there are several practical barriers to successful gene delivery. In this review we discuss the properties of NPs and overview in vitro and preclinical studies of organic NPs over the past 5 years. The results of recent clinical trials suggest that the NP field holds significant promise. The annual market value for successful gene delivery could exceed US$30 billion, and this will encourage researchers to study the application of NPs to therapeutic gene delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. In vivo gene delivery by cationic tetraamino fullerene.

    Science.gov (United States)

    Maeda-Mamiya, Rui; Noiri, Eisei; Isobe, Hiroyuki; Nakanishi, Waka; Okamoto, Koji; Doi, Kent; Sugaya, Takeshi; Izumi, Tetsuro; Homma, Tatsuya; Nakamura, Eiichi

    2010-03-23

    Application of nanotechnology to medical biology has brought remarkable success. Water-soluble fullerenes are molecules with great potential for biological use because they can endow unique characteristics of amphipathic property and form a self-assembled structure by chemical modification. Effective gene delivery in vitro with tetra(piperazino)fullerene epoxide (TPFE) and its superiority to Lipofectin have been described in a previous report. For this study, we evaluated the efficacy of in vivo gene delivery by TPFE. Delivery of enhanced green fluorescent protein gene (EGFP) by TPFE on pregnant female ICR mice showed distinct organ selectivity compared with Lipofectin; moreover, higher gene expression by TPFE was found in liver and spleen, but not in the lung. No acute toxicity of TPFE was found for the liver and kidney, although Lipofectin significantly increased liver enzymes and blood urea nitrogen. In fetal tissues, neither TPFE nor Lipofectin induced EGFP gene expression. Delivery of insulin 2 gene to female C57/BL6 mice increased plasma insulin levels and reduced blood glucose concentrations, indicating the potential of TPFE-based gene delivery for clinical application. In conclusion, this study demonstrated effective gene delivery in vivo for the first time using a water-soluble fullerene.

  18. A new electrospray method for targeted gene delivery.

    Science.gov (United States)

    Boehringer, Stephan; Ruzgys, Paulius; Tamò, Luca; Šatkauskas, Saulius; Geiser, Thomas; Gazdhar, Amiq; Hradetzky, David

    2018-03-05

    A challenge for gene therapy is absence of safe and efficient local delivery of therapeutic genetic material. An efficient and reproducible physical method of electrospray for localized and targeted gene delivery is presented. Electrospray works on the principle of coulombs repulsion, under influence of electric field the liquid carrying genetic material is dispersed into micro droplets and is accelerated towards the targeted tissue, acting as a counter electrode. The accelerated droplets penetrate the targeted cells thus facilitating the transfer of genetic material into the cell. The work described here presents the principle of electrospray for gene delivery, the basic instrument design, and the various optimized parameters to enhance gene transfer in vitro. We estimate a transfection efficiency of up to 60% was achieved. We describe an efficient gene transfer method and a potential electrospray-mediated gene transfer mechanism.

  19. Effects of AC/DC magnetic fields, frequency, and nanoparticle aspect ratio on cellular transfection of gene vectors

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    Ford, Kris; Mair, Lamar; Fisher, Mike; Rowshon Alam, Md.; Juliano, Rudolph; Superfine, Richard

    2008-10-01

    In order to make non-viral gene delivery a useful tool in the study and treatment of genetic disorders, it is imperative that these methodologies be further refined to yield optimal results. Transfection of magnetic nanoparticles and nanorods are used as non-viral gene vectors to transfect HeLa EGFP-654 cells that stably express a mutated enhanced green fluorescent protein (EGFP) gene. We deliver antisense oligonucleotides to these cells designed to correct the aberrant splicing caused by the mutation in the EGFP gene. We also transfect human bronchial endothelial cells and immortalized WI-38 lung cells with pEGFP-N1 vectors. To achieve this we bind the genes to magnetic nanoparticles and nanorods and introduce magnetic fields to effect transfection. We wish to examine the effects of magnetic fields on the transfection of these particles and the benefits of using alternating (AC) magnetic fields in improving transfection rates over direct (DC) magnetic fields. We specifically look at the frequency dependence of the AC field and particle aspect ratio as it pertains to influencing transfection rate. We posit that the increase in angular momentum brought about by the AC field and the high aspect ratio of the nanorod particles, is vital to generating the force needed to move the particle through the cell membrane.

  20. A novel DC therapy with manipulation of MKK6 gene on nickel allergy in mice.

    Directory of Open Access Journals (Sweden)

    Megumi Watanabe

    Full Text Available BACKGROUND: Although the activation of dermal dendritic cells (DCs or Langerhans cells (LCs via p38 mitogen-activated protein kinase (MAPK plays a crucial role in the pathogenesis of metal allergy, the in vivo molecular mechanisms have not been identified and a possible therapeutic strategy using the control of dermal DCs or LCs has not been established. In this study, we focused on dermal DCs to define the in vivo mechanisms of metal allergy pathogenesis in a mouse nickel (Ni allergy model. The effects of DC therapy on Ni allergic responses were also investigated. METHODS AND FINDING: The activation of dermal DCs via p38 MAPK triggered a T cell-mediated allergic immune response in this model. In the MAPK signaling cascade in DCs, Ni potently phosphorylated MAP kinase kinase 6 (MKK6 following increased DC activation. Ni-stimulated DCs could prime T cell activation to induce Ni allergy. Interestingly, when MKK6 gene-transfected DCs were transferred into the model mice, a more pronounced allergic reaction was observed. In addition, injection of short interfering (si RNA targeting the MKK6 gene protected against a hypersensitivity reaction after Ni immunization. The cooperative action between T cell activation and MKK6-mediated DC activation by Ni played an important role in the development of Ni allergy. CONCLUSIONS: DC activation by Ni played an important role in the development of Ni allergy. Manipulating the MKK6 gene in DCs may be a good therapeutic strategy for dermal Ni allergy.

  1. A novel DC therapy with manipulation of MKK6 gene on nickel allergy in mice.

    Science.gov (United States)

    Watanabe, Megumi; Ishimaru, Naozumi; Ashrin, Meinar Nur; Arakaki, Rieko; Yamada, Akiko; Ichikawa, Tetsuo; Hayashi, Yoshio

    2011-04-22

    Although the activation of dermal dendritic cells (DCs) or Langerhans cells (LCs) via p38 mitogen-activated protein kinase (MAPK) plays a crucial role in the pathogenesis of metal allergy, the in vivo molecular mechanisms have not been identified and a possible therapeutic strategy using the control of dermal DCs or LCs has not been established. In this study, we focused on dermal DCs to define the in vivo mechanisms of metal allergy pathogenesis in a mouse nickel (Ni) allergy model. The effects of DC therapy on Ni allergic responses were also investigated. The activation of dermal DCs via p38 MAPK triggered a T cell-mediated allergic immune response in this model. In the MAPK signaling cascade in DCs, Ni potently phosphorylated MAP kinase kinase 6 (MKK6) following increased DC activation. Ni-stimulated DCs could prime T cell activation to induce Ni allergy. Interestingly, when MKK6 gene-transfected DCs were transferred into the model mice, a more pronounced allergic reaction was observed. In addition, injection of short interfering (si) RNA targeting the MKK6 gene protected against a hypersensitivity reaction after Ni immunization. The cooperative action between T cell activation and MKK6-mediated DC activation by Ni played an important role in the development of Ni allergy. DC activation by Ni played an important role in the development of Ni allergy. Manipulating the MKK6 gene in DCs may be a good therapeutic strategy for dermal Ni allergy.

  2. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

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    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  3. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery.

    Science.gov (United States)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-30

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds' escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds' cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  4. Topical Non-Invasive Gene Delivery using Gemini Nanoparticles in Interferon-gamma-deficient Mice

    Energy Technology Data Exchange (ETDEWEB)

    Badea,I.; Wettig, S.; Verrall, R.; Foldvari, M.

    2007-01-01

    Cutaneous gene therapy, although a promising approach for many dermatologic diseases, has not progressed to the stage of clinical trials, mainly due to the lack of an effective gene delivery system. The main objective of this study was to construct and evaluate gemini nanoparticles as a topical formulation for the interferon gamma (IFN-{gamma}) gene in an IFN-{gamma}-deficient mouse model. Nanoparticles based on the gemini surfactant 16-3-16 (NP16-DNA) and another cationic lipid cholesteryl 3{beta}-(-N-[dimethylamino-ethyl] carbamate) [Dc-chol] (NPDc-DNA) were prepared and characterized. Zetasizer measurement indicated a bimodal distribution of 146 and 468 nm average particle sizes for the NP16-DNA ({zeta}-potential +51 mV) nanoparticles and monomodal distribution of 625 nm ({zeta}-potential +44 mV) for the NPDc-DNA. Circular dichroism studies showed that the gemini surfactant compacted the plasmid more efficiently compared to the Dc-chol. Small-angle X-ray scattering measurements revealed structural polymorphism in the NP16-DNA nanoparticles, with lamellar and Fd3m cubic phases present, while for the NPDc-DNA two lamellar phases could be distinguished. In vivo, both topically applied nanoparticles induced higher gene expression compared to untreated control and naked DNA (means of 0.480 and 0.398 ng/cm{sup 2} vs 0.067 and 0.167 ng/cm{sup 2}). However, treatment with NPDc-DNA caused skin irritation, and skin damage, whereas NP16-DNA showed no skin toxicity. In this study, we demonstrated that topical cutaneous gene delivery using gemini surfactant-based nanoparticles in IFN-{gamma}-deficient mice was safe and may provide increased gene expression in the skin due to structural complexity of NP16 nanoparticles (lamellar-cubic phases).

  5. Gene delivery with viral vectors for cerebrovascular diseases

    Science.gov (United States)

    Gan, Yu; Jing, Zheng; Stetler, R. Anne; Cao, Guodong

    2017-01-01

    Recent achievements in the understanding of molecular events involved in the pathogenesis of central nervous system (CNS) injury have made gene transfer a promising approach for various neurological disorders, including cerebrovascular diseases. However, special obstacles, including the post-mitotic nature of neurons and the blood-brain barrier (BBB), constitute key challenges for gene delivery to the CNS. Despite the various limitations in current gene delivery systems, a spectrum of viral vectors has been successfully used to deliver genes to the CNS. Furthermore, recent advancements in vector engineering have improved the safety and delivery of viral vectors. Numerous viral vector-based clinical trials for neurological disorders have been initiated. This review will summarize the current implementation of viral gene delivery in the context of cerebrovascular diseases including ischemic stroke, hemorrhagic stroke and subarachnoid hemorrhage (SAH). In particular, we will discuss the potentially feasible ways in which viral vectors can be manipulated and exploited for use in neural delivery and therapy. PMID:23276981

  6. Gene Delivery for Metastatic Prostate Cancer Cells

    National Research Council Canada - National Science Library

    Pang, Shen

    2001-01-01

    .... Enhanced by the bystander effect, the specific expression of the DTA gene causes significant cell death in prostate cancer cell cultures, with very low background cell eradication in control cell lines...

  7. Engineered nonviral nanocarriers for intracellular gene delivery applications

    International Nuclear Information System (INIS)

    Ojea-Jiménez, Isaac; Puntes, Victor F; Tort, Olivia; Lorenzo, Julia

    2012-01-01

    The efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biology and of medical applications, including cancer therapy and tissue engineering. Non-viral chemical methods have been received with great interest for transfecting cells. However, further development of nanocarriers that are biocompatible, efficient and suitable for clinical applications is still required. In this paper, the different material platforms for gene delivery are comparatively addressed, and the mechanisms of interaction with biological systems are discussed carefully. (paper)

  8. Selenium nanoparticles: potential in cancer gene and drug delivery.

    Science.gov (United States)

    Maiyo, Fiona; Singh, Moganavelli

    2017-05-01

    In recent decades, colloidal selenium nanoparticles have emerged as exceptional selenium species with reported chemopreventative and therapeutic properties. This has sparked widespread interest in their use as a carrier of therapeutic agents with results displaying synergistic effects of selenium with its therapeutic cargo and improved anticancer activity. Functionalization remains a critical step in selenium nanoparticles' development for application in gene or drug delivery. In this review, we highlight recent developments in the synthesis and functionalization strategies of selenium nanoparticles used in cancer drug and gene delivery systems. We also provide an update of recent preclinical studies utilizing selenium nanoparticles in cancer therapeutics.

  9. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  10. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

    Energy Technology Data Exchange (ETDEWEB)

    Fillat, Cristina, E-mail: cristina.fillat@crg.es; Jose, Anabel; Ros, Xavier Bofill-De; Mato-Berciano, Ana; Maliandi, Maria Victoria; Sobrevals, Luciano [Programa Gens i Malaltia, Centre de Regulació Genòmica-CRG, UPF, Parc de Recerca Biomedica de Barcelona-PRBB and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona (Spain)

    2011-01-18

    The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

  11. Physical non-viral gene delivery methods for tissue engineering.

    Science.gov (United States)

    Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

    2013-03-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

  12. Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers

    Science.gov (United States)

    Zhou, Qiu-Lan; Chen, Zhi-Yi; Yang, Feng

    2014-01-01

    With the development of nanotechnology, nanocarriers have been increasingly used for curative drug/gene delivery. Various nanocarriers are being introduced and assessed, such as polymer nanoparticles, liposomes, and micelles. As a novel theranostic system, nanocarriers hold great promise for ultrasound molecular imaging, targeted drug/gene delivery, and therapy. Nanocarriers, with the properties of smaller particle size, and long circulation time, would be advantageous in diagnostic and therapeutic applications. Nanocarriers can pass through blood capillary walls and cell membrane walls to deliver drugs. The mechanisms of interaction between ultrasound and nanocarriers are not clearly understood, which may be related to cavitation, mechanical effects, thermal effects, and so forth. These effects may induce transient membrane permeabilization (sonoporation) on a single cell level, cell death, and disruption of tissue structure, ensuring noninvasive, targeted, and efficient drug/gene delivery and therapy. The system has been used in various tissues and organs (in vitro or in vivo), including tumor tissues, kidney, cardiac, skeletal muscle, and vascular smooth muscle. In this review, we explore the research progress and application of ultrasound-mediated local drug/gene delivery with nanocarriers. PMID:25202710

  13. Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers

    Directory of Open Access Journals (Sweden)

    Qiu-Lan Zhou

    2014-01-01

    Full Text Available With the development of nanotechnology, nanocarriers have been increasingly used for curative drug/gene delivery. Various nanocarriers are being introduced and assessed, such as polymer nanoparticles, liposomes, and micelles. As a novel theranostic system, nanocarriers hold great promise for ultrasound molecular imaging, targeted drug/gene delivery, and therapy. Nanocarriers, with the properties of smaller particle size, and long circulation time, would be advantageous in diagnostic and therapeutic applications. Nanocarriers can pass through blood capillary walls and cell membrane walls to deliver drugs. The mechanisms of interaction between ultrasound and nanocarriers are not clearly understood, which may be related to cavitation, mechanical effects, thermal effects, and so forth. These effects may induce transient membrane permeabilization (sonoporation on a single cell level, cell death, and disruption of tissue structure, ensuring noninvasive, targeted, and efficient drug/gene delivery and therapy. The system has been used in various tissues and organs (in vitro or in vivo, including tumor tissues, kidney, cardiac, skeletal muscle, and vascular smooth muscle. In this review, we explore the research progress and application of ultrasound-mediated local drug/gene delivery with nanocarriers.

  14. Delivery of gene-expressing fragments using quantum dot

    Science.gov (United States)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Hanada, Sanshiro; Fujioka, Kouki; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2009-02-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes do. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced the gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/geneconstruct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as 1) the reading direction of gene fragments, 2) the quantity of gene fragments attached on the surface of QD-constructs, 3) the surface electronic charges varied according to the structure of QD/gene-constructs, and 4) the particle size of QD/gene complex varied according to the structure and amounts of gene fragments. Using this QD/geneconstruct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs was disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes. These results suggest that inappropriate treatment and disposal of QDs may still have risks to the environmental pollution including human health under certain conditions. Here we propose the further research for the immune and physiological responses in not only immune cells but also other cells, in order to clear the effect of all other nanoscale products as well as nanocrystal

  15. Gene therapy for meningioma: improved gene delivery with targeted adenoviruses

    NARCIS (Netherlands)

    Dirven, Clemens M. F.; Grill, Jacques; Lamfers, Martine L. M.; van der Valk, Paul; Leonhart, Angelique M.; van Beusechem, Victor W.; Haisma, Hidde J.; Pinedo, Herbert M.; Curiel, David T.; Vandertop, W. Peter; Gerritsen, Winald R.

    2002-01-01

    OBJECT: Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene

  16. Gene therapy for meningioma : improved gene delivery with targeted adenoviruses

    NARCIS (Netherlands)

    Dirven, CMF; Grill, J; Lamfers, MLM; Van der Valk, P; Leonhart, AM; Van Beusechem, VW; Haisma, HJ; Pinedo, HM; Curiel, DT; Vandertop, WP; Gerritsen, WR

    Object. Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene

  17. Wild Carrot Differentiation in Europe and Selection at DcAOX1 Gene?

    Directory of Open Access Journals (Sweden)

    Tânia Nobre

    Full Text Available By definition, the domestication process leads to an overall reduction of crop genetic diversity. This lead to the current search of genomic regions in wild crop relatives (CWR, an important task for modern carrot breeding. Nowadays massive sequencing possibilities can allow for discovery of novel genetic resources in wild populations, but this quest could be aided by the use of a surrogate gene (to first identify and prioritize novel wild populations for increased sequencing effort. Alternative oxidase (AOX gene family seems to be linked to all kinds of abiotic and biotic stress reactions in various organisms and thus have the potential to be used in the identification of CWR hotspots of environment-adapted diversity. High variability of DcAOX1 was found in populations of wild carrot sampled across a West-European environmental gradient. Even though no direct relation was found with the analyzed climatic conditions or with physical distance, population differentiation exists and results mainly from the polymorphisms associated with DcAOX1 exon 1 and intron 1. The relatively high number of amino acid changes and the identification of several unusually variable positions (through a likelihood ratio test, suggests that DcAOX1 gene might be under positive selection. However, if positive selection is considered, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations given the observed high genetic diversity. We were able to identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity.

  18. Application of Ferriferous Oxide Modified by Chitosan in Gene Delivery

    Directory of Open Access Journals (Sweden)

    Yu Kuang

    2012-01-01

    Full Text Available New approaches to improve the traditional gene carriers are still required. Here we explore Fe3O4 modified with degradable polymers that enhances gene delivery and target delivery using permanent magnetic field. Two magnetic Fe3O4 nanoparticles coated with chitosan (CTS and polyethylene glycol (PEG were synthesized by means of controlled chemical coprecipitation. Plasmid pEGFP was encapsulated as a reported gene. The ferriferous oxide complexes were approximately spherical; surface charge of CTS-Fe3O4 and PEG-Fe3O4 was about 20 mv and 0 mv, respectively. The controlled release of DNA from the CTS-Fe3O4 nanoparticles was observed. Concurrently, a desired Fe3O4 concentration of less than 2 mM was verified as safe by means of a cytotoxicity test in vitro. Presence of the permanent magnetic field significantly increased the transfection efficiency. Furthermore, the passive target property and safety of magnetic nanoparticles were also demonstrated in an in vivo test. The novel gene delivery system was proved to be an effective tool required for future target expression and gene therapy in vivo.

  19. Theory and in vivo application of electroporative gene delivery.

    Science.gov (United States)

    Somiari, S; Glasspool-Malone, J; Drabick, J J; Gilbert, R A; Heller, R; Jaroszeski, M J; Malone, R W

    2000-09-01

    Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

  20. Overexpression of a New Chitinase Gene EuCHIT2 Enhances Resistance to Erysiphe cichoracearum DC in Tobacco Plants.

    Science.gov (United States)

    Dong, Xuan; Zhao, Yichen; Ran, Xin; Guo, Linxia; Zhao, De-Gang

    2017-11-07

    In this study, we cloned a new chitinase gene, EuCHIT2 , from Eucommia ulmoides Oliver ( E. ulmoides ) using rapid amplification of cDNA ends (RACE) technology and constructed an overexpression vector, pSH-35S- EuCHIT 2, to introduce it into tobacco ( Nicotiana tabacum cv. Xanthi) . Resistance to Erysiphe cichoracearum de Candolle (E.cichoracearum DC) and molecular mechanisms in the transgenic tobacco were determined by drop inoculation, spore counting, determination of physicochemical indicators, and analysis of gene expression. The chitinase activity and resistance to E. cichoracearum DC were significantly higher in the transgenic tobacco than in wild-type tobacco (p cichoracearum DC, were higher in the transgenic tobacco than in the wild-type. Conversely, the malondialdehyde (MDA) content was significantly lower in the transgenic tobacco than the wild-type before and after inoculation. In addition, our study also indicated that the resistance to E. cichoracearum DC might involve the salicylic acid (SA) and jasmonic acid (JA) pathways, because the expression levels of pathogenesis-related gene 1 ( PR-1a ) and coronatine-insensitive 1 ( COI1 ) were significantly increased and decreased, respectively, after inoculation with E. cichoracearum DC. The present study supports the notion that PR-1a and POD participate in resistance to E. cichoracearum DC in the transgenic tobacco plants.

  1. Overexpression of a New Chitinase Gene EuCHIT2 Enhances Resistance to Erysiphe cichoracearum DC. in Tobacco Plants

    Directory of Open Access Journals (Sweden)

    Xuan Dong

    2017-11-01

    Full Text Available In this study, we cloned a new chitinase gene, EuCHIT2, from Eucommia ulmoides Oliver (E. ulmoides using rapid amplification of cDNA ends (RACE technology and constructed an overexpression vector, pSH-35S-EuCHIT2, to introduce it into tobacco (Nicotiana tabacum cv. Xanthi. Resistance to Erysiphe cichoracearum de Candolle (E.cichoracearum DC and molecular mechanisms in the transgenic tobacco were determined by drop inoculation, spore counting, determination of physicochemical indicators, and analysis of gene expression. The chitinase activity and resistance to E. cichoracearum DC were significantly higher in the transgenic tobacco than in wild-type tobacco (p < 0.05. The activities of peroxidase (POD and catalase (CAT, after inoculation with E. cichoracearum DC, were higher in the transgenic tobacco than in the wild-type. Conversely, the malondialdehyde (MDA content was significantly lower in the transgenic tobacco than the wild-type before and after inoculation. In addition, our study also indicated that the resistance to E. cichoracearum DC might involve the salicylic acid (SA and jasmonic acid (JA pathways, because the expression levels of pathogenesis-related gene 1 (PR-1a and coronatine-insensitive 1 (COI1 were significantly increased and decreased, respectively, after inoculation with E. cichoracearum DC. The present study supports the notion that PR-1a and POD participate in resistance to E. cichoracearum DC in the transgenic tobacco plants.

  2. Layered double hydroxide nanoparticles in gene and drug delivery.

    Science.gov (United States)

    Ladewig, Katharina; Xu, Zhi Ping; Lu, Gao Qing Max

    2009-09-01

    Layered double hydroxides (LDHs) have been known for many decades as catalyst and ceramic precursors, traps for anionic pollutants, catalysts and additives for polymers, but their successful synthesis on the nanometer scale a few years ago opened up a whole new field for their application in nanomedicine. The delivery of drugs and other therapeutic/bioactive molecules (e.g., peptides, proteins, nucleic acids) to mammalian cells is an area of research that is of tremendous importance to medicine and provides manifold applications for any new developments in the area of nanotechnology. Among the many different nanoparticles that have been shown to facilitate gene and/or drug delivery, LDH nanoparticles have attracted particular attention owing to their many desirable properties. This review aims to report recent progress in gene and drug delivery using LDH nanoparticles. It summarizes the advantages and disadvantages of using LDH nanoparticles as carriers for nucleic acids and drugs against the general background of bottlenecks that are encountered by cellular delivery systems. It describes further the models that have been proposed for the internalization of LDH nanoparticles into cells so far and discusses the intracellular fate of the particles and their cargo. The authors offer some remarks on how this field of research will progress in the near future and which challenges need to be overcome before LDH nanoparticles can be used in a clinical setting.

  3. A variable gene delivery carrier-biotinylated chitosan/polyethyleneimine

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Yi-Chen; Young, Tai-Horng [Institute of Polymer Science and Engineering, College of Engineering, National Taiwan University, Taipei 106, Taiwan (China); Chang, Fu-Hsiung [Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Wei, Ming-Feng, E-mail: thyoung@ntu.edu.t [Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan (China)

    2010-12-15

    A variable gene delivery system has been developed based on conjugating chitosan to biotin through a functionalized poly(ethylene glycol) (PEG) spacer, which can be used to further bind different molecules on the outer layer of a polymer/DNA complex by streptavidin (SA)-biotin linkage. In this study, TAT-conjugated SA was used as the model molecule to prove the conjugation function of the prepared complex. In addition, low-molecular-weight poly(ethyleneimine) (PEI) was added into the polymer/DNA complex to increase the transfection efficiency. The results of the luciferase assay show that the transfection efficiency of the prepared complex was significantly correlated with the amount of PEI and was further enhanced when TAT was conjugated to the complex by SA-biotin linkage. Considered to have negligible cytotoxic effects, the variable gene delivery complex prepared in this study would be of considerable potential as carriers for in vitro applications.

  4. Membrane-Mimic Nanoparticles for Drug and Gene Delivery

    KAUST Repository

    Alamoudi, Kholod

    2017-12-01

    Nanoscale organic particles have gained a prominent role in drug and gene delivery field. As the nature of the nanoparticle’s (NPs) surface plays a major role in their targeting efficiency, bioavailability, and cytotoxicity, membrane-mimic nanoparticles are considered highly attractive materials for in vivo and in vitro applications. Synthetic membrane vesicles (liposomes) and nanoconstructs built with native cancer cellular membrane are excellent scaffolds to improve cellular delivery. Liposomes have been extensively used due to their high loading capacity, biocompatibility and biodegradability. However, modifications with stimuli responsive materials are highly needed to improve their stability and turn them active participants in controlled delivery. Towards a nature inspired approach, reconstructed bilayers from cell membrane are a good candidate to enhance NP’s targeting ability and biocompatibility. The primary focus of this research is to develop smart responsive (lipid) membrane coated NPs with surface modifications for controlled and targeted drug and/or gene delivery for application in cancer therapy. Three approaches have been developed, namely i) liposomes as thermoresponsive nanocarriers for the delivery of genetic material; ii) magnetically photosensitive liposome hybrids and iii) biomimetic periodic mesoporous organo silica engineered for better a biocompatibility and targeting capabilities. In the first project synthetic liposomes were loaded with ammonium bicarbonate salt (ABC) and siRNA. The combination of lipids chosen and the relative ratios allowed the rapid release of the genetic material to the multi drug resistant cancer cells studied, upon external heat trigger. This design has improved the gene silencing efficiency via successful endosomal escape. In the second project, SPIO@Au nanoparticles were imbedded in the lipid bilayer to produce a photo/thermal responsive carrier that could be also used in cell imaging besides gene transfection

  5. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

    Directory of Open Access Journals (Sweden)

    Maria Victoria Maliandi

    2011-01-01

    Full Text Available The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

  6. Mannan-Modified PLGA Nanoparticles for Targeted Gene Delivery

    Directory of Open Access Journals (Sweden)

    Fansheng Kong

    2012-01-01

    Full Text Available The studies of targeted gene delivery nanocarriers have gained increasing attention during the past decades. In this study, mannan modified DNA loaded bioadhesive PLGA nanoparticles (MAN-DNA-NPs were investigated for targeted gene delivery to the Kupffer cells (KCs. Bioadhesive PLGA nanoparticles were prepared and subsequently bound with pEGFP. Following the coupling of the mannan-based PE-grafted ligands (MAN-PE with the DNA-NPs, the MAN-DNA-NPs were delivered intravenously to rats. The transfection efficiency was determined from the isolated KCs and flow cytometry was applied for the quantitation of gene expression after 48 h post transfection. The size of the MAN-DNA-NPs was found to be around 190 nm and the Zeta potential was determined to be −15.46mV. The pEGFP binding capacity of MAN-DNA-NPs was (88.9±5.8% and the in vitro release profiles of the MAN-DNA-NPs follow the Higuchi model. When compared with non-modified DNA-NPs and Lipofectamine 2000-DNA, MAN-DNA-NPs produced the highest gene expressions, especially in vivo. The in vivo data from flow cytometry analysis showed that MAN-DNA-NPs displayed a remarkably higher transfection efficiency (39% than non-modified DNA-NPs (25% and Lipofectamine 2000-DNA (23% in KCs. The results illustrate that MAN-DNA-NPs have the ability to target liver KCs and could function as promising active targeting drug delivery vectors.

  7. Molecular Cloning and Expression Analysis of Cu/Zn SOD Gene from Gynura bicolor DC.

    Directory of Open Access Journals (Sweden)

    Xin Xu

    2017-01-01

    Full Text Available Superoxide dismutase is an important antioxidant enzyme extensively existing in eukaryote, which scavenges reactive oxygen species (ROS and plays an essential role in stress tolerance of higher plants. A full-length cDNA encoding Cu/Zn SOD was cloned from leaves of Gynura bicolor DC. by rapid amplification of cDNA ends (RACE. The full-length cDNA of Cu/Zn SOD is 924 bp and has a 681 bp open reading frame encoding 227 amino acids. Bioinformatics analysis revealed that belonged to the plant SOD super family. Cu/Zn SODs of the Helianthus annuus, Mikania micrantha, and Solidago canadensis var. scabra all have 86% similarity to the G. bicolor Cu/Zn SOD. Analysis of the expression of Cu/Zn SOD under different treatments revealed that Cu/Zn SOD was a stress-responsive gene, especially to 1-MCP. It indicates that the Cu/Zn SOD gene would be an important gene in the resistance to stresses and will be helpful in providing evidence for future research on underlying molecular mechanism and choosing proper postharvest treatments for G. bicolor.

  8. Hydrogels: a journey from diapers to gene delivery.

    Science.gov (United States)

    Chawla, Pooja; Srivastava, Alok Ranjan; Pandey, Priyanka; Chawla, Viney

    2014-02-01

    Hydrogels are the biomaterials comprising network of natural or synthetic polymers capable of absorbing large amount of water. Hydrogels are "Smart Gels" or "Intelligent Gels" which can be made to respond to the various environmental conditions like temperature, pH, magnetic/electric field, ionic strength, inflammation, external stress etc. There are numerous potential applications of hydrogels in modern day life ranging from a diaper to gene delivery. This review succinctly describes the classification, properties and preparation methods along with numerous diverse applications of hydrogels like agricultural hydrogels, hydrogel for drug delivery, sensing, dental adhesives, wound healing and tissue regeneration, diet aid and gastric retention and in tissue engineering etc. Hydrogels can be regarded as highly valuable biomaterials for human-beings.

  9. Long circulating polymeric nanoparticles for gene/drug delivery.

    Science.gov (United States)

    Hu, Jiaming; Sheng, Yan; Shi, Junfeng; Yu, Bohao; Yu, Zhiqiang; Liao, Guochao

    2017-12-07

    The major limitation in the improving polymeric nanoparticles into an efficient gene/drug delivery carrier is the rapid opsonization, phagocytic uptake by mononuclear phagocyte system and subsequent clearance from the bloodstream. The prolonged circulation time of nanoparticles in the blood is a prerequisite to realizing a controlled and targeted (passive or active targeting) release of the encapsulated gene/drug at the desired site of action. In this review, the factors such as biological barriers and physical barriers including particle size, shape, zeta potential, and hydrophilicity will be discussed, which can cause effects on blood clearance and organ accumulation. Some natural and synthetic polymers utilized in long-circulating nanoparticles will also be discussed. The most popular method to mask or camouflage nanoparticles is the adsorbed, grafted or conjugated of poly (ethylene glycol) (PEG) or other hydrophilic polymers (e.g. polysaccharides) to the particle surface. Surface modification of nanoparticles with these polymers results in an increased blood circulation time by several orders of magnitude in comparison to the bare nanoparticles. However, the circulation half-life of nanoparticles still cannot satisfy the need for clinical use. At present, identification of novel potential coating materials is an emerging field of interest in the design of long-circulating polymer-based nanoparticulate gene/drug delivery. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Biolistic DNA delivery to mice with the low pressure gene gun.

    Science.gov (United States)

    Yen, Meng-Chi; Lai, Ming-Derg

    2013-01-01

    Biolistic DNA delivery is an approach to deliver plasmid to culture cells, plants, or animals. Plasmid DNA is usually transferred through bombardment of DNA-coated particles by highly pressurized gas in various kinds of delivery vehicles. The low pressure gene gun can deliver plasmid at lower pressure. Here, we describe methods of biolistic DNA delivery to mice using the low pressure gene gun.

  11. Efficient gene delivery using chitosan-polyethylenimine hybrid systems

    International Nuclear Information System (INIS)

    Jiang, Hu-Lin; Kim, Tae-Hee; Kim, You-Kyoung; Park, In-Young; Cho, Chong-Su; Cho, Myung-Haing

    2008-01-01

    Chitosan and chitosan derivatives have been investigated as non-viral vectors because they have several advantages, such as biocompatibility, biodegradability, low cytotoxicity and low immunogenicity. However, low transfection efficiency and low cell specificity must be solved for their use in clinical trials. In this paper, chitosan-polyethylenimine (PEI) hybrid systems such as chitosan/PEI blend and chitosan-graft-PEI are described for efficient gene delivery because the PEI has high transfection efficiency owing to a proton sponge effect and chitosan has biocompatibility. Also, hepatocyte specificity of the galactosylated chitosan is explained after combination with PEI

  12. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    Science.gov (United States)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  13. Human gene therapy: novel approaches to improve the current gene delivery systems.

    Science.gov (United States)

    Cucchiarini, Magali

    2016-06-01

    Even though gene therapy made its way through the clinics to treat a number of human pathologies since the early years of experimental research and despite the recent approval of the first gene-based product (Glybera) in Europe, the safe and effective use of gene transfer vectors remains a challenge in human gene therapy due to the existence of barriers in the host organism. While work is under active investigation to improve the gene transfer systems themselves, the use of controlled release approaches may offer alternative, convenient tools of vector delivery to achieve a performant gene transfer in vivo while overcoming the various physiological barriers that preclude its wide use in patients. This article provides an overview of the most significant contributions showing how the principles of controlled release strategies may be adapted for human gene therapy.

  14. Approaches to gene pool conservation of medicinal plant Oxytropis lanata (Pall. DC. (Fabaceae

    Directory of Open Access Journals (Sweden)

    A. B. Kholina

    2015-05-01

    Full Text Available In order to preserve the gene pool of medicinal plant Oxytropis lanata (Pall. DC. we analyzed allozyme polymorphism and identified reliable and informative marker enzyme systems of this species; also we studied the response of seeds to deep freezing in liquid nitrogen (–196 ºС. Population has an average level of polymorphism (P95 = 41,2 %, P99 = 52,9 %, A = 1,58, Ho = 0,158, He = 0,171 in general typical for herbaceous legumes, and can serve as a source of material for gene pool conservation of the species. Deep freezing has not led to the death of the seeds; it was marked stimulatory effect of ultralow temperatures, expressed as an acceleration of germination and sharp increase of germinability (98,6 ± 2,3 % compared to the control (12,0 ± 3,5 % that is associated with overcoming physical dormancy. There were no abnormalities in the development of seedlings from seeds passed cryopreservation.

  15. Ideas on DC-DC Converters for Delivery of Low Voltage and High Currents for the SLHC / ILC Detector Electronics in Magnetic field and Radiation environments

    CERN Document Server

    Dhawan, Satish; Neal, H; Sumner, R; Weber, M; Weber, R

    2007-01-01

    For more efficient power transport to the electronics embedded inside large colliding beam detectors, we explore the feasibility of supplying 48 Volts DC and using local DCDC conversion to 2 V (or lower, depending upon on the lithography of the embedded electronics) using switch mode regulators located very close to the front end electronics. These devices will be exposed to high radiation and high magnetic fields, 10 – 100 Mrads and 2 - 4 Tesla at the SLHC, and 20 Krads and 6 Tesla at the ILC.

  16. Parallel synthesis and screening of polymers for nonviral gene delivery.

    Science.gov (United States)

    Barua, Sutapa; Joshi, Amit; Banerjee, Akhilesh; Matthews, Dana; Sharfstein, Susan T; Cramer, Steven M; Kane, Ravi S; Rege, Kaushal

    2009-01-01

    We describe the parallel synthesis and in vitro evaluation of a cationic polymer library for the discovery of nonviral gene delivery vectors. The library was synthesized based on the ring-opening polymerization reaction between epoxide groups of diglycidyl ethers and the amines of (poly)amines. Parallel screening of soluble library constituents led to the identification of lead polymers with high DNA-binding efficacies. Transfection efficacies of lead polymers were evaluated using PC3-PSMA human prostate cancer cells and murine osteoblasts in the absence and presence of serum. In vitro experiments resulted in the identification of a candidate polymer that demonstrated significantly higher transfection efficacies and lower cytotoxicities than poly(ethyleneimine) (pEI), the current standard for polymeric transfection agents. In addition, polymers that demonstrated moderately higher and comparable transfection efficacies with respect to pEI were also identified. Our results demonstrate that high-throughput synthesis and screening of polymers is a powerful approach for the identification of novel nonviral gene delivery agents.

  17. Self-Assembling Multifunctional Peptide Dimers for Gene Delivery Systems

    Directory of Open Access Journals (Sweden)

    Kitae Ryu

    2015-01-01

    Full Text Available Self-assembling multifunctional peptide was designed for gene delivery systems. The multifunctional peptide (MP consists of cellular penetrating peptide moiety (R8, matrix metalloproteinase-2 (MMP-2 specific sequence (GPLGV, pH-responsive moiety (H5, and hydrophobic moiety (palmitic acid (CR8GPLGVH5-Pal. MP was oxidized to form multifunctional peptide dimer (MPD by DMSO oxidation of thiols in terminal cysteine residues. MPD could condense pDNA successfully at a weight ratio of 5. MPD itself could self-assemble into submicron micelle particles via hydrophobic interaction, of which critical micelle concentration is about 0.01 mM. MPD showed concentration-dependent but low cytotoxicity in comparison with PEI25k. MPD polyplexes showed low transfection efficiency in HEK293 cells expressing low level of MMP-2 but high transfection efficiency in A549 and C2C12 cells expressing high level of MMP-2, meaning the enhanced transfection efficiency probably due to MMP-induced structural change of polyplexes. Bafilomycin A1-treated transfection results suggest that the transfection of MPD is mediated via endosomal escape by endosome buffering ability. These results show the potential of MPD for MMP-2 targeted gene delivery systems due to its multifunctionality.

  18. Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery

    Science.gov (United States)

    Huang, Xianzhang; Shen, Sujing; Zhang, Zhanfeng; Zhuang, Junhua

    2014-01-01

    The high transfection efficiency of polyethylenimine (PEI) makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP). We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR) spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase) I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site–enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin)-labeled small interfering ribonucleic acids (siRNAs) was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA) and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked) than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA) tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene delivery. PMID:25342902

  19. Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy.

    Science.gov (United States)

    Bakhtiar, Athirah; Sayyad, Mustak; Rosli, Rozita; Maruyama, Atsushi; Chowdhury, Ezharul H

    2014-01-01

    Conventional therapies for malignant cancer such as chemotherapy and radiotherapy are associated with poor survival rates owing to the development of cellular resistance to cancer drugs and the lack of targetability, resulting in unwanted adverse effects on healthy cells and necessitating the lowering of therapeutic dose with consequential lower efficacy of the treatment. Gene therapy employing different types of viral and non-viral carriers to transport gene(s) of interest and facilitating production of the desirable therapeutic protein(s) has tremendous prospects in cancer treatments due to the high-level of specificity in therapeutic action of the expressed protein(s) with diminished off-target effects, although cancer cell-specific delivery of transgene(s) still poses some challenges to be addressed. Depending on the potential therapeutic target genes, cancer gene therapy could be categorized into tumor suppressor gene replacement therapy, immune gene therapy and enzyme- or prodrug-based therapy. This review would shed light on the current progress of delivery of potentially therapeutic genes into various cancer cells in vitro and animal models utilizing a variety of viral and non-viral vectors.

  20. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

    Science.gov (United States)

    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Gene delivery to the lungs: pulmonary gene therapy for cystic fibrosis.

    Science.gov (United States)

    Villate-Beitia, Ilia; Zarate, Jon; Puras, Gustavo; Pedraz, José Luis

    2017-07-01

    Cystic fibrosis (CF) is a monogenic autosomal recessive disorder where the defective gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is well identified. Moreover, the respiratory tract can be targeted through noninvasive aerosolized formulations for inhalation. Therefore, gene therapy is considered a plausible strategy to address this disease. Conventional gene therapy strategies rely on the addition of a correct copy of the CFTR gene into affected cells in order to restore the channel activity. In recent years, genome correction strategies have emerged, such as zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases. These gene editing tools aim to repair the mutated gene at its original genomic locus with high specificity. Besides, the success of gene therapy critically depends on the nucleic acids carriers. To date, several clinical studies have been carried out to add corrected copies of the CFTR gene into target cells using viral and non-viral vectors, some of them with encouraging results. Regarding genome editing systems, preliminary in vitro studies have been performed in order to repair the CFTR gene. In this review, after briefly introducing the basis of CF, we discuss the up-to-date gene therapy strategies to address the disease. The review focuses on the main factors to take into consideration when developing gene delivery strategies, such as the design of vectors and plasmid DNA, in vitro/in vivo tests, translation to human use, administration methods, manufacturing conditions and regulatory issues.

  2. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, S.W.; Liu, G. [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); Hong, R.Y., E-mail: rhong@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, H.Z. [State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, Y.G., E-mail: ilguoliang@sohu.com [Department of radiology, the First Affiliated Hospital of Soochow University, Suzhou 215007 (China); Wei, D.G., E-mail: dougwei@deas.harvard.edu [Center for Nanoscale Systems, School of Engineering and Applied Science, Harvard University, 11 Oxford Street, Cambridge, MA 02139 (United States)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer PEI is ideal candidate polymer for the design of gene delivery systems. Black-Right-Pointing-Pointer PEI-CMD-MNPs exhibited a typical superparamagnetic behavior. Black-Right-Pointing-Pointer PEI-CMD-MNPs were well stable over the entire range of pH and NaCl concentration. Black-Right-Pointing-Pointer DNA-PEI-CMD-MNPs transfected cells by a magnet have higher transfection efficiency and gene expression efficiency. - Abstract: The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe{sub 3}O{sub 4} nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  3. Functionalized nanoparticles for AMF-induced gene and drug delivery

    Science.gov (United States)

    Biswas, Souvik

    The properties and broad applications of nano-magnetic colloids have generated much interest in recent years. Specially, Fe3O4 nanoparticles have attracted a great deal of attention since their magnetic properties can be used for hyperthermia treatment or drug targeting. For example, enhanced levels of intracellular gene delivery can be achieved using Fe3O4 nano-vectors in the presence of an external magnetic field, a process known as 'magnetofection'. The low cytotoxicity, tunable particle size, ease of surface functionalization, and ability to generate thermal energy using an external alternating magnetic field (AMF) are properties have propelled Fe3O4 research to the forefront of nanoparticle research. The strategy of nanoparticle-mediated, AMF-induced heat generation has been used to effect intracellular hyperthermia. One application of this 'magnetic hyperthermia' is heat activated local delivery of a therapeutic effector (e.g.; drug or polynucleotide). This thesis describes the development of a magnetic nano-vector for AMF-induced, heat-activated pDNA and small molecule delivery. The use of heat-inducible vectors, such as heat shock protein ( hsp) genes, is a promising mode of gene therapy that would restrict gene expression to a local region by focusing a heat stimulus only at a target region. We thus aimed to design an Fe3O4 nanoparticle-mediated gene transfer vehicle for AMF-induced localized gene expression. We opted to use 'click' oximation techniques to assemble the magnetic gene transfer vector. Chapter 2 describes the synthesis, characterization, and transfection studies of the oxime ether lipid-based nano-magnetic vectors MLP and dMLP. The synthesis and characterization of a novel series of quaternary ammonium aminooxy reagents (2.1--2.4) is described. These cationic aminooxy compounds were loaded onto nanoparticles for ligation with carbonyl groups and also to impart a net positive charge on the nanoparticle surface. Our studies indicated that the

  4. Starburst low-molecular weight polyethylenimine for efficient gene delivery.

    Science.gov (United States)

    Zhao, Yanjun; Yang, Rulei; Liu, Dong; Sun, Mingjing; Zhou, Lijun; Wang, Zheng; Wan, Ying

    2012-01-01

    Low-molecular weight polyethylenimine (LMW PEI) shows the advantage of low-cytotoxicity, but has been inefficient in gene delivery as a consequence of the low-charge density. A number of previous studies employed the approach of crosslinking to solve this problem. In this study, a starburst LMW PEI gene vector has been developed. It has a polyamidoamine (PAMAM) core conjugated with a shell composed of LWM PEI and polyethylene glycol (PEG), that is PAMAM-PEI-PEG. Plasmid DNA (pEGFP-N1) and human cervix epithelial carcinoma (HeLa) cells were used in the study. The results showed that the starburst LMW PEI could effectively condense DNA at N/P above 5. The polyplexes had a size of about 500 nm and a nearly neutral surface because of the PEG shielding effect. This novel gene vector is able to maintain the low-cytotoxicity of LMW PEI, whereas its transfection efficiency was significantly improved. Copyright © 2011 Wiley Periodicals, Inc.

  5. Perinatal systemic gene delivery using adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda

    2014-11-01

    Full Text Available Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

  6. Optimization of conditions for gene delivery system based on PEI

    Directory of Open Access Journals (Sweden)

    Roya Cheraghi

    2017-01-01

    Full Text Available Objective(s: PEI based nanoparticle (NP due to dual capabilities of proton sponge and DNA binding is known as powerful tool for nucleic acid delivery to cells. However, serious cytotoxicity and complicated conditions, which govern NPs properties and its interactions with cells practically, hindered achievement to high transfection efficiency. Here, we have tried to optimize the properties of PEI/ firefly luciferase plasmid complexes and cellular condition to improve transfection efficiency. Materials and Methods: For this purpose, firefly luciferase, as a robust gene reporter, was complexed with PEI to prepare NPs with different size and charge. The physicochemical properties of nanoparticles were evaluated using agarose gel retardation and dynamic light scattering.  MCF7 and BT474 cells at different confluency were also transfected with prepared nanoparticles at various concentrations for short and long times. Results: The branched PEI can instantaneously bind to DNA and form cationic NPs. The results demonstrated the production of nanoparticles with size about 100-500 nm dependent on N/P ratio. Moreover, increase of nanoparticles concentration on the cell surface drastically improved the transfection rate, so at a concentration of 30 ng/ìl, the highest transfection efficiency was achieved. On the other side, at confluency between 40-60%, the maximum efficiency was obtained. The result demonstrated that N/P ratio of 12 could establish an optimized ratio between transfection efficiency and cytotoxicity of PEI/plasmid nanoparticles. The increase of NPs N/P ratio led to significant cytotoxicity. Conclusion: Obtained results verified the optimum conditions for PEI based gene delivery in different cell lines.

  7. Design of novel polysaccharidic nanostructures for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Fuente, M de la; Seijo, B; Alonso, M J [Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, Campus sur s/n, E-15782 Santiago de Compostela (Spain)], E-mail: ffmjalon@usc.es

    2008-02-20

    The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10 kDa (HAO) and chitosan 125 (CS) or 10-12 (CSO) kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230 nm, a positive zeta potential of +10 to +32 mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK 293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In conclusion, HA:CS nanoparticles are promising carriers for gene delivery.

  8. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    Science.gov (United States)

    Zheng, S. W.; Liu, G.; Hong, R. Y.; Li, H. Z.; Li, Y. G.; Wei, D. G.

    2012-10-01

    The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe3O4 nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  9. Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery

    Directory of Open Access Journals (Sweden)

    Huang XZ

    2014-10-01

    Full Text Available Xianzhang Huang,1 Sujing Shen,2 Zhanfeng Zhang,1 Junhua Zhuang1 1Department of Laboratory Science, Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 2Department of Laboratory Science, Guangdong Second Provincial Traditional Chinese Medicine Hospital, Guangzhou, People’s Republic of China Abstract: The high transfection efficiency of polyethylenimine (PEI makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP. We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site–enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin-labeled small interfering ribonucleic acids (siRNAs was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene

  10. Overexpression of a New Chitinase Gene EuCHIT2 Enhances Resistance to Erysiphe cichoracearum DC. in Tobacco Plants

    OpenAIRE

    Dong, Xuan; Zhao, Yichen; Ran, Xin; Guo, Linxia; Zhao, De-Gang

    2017-01-01

    In this study, we cloned a new chitinase gene, EuCHIT2, from Eucommia ulmoides Oliver (E. ulmoides) using rapid amplification of cDNA ends (RACE) technology and constructed an overexpression vector, pSH-35S-EuCHIT2, to introduce it into tobacco (Nicotiana tabacum cv. Xanthi). Resistance to Erysiphe cichoracearum de Candolle (E. cichoracearum DC.) and molecular mechanisms in the transgenic tobacco were determined by drop inoculation, spore counting, determination of physicochemical indicators,...

  11. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing.

    Science.gov (United States)

    Mout, Rubul; Ray, Moumita; Yesilbag Tonga, Gulen; Lee, Yi-Wei; Tay, Tristan; Sasaki, Kanae; Rotello, Vincent M

    2017-03-28

    Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

  12. 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

  13. A sight on the current nanoparticle-based gene delivery vectors

    Science.gov (United States)

    Dizaj, Solmaz Maleki; Jafari, Samira; Khosroushahi, Ahmad Yari

    2014-05-01

    Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/ in vivo transformation efficiency.

  14. PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

    Directory of Open Access Journals (Sweden)

    Marxa Figueiredo

    2012-01-01

    Full Text Available This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid (PLGA or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound composed either of polymers (PLGA, polystyrene or other contrast agent materials (Optison, SonoVue microbubbles. The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery.

  15. Polyethylenimine-mediated gene delivery to the lung and therapeutic applications

    Directory of Open Access Journals (Sweden)

    Sante Di Gioia

    2008-09-01

    Full Text Available Sante Di Gioia, Massimo ConeseDepartment of Biomedical Sciences, University of Foggia, Foggia, ItalyAbstract: Nonviral gene delivery is now considered a promising alternative to viral vectors. Among nonviral gene delivery agents, polyethylenimine (PEI has emerged as a potent candidate for gene delivery to the lung. PEI has some advantages over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. However, intracellular (mainly the nuclear membrane and extracellular obstacles still hamper its efficiency in vitro and in vivo, depending on the route of administration and the type of PEI. Nuclear delivery has been increased by adding nuclear localization signals. To overcome nonspecific interactions with biological fluids, extracellular matrix components and nontarget cells, strategies have been developed to protect polyplexes from these interactions and to increase target specificity and gene expression. When gene delivery into airway epithelial cells of the conducting airways is necessary, aerosolization of complexes seems to be better suited to guarantee higher transgene expression in the airway epithelial cells with lower toxicity than observed with either intratracheal or intravenous administration. Aerosolization, indeed, is useful to target the alveolar epithelium and pulmonary endothelium. Proof-of-principle that PEI-mediated gene delivery has therapeutic application to some genetic and acquired lung disease is presented, using as genetic material either plasmidic DNA or small-interfering RNA, although optimization of formulation and delivery protocols and limitation of toxicity need further studies.Keywords: gene transfer, gene therapy, polyethylenimine, airway epithelial cells, lung, RNA interference

  16. Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer—The Case for Combined Delivery?

    Directory of Open Access Journals (Sweden)

    Rita Mendes

    2017-03-01

    Full Text Available Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics.

  17. Six HLA-D region alpha-chain genes on human chromosome 6: polymorphisms and associations of DC alpha-related sequences with DR types.

    OpenAIRE

    Spielman, R S; Lee, J; Bodmer, W F; Bodmer, J G; Trowsdale, J

    1984-01-01

    Analysis of cosmid clones containing genes related to the HLA-DR alpha chain calls for at least six HLA-D region alpha-chain coding sequences in man; namely, DR alpha, DC alpha, DX alpha (very closely related to DC alpha), SB alpha 1, SB alpha 2 (two closely linked genes on the same cosmid clones), and DZ alpha. The first four genes have been described previously. SB alpha 2 and DZ alpha are recently identified genes, characterized by their unique and, from a limited study, nonpolymorphic ban...

  18. 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

  19. Image-Guided Hydrodynamic Gene Delivery: Current Status and Future Directions

    Directory of Open Access Journals (Sweden)

    Kenya Kamimura

    2015-08-01

    Full Text Available Hydrodynamics-based delivery has been used as an experimental tool to express transgene in small animals. This in vivo gene transfer method is useful for functional analysis of genetic elements, therapeutic effect of oligonucleotides, and cancer cells to establish the metastatic cancer animal model for experimental research. Recent progress in the development of image-guided procedure for hydrodynamics-based gene delivery in large animals directly supports the clinical applicability of this technique. This review summarizes the current status and recent progress in the development of hydrodynamics-based gene delivery and discusses the future directions for its clinical application.

  20. Complexation of oppositely charged polyelectrolytes in gene delivery and biology

    Science.gov (United States)

    Shklovskii, Boris

    2009-03-01

    Charge inversion of a DNA double helix by a positively charged flexible polymer (polyelectrolyte) is widely used to facilitate DNA contact with negative cell membranes for gene delivery. Motivated by this application in the first part of the talk I study the phase diagram a solution of long polyanions (PA) with a shorter polycations (PC) as a function the ratio of total charges of PC and PA in the solution, x, and the concentration of monovalent salt. Each PA attracts many PCs to form a complex. When x= 1, the complexes are neutral and condense in a macroscopic drop. When x is far away from 1, complexes are strongly charged and stable. PA are overcharged by PC at x > 1 and undercharged by PC at x < 1. As x approaches 1, PCs attached to PA disproportionate between complexes. Some complexes become neutral and condensed in a macroscopic drop while others become even stronger charged and stay free. The second part of the talk deals with biological example of PA -PC complexes namely self-assembly of vegetable viruses from long ss-RNA molecule paying role of scaffold and identical capsid proteins with long positive tails. I show that optimization Coulomb energy of the virus leads to the charge of RNA twice larger than the total charge of the capsid, in agreement with the experimental data. Then I discuss kinetics of the Coulomb complexation driven virus self-assembly. Capsid proteins stick to unassembled chain of ss RNA (which we call ``antenna'') and slide on it towards the assembly site. I show that at excess of capsid proteins such one-dimensional diffusion accelerates self-assembly more than ten times. On the other hand at excess of ss-RNA, antenna slows self-assembly down. Several experiments are proposed to verify the role of ss-RNA antenna in self-assembly.

  1. Molecular Imaging of Biological Gene Delivery Vehicles for Targeted Cancer Therapy: Beyond Viral Vectors

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Nguyen, Vu H. [Chonnam National University Medical School, Gwangju (Korea, Republic of); Gambhir, Sanjiv S. [Stanford University, California(United States)

    2010-04-15

    Cancer persists as one of the most devastating diseases in the world. Problems including metastasis and tumor resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of present clinical treatments. To overcome these limitations, cancer gene therapy has been developed over the last two decades for a broad spectrum of applications, from gene replacement and knockdown to vaccination, each with different requirements for gene delivery. So far, a number of genes and delivery vectors have been investigated, and significant progress has been made with several gene therapy modalities in clinical trials. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications. However, both have limitations and risks that restrict gene therapy applications, including the complexity of production, limited packaging capacity, and unfavorable immunological features. While continuing to improve these vectors, it is important to investigate other options, particularly nonarrival biological agents such as bacteria, bacteriophages, and bacteria-like particles. Recently, many molecular imaging techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been employed to assess vector-mediated gene expression in living subjects. In this review, molecular imaging techniques for monitoring biological gene delivery vehicles are described, and the specific use of these methods at different steps is illustrated. Linking molecular imaging to gene therapy will eventually help to develop novel gene delivery vehicles for preclinical study and support the development of future human applications.

  2. Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery

    NARCIS (Netherlands)

    Christensen, Lane V.; Christensen, L.; Chang, Chien-Wen; Kim, Won Jong; Kim, Sung Wan; Zhong, Zhiyuan; Lin, C.; Engbersen, Johannes F.J.; Feijen, Jan

    2006-01-01

    Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More

  3. Non-electrostatic complexes with DNA: towards novel synthetic gene delivery systems.

    Science.gov (United States)

    Soto, J; Bessodes, M; Pitard, B; Mailhe, P; Scherman, D; Byk, G

    2000-05-01

    We have developed new DNA complexing amphiphile based on Hoechst 33258 interaction with DNA grooves. The synthesis and physicochemical characterisation of lipid/DNA complexes, as compared to that of classical lipopolyamine for gene delivery, are described and discussed.

  4. Nano-scale gene delivery systems; current technology, obstacles, and future directions.

    Science.gov (United States)

    Garcia-Guerra, Antonio; Dunwell, Thomas L; Trigueros, Sonia

    2018-01-07

    Within the different applications of nanomedicine currently being developed, nano-gene delivery is appearing as an exciting new technique with the possibility to overcome recognised hurdles and fulfill several biological and medical needs. The central component of all delivery systems is the requirement for the delivery of genetic material into cells, and for them to eventually reside in the nucleus where their desired function will be exposed. However, genetic material does not passively enter cells; thus, a delivery system is necessary. The emerging field of nano-gene delivery exploits the use of new materials and the properties that arise at the nanometre-scale to produce delivery vectors that can effectively deliver genetic material into a variety of different types of cells. The novel physicochemical properties of the new delivery vectors can be used to address the current challenges existing in nucleic acid delivery in vitro and in vivo. While there is a growing interest in nanostructure-based gene delivery, the field is still in its infancy, and there is yet much to discover about nanostructures and their physicochemical properties in a biological context. We carry out an organized and focused search of bibliographic databases. Our results suggest that despite new breakthroughs in nanostructure synthesis and advanced characterization techniques, we still face many barriers in producing highly efficient and non-toxic delivery systems. In this review, we overview the types of systems currently used for clinical and biomedical research applications along with their advantages and disadvantages, as well as discussing barriers that arise from nano-scale interactions with biological material. In conclusion, we hope that by bringing the far reaching multidisciplinary nature of nano-gene delivery to light, new targeted nanotechnology-bases strategies are developed to overcome the major challenges covered in this review. Copyright© Bentham Science Publishers; For

  5. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    Science.gov (United States)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2008-12-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  6. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    International Nuclear Information System (INIS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yamamoto, Kenji; Yasuhara, Masato; Kondo, Akihiko

    2008-01-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  7. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    International Nuclear Information System (INIS)

    Yuan, Chenyan; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng; An, Yanli

    2014-01-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression. (paper)

  8. 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

  9. Cell based-gene delivery approaches for the treatment of spinal cord injury and neurodegenerative disorders.

    Science.gov (United States)

    Taha, Masoumeh Fakhr

    2010-03-01

    Cell based-gene delivery has provided an important therapeutic strategy for different disorders in the recent years. This strategy is based on the transplantation of genetically modified cells to express specific genes and to target the delivery of therapeutic factors, especially for the treatment of cancers and neurological, immunological, cardiovascular and heamatopoietic disorders. Although, preliminary reports are encouraging, and experimental studies indicate functionally and structurally improvements in the animal models of different disorders, universal application of this strategy for human diseases requires more evidence. There are a number of parameters that need to be evaluated, including the optimal cell source, the most effective gene/genes to be delivered, the optimal vector and method of gene delivery into the cells and the most efficient route for the delivery of genetically modified cells into the patient. Also, some obstacles have to be overcome, including the safety and usefulness of the approaches and the stability of the improvements. Here, recent studies concerning with the cell-based gene delivery for spinal cord injury and some neurodegenerative disorders such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease are briefly reviewed, and their exciting consequences are discussed.

  10. Advanced DC/DC converters

    CERN Document Server

    Luo, Fang Lin

    2003-01-01

    DC/DC conversion techniques have undergone rapid development in recent decades. With the pioneering work of these authors, DC/DC converters have now moved into their sixth generation. This book offers a concise, practical presentation of DC/DC converters, summarizing the spectrum of conversion tecnologies and presentingmany new ideas and more than 100 new topologies. Nowhere else in the literature are DC/DC converters so logically sorted and systematically introduced, and nowhere else can readers find detailed information on prototype topologies that represent a major contribution to modern power engineering. More than 320 figures, 60 tables, and 500 formulae facilitate understand and provide precise data.

  11. Self-assembled pentablock copolymers for selective and sustained gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bingqi [Iowa State Univ., Ames, IA (United States)

    2011-05-15

    The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomes of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.

  12. Safety assessment of liver-targeted hydrodynamic gene delivery in dogs.

    Directory of Open Access Journals (Sweden)

    Kenya Kamimura

    Full Text Available Evidence in support of safety of a gene delivery procedure is essential toward gene therapy. Previous studies using the hydrodynamics-based procedure primarily focus on gene delivery efficiency or gene function analysis in mice. The current study focuses on an assessment of the safety of computer-controlled and liver-targeted hydrodynamic gene delivery in dogs as the first step toward hydrodynamic gene therapy in clinic. We demonstrate that the impacts of the hydrodynamic procedure were limited in the injected region and the influences were transient. Histological examination and the hepatic microcirculation measurement using reflectance spectrophotometry reveal that the liver-specific impact of the procedure involves a transient expansion of the liver sinusoids. No systemic damage or toxicity was observed. Physiological parameters, including electrocardiogram, heart rate, blood pressure, oxygen saturation, and body temperature, remained in normal ranges during and after hydrodynamic injection. Body weight was also examined to assess the long-term effects of the procedure in animals who underwent 3 hydrodynamic injections in 6 weeks with 2-week time interval in between. Serum biochemistry analysis showed a transient increase in liver enzymes and a few cytokines upon injection. These results demonstrate that image-guided, liver-specific hydrodynamic gene delivery is safe.

  13. Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery.

    Science.gov (United States)

    Collins, Louise; Kaszuba, Michael; Fabre, John W

    2004-04-07

    The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)(16)-containing peptide/DNA particles. Eight (Lys)(16)-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to - charge ratio of 2.8:1 with 10 microg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate approximately 60 peptide/DNA nanoparticles per target cell, each containing approximately 70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.

  14. Antibody-IL2 Fusion Protein Delivery by Gene Transfer

    National Research Council Canada - National Science Library

    Nicolet, Charles

    1997-01-01

    The purpose of the work described is to assess the feasibility of a gene therapy approach to deliver a specific antibody cytokine fusion protein called CC49-1L2 to a tumor expressing antigen reactive with the antibody...

  15. DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy.

    Science.gov (United States)

    Angell, Chava; Xie, Sibai; Zhang, Liangfang; Chen, Yi

    2016-03-02

    Nanomedicine has been growing exponentially due to its enhanced drug targeting and reduced drug toxicity. It uses the interactions where nanotechnological components and biological systems communicate with each other to facilitate the delivery performance. At this scale, the physiochemical properties of delivery systems strongly affect their capacities. Among current delivery systems, DNA nanotechnology shows many advantages because of its unprecedented engineering abilities. Through molecular recognition, DNA nanotechnology can be used to construct a variety of nanostructures with precisely controllable size, shape, and surface chemistry, which can be appreciated in the delivery process. In this review, different approaches that are currently used for the construction of DNA nanostructures are reported. Further, the utilization of these DNA nanostructures with the well-defined parameters for the precise control in drug delivery and gene therapy is discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Translational Advancement of Somatostatin Gene Delivery for Disease Modification and Cognitive Sparing in Intractable Epilepsy

    Science.gov (United States)

    2015-09-01

    efficacy against seizures. 15. SUBJECT TERMS Epilepsy; seizure; kindling; somatatostatin; traumatic brain injury; gene delivery; adeno­ associated  viral ...vector; neurogenesis; inflammation; neurodegeneration; hippocampus, memory 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...delivery using adeno-associated viral vectors for SST or inactive control protein will be tested for ability to suppress seizure susceptibility and

  17. Self-assembling polycations for gene delivery: Effects of polymer structure and environmental pH

    OpenAIRE

    Samsonova, Olga

    2012-01-01

    SUMMARY In this thesis, novel polycationic vectors for therapeutic gene delivery were introduced with the focus on structure-function relationship in both physicochemical and biological aspects. First the transfection related differences of classical vectors with high and low branching grade and charge density: PEI and PLL, were examined under pH-alternating tumor tissue conditions. Further structural design for siRNA delivery was...

  18. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

    Directory of Open Access Journals (Sweden)

    Atsushi Miyanohara

    2016-01-01

    Full Text Available Effective in vivo use of adeno-associated virus (AAV-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal. Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii potent retrograde transgene expression in brain motor centers (motor cortex and brain stem; and (iv the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

  19. Ligand-Modified Human Serum Albumin Nanoparticles for Enhanced Gene Delivery.

    Science.gov (United States)

    Look, Jennifer; Wilhelm, Nadine; von Briesen, Hagen; Noske, Nadja; Günther, Christine; Langer, Klaus; Gorjup, Erwin

    2015-09-08

    The development of nonviral gene delivery systems is a great challenge to enable safe gene therapy. In this study, ligand-modified nanoparticles based on human serum albumin (HSA) were developed and optimized for an efficient gene therapy. Different glutaraldehyde cross-linking degrees were investigated to optimize the HSA nanoparticles for gene delivery. The peptide sequence arginine-glycine-aspartate (RGD) and the HIV-1 transactivator of transduction sequence (Tat) are well-known as promising targeting ligands. Plasmid DNA loaded HSA nanoparticles were covalently modified on their surface with these different ligands. The transfection potential of the obtained plasmid DNA loaded RGD- and Tat-modified nanoparticles was investigated in vitro, and optimal incubation conditions for these preparations were studied. It turned out that Tat-modified HSA nanoparticles with the lowest cross-linking degree of 20% showed the highest transfection potential. Taken together, ligand-functionalized HSA nanoparticles represent promising tools for efficient and safe gene therapy.

  20. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

    Science.gov (United States)

    Liu, Jia; Shui, Sai-Lan

    2016-12-28

    The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Prodrug converting enzyme gene delivery by L. monocytogenes

    Directory of Open Access Journals (Sweden)

    Pilgrim Sabine

    2008-04-01

    Full Text Available Abstract Background Listeria monocytogenes is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that L. monocytogenes is able to colonize and replicate within solid tumors after local or even systemic injection. Methods Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1 into cancer cells in culture by L. monocytogenes. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria. Results The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although L. monocytogenes-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively PCMV-promoter with the melanoma specific P4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies. Conclusion These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors.

  2. Artificial Virus as Trump-card to Resolve Exigencies in Targeted Gene Delivery.

    Science.gov (United States)

    Ajithkumar, K C; Pramod, Kannissery

    2018-01-01

    Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized to successfully deliver the genetic material. However, the use of virus mediated gene delivery is considered divisive, because the potentially replicable genomes recombine or integrate with the cell DNA resulting in immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective non-viral gene delivery vehicles arises. Non-viral vectors, protein only particles and virus like particles (VLP) can be constructed which contain all the necessary functional moieties. These resemble viruses and are called artificial or synthetic virus. The artificial virus eliminates the disadvantages of viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because incorporation of requisite agents such as cell ligands, membrane active peptides, etc. into proteins is possible. The protein- DNA complexes resemble bacterial inclusion bodies. Nucleic acids influence conformation of protein units which subsequently result in cell uptake and finally to the cell nucleus. Such tunable systems mimic the activities of infected viruses and are used for the safe and effective delivery of drugs and genetic material in gene therapy. The versatility, stability and biocompatible nature of artificial virus along with high transfection efficacy have made it favorite for gene delivery purposes, in addition to being useful for various biomedical and drug delivery applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection.

    Science.gov (United States)

    Pacheco-Lugo, Lisandro; Díaz-Olmos, Yirys; Sáenz-García, José; Probst, Christian Macagnan; DaRocha, Wanderson Duarte

    2017-06-01

    New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Non-viral gene delivery strategies for cancer therapy, tissue engineering and regenerative medicine

    Science.gov (United States)

    Bhise, Nupura S.

    Gene therapy involves the delivery of deoxyribonucleic acid (DNA) into cells to override or replace a malfunctioning gene for treating debilitating genetic diseases, including cancer and neurodegenerative diseases. In addition to its use as a therapeutic, it can also serve as a technology to enable regenerative medicine strategies. The central challenge of the gene therapy research arena is developing a safe and effective delivery agent. Since viral vectors have critical immunogenic and tumorogenic safety issues that limit their clinical use, recent efforts have focused on developing non-viral biomaterial based delivery vectors. Cationic polymers are an attractive class of gene delivery vectors due to their structural versatility, ease of synthesis, biodegradability, ability to self-complex into nanoparticles with negatively charged DNA, capacity to carry large cargo, cellular uptake and endosomal escape capacity. In this thesis, we hypothesized that developing a biomaterial library of poly(betaamino esters) (PBAE), a newer class of cationic polymers consisting of biodegradable ester groups, would allow investigating vector design parameters and formulating effective non-viral gene delivery strategies for cancer drug delivery, tissue engineering and stem cell engineering. Consequently, a high-throughput transfection assay was developed to screen the PBAE-based nanoparticles in hard to transfect fibroblast cell lines. To gain mechanistic insights into the nanoparticle formulation process, biophysical properties of the vectors were characterized in terms of molecular weight (MW), nanoparticle size, zeta potential and plasmid per particle count. We report a novel assay developed for quantifying the plasmid per nanoparticle count and studying its implications for co-delivery of multiple genes. The MW of the polymers ranged from 10 kDa to 100 kDa, nanoparticle size was about 150 run, zeta potential was about 30 mV in sodium acetate buffer (25 mM, pH 5) and 30 to 100

  5. Electric pulse-mediated gene delivery to various animal tissues

    DEFF Research Database (Denmark)

    Mir, Lluis M; Moller, Pernille H; André, Franck

    2005-01-01

    therapy, termed electrogenetherapy (EGT as well). By transfecting cells with a long lifetime, such as muscle fibers, a very long-term expression of genes can be obtained. A great variety of tissues have been transfected successfully, from muscle as the most extensively used, to both soft (e.g., spleen...

  6. DNA-transporting nanoparticles : design and in vitro evaluation of DNA and formulation for non-viral gene delivery

    NARCIS (Netherlands)

    van Gaal, E.V.B.

    2010-01-01

    The aim of gene therapy is to treat, cure or prevent a disease by replacing defective genes, introducing new genes or changing the expression of a person’s genes. Success of gene therapy is dependent on successful delivery of DNA from the site of administration into cell nuclei. Naturally occurring

  7. Magnetotactic Bacterial Cages as Safe and Smart Gene Delivery Vehicles

    KAUST Repository

    Alsaiari, Shahad K.

    2016-07-27

    In spite of the huge advances in the area of synthetic carriers, their efficiency still poorly compares to natural vectors. Herein, we report the use of unmodified magnetotactic bacteria as a guidable delivery vehicle for DNA functionalized gold nanoparticles (AuNPs). High cargo loading is established under anaerobic conditions (bacteria is alive) through endocytosis where AuNPs are employed as transmembrane proteins mimics (facilitate endocytosis) as well as imaging agents to verify and quantify loading and release. The naturally bio-mineralized magnetosomes, within the bacteria, induce heat generation inside bacteria through magnetic hyperthermia. Most importantly after exposing the system to air (bacteria is dead) the cell wall stays intact providing an efficient bacterial vessel. Upon incubation with THP-1 cells, the magnetotactic bacterial cages (MBCs) adhere to the cell wall and are directly engulfed through the phagocytic activity of these cells. Applying magnetic hyperthermia leads to the dissociation of the bacterial microcarrier and eventual release of cargo.

  8. Vascular gene transfer and drug delivery in vitro using low-frequency ultrasound and microbubbles

    Science.gov (United States)

    Yang, Hong; Liu, Zhong-hua; Liu, Yi-yao; Lou, Chang-chun; Ren, Zheng-long; Miyoshi, Hirokazu

    2010-01-01

    Aim: To determine the effects of ultrasound exposure in combination with a microbubble contrast agent (SonoVue) on the cellular uptake and delivery of drugs/genes into human umbilical vein endothelial cells (HUVECs) as well as their biological effects on migration. Methods: HUVECs in suspension were exposed to pulsed ultrasound with a 10% duty cycle in combination with various concentrations of a microbubble contrast agent (SonoVue) using a digital sonifier at a frequency of 20 kHz and an intensity of 3.77 W/cm2 on the surface of a horn tip. Cell culture inserts were used to determine the cell migration ability. Results: Exposure to pulsed ultrasound resulted in enhanced green fluorescent protein (EGFP) gene transfection efficiencies ranging from 0.2% to 2%. The transfection efficiency of HUVECs was approximately 3-fold higher in the presence of SonoVue than in its absence at the effective exposure time of 6 s. For drug delivery to HUVECs using ultrasound, the delivery efficiencies of a low-molecular-weight model drug (TO-PRO®−1, MW 645.38) were significantly higher when compared to drug delivery without ultrasound, with a maximum efficiency of approximately 34%. However, the delivery efficiencies of a high-molecular-weight model drug (Dextran-Rhodamine B, MW 70 000) were low, with a maximum delivery efficiency of nearly 0.5%, and gene transfection results were similarly poor. The migration ability of HUVECs exposed to ultrasound was also lower than that of the control (no exposure). Conclusion: The use of low-frequency and low-energy ultrasound in combination with microbubbles could be a potent physical method of increasing drug/gene delivery efficiency. This technique is a promising nonviral approach that can be used in cardiovascular disease therapy. PMID:20348943

  9. Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in

  10. Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2011-04-01

    Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in

  11. Effect of adrenomedullin gene delivery on insulin resistance in type 2 diabetic rats

    Directory of Open Access Journals (Sweden)

    Hoda Y. Henein

    2011-01-01

    Full Text Available Type 2 diabetes mellitus is one of the common metabolic disorders that ultimately afflicts large number of individuals. Adrenomedullin (AM is a potent vasodilator peptide; previous studies reported development of insulin resistance in aged AM deficient mice. In this study, we employed a gene delivery approach to explore its potential role in insulin resistance. Four groups were included: control, diabetic, non-diabetic injected with the AM gene and diabetic injected with the AM gene. One week following gene delivery, serum glucose, insulin, triglycerides, leptin, adiponectin and corticosterone were measured as well as the insulin resistance index (HOMA-IR. Soleus muscle glucose uptake and RT-PCR of both AM and glucose transporter-4 (GLUT 4 gene expressions were assessed. A single tail vein injection of adrenomedullin gene in type 2 diabetic rats improved skeletal muscle insulin responsiveness with significant improvement of soleus muscle glucose uptake, HOMA-IR, serum glucose, insulin and triglycerides and significant increase in muscle GLUT 4 gene expression (P < 0.05 compared with the non-injected diabetic rats. The beneficial effects of AM gene delivery were accompanied by a significant increase in the serum level of adiponectin (2.95 ± 0.09 versus 2.33 ± 0.17 μg/ml in the non-injected diabetic group as well as a significant decrease in leptin and corticosterone levels (7.51 ± 0.51 and 262.88 ± 10.34 versus 10.63 ± 1.4 and 275.86 ± 11.19 ng/ml respectively in the non-injected diabetic group. The conclusion of the study is that AM gene delivery can improve insulin resistance and may have significant therapeutic applications in type 2 diabetes mellitus.

  12. Non-viral gene delivery systems: hurdles for bench-to-bedside transformation.

    Science.gov (United States)

    Helal, N A; Osami, A; Helmy, A; McDonald, T; Shaaban, L A; Nounou, M I

    2017-11-01

    Biologist and Nobel Prize winner James Watson's quote, "We used to think that our fate was in our stars, but now we know that, in large measure, our fate is in our genes", represents the initial food for thought that revolutionized the way medications and active pharmaceutical ingredients are defined (Rocholl 1996). This fate engraved in the genetic material, as mentioned in Watson's quote, fueled a tremendous revolution wave in gene therapy. Gene therapy is a promising technology for treating genetic and acquired diseases by modulating the expression of a specific gene in the pathological cells. This is achieved by introducing a DNA sequence or other nucleic acid material or oligonucleotides to the target cell (Kay, 2011). Moreover, gene therapy contributes to correction of genetic defects, expression of therapeutic proteins, and inhibition of the synthesis of malignant proteins. In this review article, different non-viral gene delivery systems and their applications are discussed in detail. We reviewed and tabulated over 90 papers and 50 patents from 2006 to date discussing non-viral gene delivery technologies, innovation, and bench-to-bedside transformation. Furthermore, we are going to shed light on the lack of standardization in the design and characterization of non-viral gene delivery systems worldwide, which is a major concern in this research's field. This review would aid in getting an eagle eye view through non-viral gene delivery technologies during the past 20 years. Such a view, capturing the advances, the hurdles, and experimental details, would aid expert researchers in tuning their experimentation strategies and help newcomers better initially design their studies to generate solid and comprehensive results that can be reliable and reproducible.

  13. Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems.

    Science.gov (United States)

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2014-10-01

    The identification of more efficient gene delivery vehicles (GDVs) is essential to fulfill the expectations of clinical gene therapy. Bacteriophages, due to their excellent safety profile, extreme stability under a variety of harsh environmental conditions and the capability for being genetically manipulated, have drawn a flurry of interest to be applied as a newly arisen category of gene delivery platforms. The incessant evolutionary interaction of bacteriophages with human cells has turned them into a part of our body's natural ecosystem. However, these carriers represent several barriers to gene transduction of mammalian cells. The lack of evolvement of specialized machinery for targeted cellular internalization, endosomal, lysosomal and proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear transcription poses major challenges to the expression of the phage-carried gene. In this review, we describe pros and cons of bacteriophages as GDVs, provide an insight into numerous barriers that bacteriophages face for entry into and subsequent trafficking inside mammalian cells and elaborate on the strategies used to bypass these barriers. Tremendous genetic flexibility of bacteriophages to undergo numerous surface modifications through phage display technology has proven to be a turning point in the uncompromising efforts to surmount the limitations of phage-mediated gene expression. The revelatory outcomes of the studies undertaken within the recent years have been promising for phage-mediated gene delivery to move from concept to reality.

  14. A Promising Combo Gene Delivery System Developed from (3-Aminopropyl)triethoxysilane-Modified Iron Oxide Nanoparticles and Cationic Polymers

    International Nuclear Information System (INIS)

    Zhang Zubin; Song Lina; Dong Jinlai; Guo Dawei; Du Xiaolin; Cao Biyin; Zhang Yu; Gu Ning; Mao Xinliang

    2013-01-01

    (3-Aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers (CPs) such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water-soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and CPs. This system significantly increased gene-binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene-carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

  15. A Promising Combo Gene Delivery System Developed from (3-Aminopropyl)triethoxysilane-Modified Iron Oxide Nanoparticles and Cationic Polymers

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Zubin [Soochow University, Cyrus Tang Hematology Center (China); Song Lina; Dong Jinlai; Guo Dawei [School of Biological Science and Medical Engineering, Southeast University, State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices (China); Du Xiaolin; Cao Biyin [Soochow University, Cyrus Tang Hematology Center (China); Zhang Yu; Gu Ning [School of Biological Science and Medical Engineering, Southeast University, State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices (China); Mao Xinliang, E-mail: xinliangmao@suda.edu.cn [Soochow University, Cyrus Tang Hematology Center (China)

    2013-05-15

    (3-Aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers (CPs) such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water-soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and CPs. This system significantly increased gene-binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene-carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

  16. Calcium gluconate in phosphate buffered saline increases gene delivery with adenovirus type 5.

    Directory of Open Access Journals (Sweden)

    Marko T Ahonen

    Full Text Available BACKGROUND: Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. METHODS/RESULTS: We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. CONCLUSION: In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.

  17. Gene Delivery Particle Engineering Strategies for Shape-dependent Targeting of Cells and Tissues.

    Science.gov (United States)

    Kozielski, Kristen L; Sitti, Metin

    2017-01-01

    Successful gene delivery requires overcoming both systemic and intracellular obstacles before the nucleic acid cargo can successfully reach its tissue and subcellular target location. Materials & Methods: Non-viral mechanisms to enable targeting while avoiding off-target delivery have arisen via biological, chemical, and physical engineering strategies. Herein we will discuss the physical parameters in particle design that promote tissue- and cell-targeted delivery of genetic cargo. We will discuss systemic concerns, such as circulation, tissue localization, and clearance, as well as cell-scale obstacles, such as cellular uptake and nucleic acid packaging. In particular, we will focus on engineering particle shape and size in order to enhance delivery and promote precise targeting. We will also address methods to program or change particle shape in situ using environmentally triggered cues. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Systemic gene delivery to the central nervous system using Adeno-associated virus

    Directory of Open Access Journals (Sweden)

    Mathieu eBOURDENX

    2014-06-01

    Full Text Available Adeno-associated virus (AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain-barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  19. Efficient Gene Delivery to Pig Airway Epithelia and Submucosal Glands Using Helper-Dependent Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Huibi Cao

    2013-01-01

    Full Text Available Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF. However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR. Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy.

  20. Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy

    NARCIS (Netherlands)

    Martens, Thomas F.; Remaut, Katrien; Deschout, Hendrik; Engbersen, Johan F J; Hennink, Wim E.; Van Steenbergen, Mies J.; Demeester, Jo; De Smedt, Stefaan C.; Braeckmans, Kevin

    2015-01-01

    Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or

  1. Effective Nanoparticle-based Gene Delivery by a Protease Triggered Charge Switch

    DEFF Research Database (Denmark)

    Gjetting, Torben; Jølck, Rasmus Irming; Andresen, Thomas Lars

    2014-01-01

    (ethylene glycol) (PEG) chain is investigated. Utilizing ethanol-mediated nucleic acid encapsulation to prepare lipo-nanoparticles (LNPs), LNPs that are stable in serum are obtained. The LNPs constitute a highly effective gene delivery systems in vitro and possess the right features for further...

  2. DC-SIGN (CD209), pentraxin 3 and vitamin D receptor gene variants associate with pulmonary tuberculosis risk in West Africans

    DEFF Research Database (Denmark)

    Olesen, R; Wejse, C; Velez, D R

    2007-01-01

    We investigated the role of DC-SIGN (CD209), long pentraxin 3 (PTX3) and vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) in susceptibility to pulmonary tuberculosis (TB) in 321 TB cases and 347 healthy controls from Guinea-Bissau. Five additional, functionally relevant SNPs...... in a nonadditive model with disease risk when analyzed in combination with ethnicity (P=0.03 for DC-SIGN and P=0.003 for VDR). In addition, PTX3 haplotype frequencies significantly differed in cases compared to controls and a protective effect was found in association with a specific haplotype (OR 0.78, 95% CI 0.......63-0.98). Our findings support previous data showing that VDR SNPs modulate the risk for TB in West Africans and suggest that variation within DC-SIGN and PTX3 also affect the disease outcome. Udgivelsesdato: 2007-Sep...

  3. Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

    Science.gov (United States)

    Sabet, Salwa; George, Marina A; El-Shorbagy, Haidan M; Bassiony, Heba; Farroh, Khaled Y; Youssef, Tareq; Salaheldin, Taher A

    2017-01-01

    Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.

  4. Well-defined block copolymers for gene delivery to dendritic cells: probing the effect of polycation chain-length.

    Science.gov (United States)

    Tang, Rupei; Palumbo, R Noelle; Nagarajan, Lakshmi; Krogstad, Emily; Wang, Chun

    2010-03-03

    The development of safe and efficient polymer carriers for DNA vaccine delivery requires mechanistic understanding of structure-function relationship of the polymer carriers and their interaction with antigen-presenting cells. Here we have synthesized a series of diblock copolymers with well-defined chain-length using atom transfer radical polymerization and characterized the influence of polycation chain-length on the physico-chemical properties of the polymer/DNA complexes as well as the interaction with dendritic cells. The copolymers consist of a hydrophilic poly(ethylene glycol) block and a cationic poly(aminoethyl methacrylate) (PAEM) block. The average degree of polymerization (DP) of the PAEM block was varied among 19, 39, and 75, with nearly uniform distribution. With increasing PAEM chain-length, polyplexes formed by the diblock copolymers and plasmid DNA had smaller average particle size and showed higher stability against electrostatic destabilization by salt and heparin. The polymers were not toxic to mouse dendritic cells (DCs) and only displayed chain-length-dependent toxicity at a high concentration (1mg/mL). In vitro gene transfection efficiency and polyplex uptake in DCs were also found to correlate with chain-length of the PAEM block with the longer polymer chain favoring transfection and cellular uptake. The polyplexes induced a modest up-regulation of surface markers for DC maturation that was not significantly dependent on PAEM chain-length. Finally, the polyplex prepared from the longest PAEM block (DP of 75) achieved an average of 20% enhancement over non-condensed anionic dextran in terms of uptake by DCs in the draining lymph nodes 24h after subcutaneous injection into mice. Insights gained from studying such structurally well-defined polymer carriers and their interaction with dendritic cells may contribute to improved design of practically useful DNA vaccine delivery systems. Copyright 2009 Elsevier B.V. All rights reserved.

  5. A magnetic nanoparticle-based multiple-gene delivery system for transfection of porcine kidney cells.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available Superparamagnetic nanoparticles are promising candidates for gene delivery into mammalian somatic cells and may be useful for reproductive cloning using the somatic cell nuclear transfer technique. However, limited investigations of their potential applications in animal genetics and breeding, particularly multiple-gene delivery by magnetofection, have been performed. Here, we developed a stable, targetable and convenient system for delivering multiple genes into the nuclei of porcine somatic cells using magnetic Fe3O4 nanoparticles as gene carriers. After surface modification by polyethylenimine, the spherical magnetic Fe3O4 nanoparticles showed strong binding affinity for DNA plasmids expressing the genes encoding a green (DNAGFP or red (DNADsRed fluorescent protein. At weight ratios of DNAGFP or DNADsRed to magnetic nanoparticles lower than or equal to 10∶1 or 5∶1, respectively, the DNA molecules were completely bound by the magnetic nanoparticles. Atomic force microscopy analyses confirmed binding of the spherical magnetic nanoparticles to stretched DNA strands up to several hundred nanometers in length. As a result, stable and efficient co-expression of GFP and DsRed in porcine kidney PK-15 cells was achieved by magnetofection. The results presented here demonstrate the potential application of magnetic nanoparticles as an attractive delivery system for animal genetics and breeding studies.

  6. VEGF targeting in mesotheliomas using an interleukin-6 signal inhibitor based on adenovirus gene delivery.

    Science.gov (United States)

    Adachi, Yasuo; Yoshio-Hoshino, Naoko; Aoki, Chieko; Nishimoto, Norihiro

    2010-06-01

    Malignant mesotheliomas reportedly secrete interleukin-6 (IL-6) which augments production of vascular endothelial growth factor (VEGF) from mesothelioma cells. We previously reported the development of a new receptor inhibitor of IL-6 (NRI) by genetically engineering tocilizumab, a humanized anti-IL-6 receptor monoclonal antibody. Since NRI is encoded on a single gene, it is easily applicable to a gene delivery system using virus vehicles. In this study, we report VEGF targeting through NRI expression based on adenovirus-mediated gene delivery in mesothelioma cells. We constructed an NRI expression vector in the context of a tropism-modified adenovirus vector that had enhanced infectivity in mesothelioma cells. This virus effectively induced NRI secretion from mesothelioma cells. This virus infection also reduced the VEGF production in mesothelioma cells. These results indicate that NRI shows potential as an agent in the treatment of mesotheliomas.

  7. PLGA-Chitosan nanoparticle-mediated gene delivery for oral cancer treatment: A brief review

    Science.gov (United States)

    Bakar, L. M.; Abdullah, M. Z.; Doolaanea, A. A.; Ichwan, S. J. A.

    2017-08-01

    Cancer becomes a serious issue on society with increasing of their growth and proliferation, either in well economic developed countries or not. Recent years, oral cancer is one of the most threatening diseases impairing the quality of life of the patient. Scientists have emphasised on application of gene therapy for oral cancer by using nanoparticle as transportation vectors as a new alternative platform in order to overcome the limitations of conventional approaches. In modern medicine, nanotechnologies’ application, such as nanoparticles-mediated gene delivery, is one of promising tool for therapeutic devices. The objective of this article is to present a brief review summarizes on the current progress of nanotechnology-based gene delivery treatment system targeted for oral cancer.

  8. Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.

    Science.gov (United States)

    Kim, Na Hyung; Provoda, Chester; Lee, Kyung-Dall

    2015-02-02

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems.

  9. Gene delivery in conjunction with gold nanoparticle and tumor treating electric field

    Science.gov (United States)

    Tiwari, Pawan K.; Soo Lee, Yeon

    2013-08-01

    The advances in electrotherapy to treat the diseased biological cell instigate its extension in gene therapy through the delivery of gene into the nucleus. The objective of this study is to investigate the application of moderate intensity alternating electric field, also known as tumor treating electric field on a carrier system consisting of a charged gene complex conjugated to the surface of a gold nanoparticle. The gene delivery mechanism relies on the magnitude and direction of the induced electric field inside the cytoplasm in presence of carrier system. The induced electric field strength is significant in breaking the gene complex-gold nanoparticle bonding, and exerting an electric force pushing the charged gene into the nucleus. The electric force orientation is dependent on the aspect ratio (AR) of the gold nanoparticle and a relationship between them is studied via Maxwell two-dimensional (2D) finite element simulation analyzer. The development of charge density on the surface of carrier system and the required electric field strength to break the bonding are investigated utilizing the Gouy-Chapman-Grahame-Stern (GCGS) theoretical model. A carrier system having the aspect ratio of the gold nanoparticle in the range 1 < AR ≤ 5 and AR = 1 are substantial delivering cationic and anionic genes into the nucleus, respectively.

  10. Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

    Directory of Open Access Journals (Sweden)

    Yoichi Negishi

    2011-01-01

    Full Text Available Recently, we have developed novel polyethylene glycol modified liposomes (bubble liposomes; BL entrapping an ultrasound (US imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes in vitro and joint synovium in vivo. Highly efficient gene transfer could be achieved in the cultured primary synoviocytes transfected with the combination of BL and US exposure, compared to treatment with plasmid DNA (pDNA alone, pDNA plus BL, or pDNA plus US. When BL was injected into the knee joints of mice, and US exposure was applied transcutaneously to the injection site, highly efficient gene expression could be observed in the knee joint transfected with the combination of BL and US exposure, compared to treatment with pDNA alone, pDNA plus BL, or pDNA plus US. The localized and prolonged gene expression was also shown by an in vivo luciferase imaging system. Thus, this local gene delivery system into joint synovium using the combination of BL and US exposure may be an effective means for gene therapy in joint disorders.

  11. An efficient parallel stochastic simulation method for analysis of nonviral gene delivery systems

    KAUST Repository

    Kuwahara, Hiroyuki

    2011-01-01

    Gene therapy has a great potential to become an effective treatment for a wide variety of diseases. One of the main challenges to make gene therapy practical in clinical settings is the development of efficient and safe mechanisms to deliver foreign DNA molecules into the nucleus of target cells. Several computational and experimental studies have shown that the design process of synthetic gene transfer vectors can be greatly enhanced by computational modeling and simulation. This paper proposes a novel, effective parallelization of the stochastic simulation algorithm (SSA) for pharmacokinetic models that characterize the rate-limiting, multi-step processes of intracellular gene delivery. While efficient parallelizations of the SSA are still an open problem in a general setting, the proposed parallel simulation method is able to substantially accelerate the next reaction selection scheme and the reaction update scheme in the SSA by exploiting and decomposing the structures of stochastic gene delivery models. This, thus, makes computationally intensive analysis such as parameter optimizations and gene dosage control for specific cell types, gene vectors, and transgene expression stability substantially more practical than that could otherwise be with the standard SSA. Here, we translated the nonviral gene delivery model based on mass-action kinetics by Varga et al. [Molecular Therapy, 4(5), 2001] into a more realistic model that captures intracellular fluctuations based on stochastic chemical kinetics, and as a case study we applied our parallel simulation to this stochastic model. Our results show that our simulation method is able to increase the efficiency of statistical analysis by at least 50% in various settings. © 2011 ACM.

  12. Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene Delivery

    Science.gov (United States)

    Hofmann, Anna T.; Slezak, Paul; Schuetzenberger, Sebastian; Kaipel, Martin; Schwartz, Ernst; Neef, Anne; Nomikou, Nikolitsa; Nau, Thomas; van Griensven, Martijn; McHale, Anthony P.; Redl, Heinz

    2014-01-01

    Abstract An ideal novel treatment for bone defects should provide regeneration without autologous or allogenous grafting, exogenous cells, growth factors, or biomaterials while ensuring spatial and temporal control as well as safety. Therefore, a novel osteoinductive nonviral in vivo gene therapy approach using sonoporation was investigated in ectopic and orthotopic models. Constitutive or regulated, doxycycline-inducible, bone morphogenetic protein 2 and 7 coexpression plasmids were repeatedly applied for 5 days. Ectopic and orthotopic gene transfer efficacy was monitored by coapplication of a luciferase plasmid and bioluminescence imaging. Orthotopic plasmid DNA distribution was investigated using a novel plasmid-labeling method. Luciferase imaging demonstrated an increased trend (61% vs. 100%) of gene transfer efficacy, and micro-computed tomography evaluation showed significantly enhanced frequency of ectopic bone formation for sonoporation compared with passive gene delivery (46% vs. 100%) dependent on applied ultrasound power. Bone formation by the inducible system (83%) was stringently controlled by doxycycline in vivo, and no ectopic bone formation was observed without induction or with passive gene transfer without sonoporation. Orthotopic evaluation in a rat femur segmental defect model demonstrated an increased trend of gene transfer efficacy using sonoporation. Investigation of DNA distribution demonstrated extensive binding of plasmid DNA to bone tissue. Sonoporated animals displayed a potentially increased union rate (33%) without extensive callus formation or heterotopic ossification. We conclude that sonoporation of BMP2/7 coexpression plasmids is a feasible, minimally invasive method for osteoinduction and that improvement of bone regeneration by sonoporative gene delivery is superior to passive gene delivery. PMID:24164605

  13. The next step in gene delivery: molecular engineering of adeno-associated virus serotypes.

    Science.gov (United States)

    Wang, Jinhui; Faust, Susan M; Rabinowitz, Joseph E

    2011-05-01

    Delivery is at the heart of gene therapy. Viral DNA delivery systems are asked to avoid the immune system, transduce specific target cell types while avoiding other cell types, infect dividing and non-dividing cells, insert their cargo within the host genome without mutagenesis or to remain episomal, and efficiently express transgenes for a substantial portion of a lifespan. These sought-after features cannot be associated with a single delivery system, or can they? The Adeno-associated virus family of gene delivery vehicles has proven to be highly malleable. Pseudotyping, using AAV serotype 2 terminal repeats to generate designer shells capable of transducing selected cell types, enables the packaging of common genomes into multiple serotypes virions to directly compare gene expression and tropism. In this review the ability to manipulate this virus will be examined from the inside out. The influence of host cell factors and organism biology including the immune response on the molecular fate of the viral genome will be discussed as well as differences in cellular trafficking patterns and uncoating properties that influence serotype transduction. Re-engineering the prototype vector AAV2 using epitope insertion, chemical modification, and molecular evolution not only demonstrated the flexibility of the best-studied serotype, but now also expanded the tool kit for molecular modification of all AAV serotypes. Current AAV research has changed its focus from examination of wild-type AAV biology to the feedback of host cell/organism on the design and development of a new generation of recombinant AAV delivery vehicles. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy". Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.

    LENUS (Irish Health Repository)

    van Pijkeren, Jan Peter

    2012-01-31

    Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to ampicillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor samples ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic ampicillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and ampicillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.

  15. Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

    Science.gov (United States)

    Shepard, Jaclyn A.; Stevans, Alyson C.; Holland, Samantha; Wang, Christine E.; Shikanov, Ariella; Shea, Lonnie D.

    2012-01-01

    Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. PMID:22038654

  16. Application of quantum dots as vectors in targeted survivin gene siRNA delivery

    Directory of Open Access Journals (Sweden)

    Zhao JJ

    2013-04-01

    Full Text Available Jianjiang Zhao, Xiaoling Qiu, Zhiping Wang, Jie Pan, Jun Chen, Jiusong Han Department of Surgery, Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou, People's Republic of China Abstract: Gene silencing using short interfering RNA (siRNA is becoming an attractive approach for probing gene function in mammalian cells. This study evaluated the specificity and efficiency of quantum dots (QDs as non-viral gene vectors for delivery of survivin siRNA and downregulation of survivin gene expression in oral squamous cell carcinoma Tca8113 cells. Water-dispersible cationically-modified QDs were electrostatically attached to anionic siRNA molecules and complexed with siRNA for downregulating expression of the survivin gene. Cellular uptake and allocation of QD–siRNA complexes in Tca8113 cells were monitored using confocal laser scanning microscopy. Real-time polymerase chain reaction (PCR was used to quantify survivin messenger RNA (mRNA levels. CdSe QDs were observed with high intensity fluorescence under confocal laser scanning microscopy. Tca8113 cells were successfully transfected by QDs with survivin siRNA, and the red fluorescence from CdSe QDs and green fluorescein amidite fluorescence from siRNA could both be easily observed after 6 hours of incubation. The release of siRNA into the cytoplasm was verified through real-time PCR quantification that showed reduced survivin mRNA levels. In this study, survivin siRNA successfully complexed with water-soluble CdSe QDs and exhibited excellent fluorescent properties and downregulated the expression of the survivin gene in oral squamous cell carcinoma Tca8113 cells. QDs are a novel non-viral gene delivery vector. Keywords: quantum dots, survivin, siRNA delivery, transfection, Tca8113, tongue cancer cells

  17. Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors

    Science.gov (United States)

    Huang, Rongqin; Liu, Shuhuan; Shao, Kun; Han, Liang; Ke, Weilun; Liu, Yang; Li, Jianfeng; Huang, Shixian; Jiang, Chen

    2010-07-01

    Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

  18. Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors

    Energy Technology Data Exchange (ETDEWEB)

    Huang Rongqin; Liu Shuhuan; Shao Kun; Han Liang; Ke Weilun; Liu Yang; Li Jianfeng; Huang Shixian; Jiang Chen, E-mail: jiangchen@shmu.edu.cn [Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203 (China)

    2010-07-02

    Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

  19. Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

    Directory of Open Access Journals (Sweden)

    Tuttle John

    2012-08-01

    Full Text Available Abstract Background We previously developed a virus-induced gene silencing (VIGS vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV. The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens. We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

  20. Bidirectional DC/DC Converter

    Science.gov (United States)

    Pedersen, F.

    2008-09-01

    The presented bidirectional DC/DC converter design concept is a further development of an already existing converter used for low battery voltage operation.For low battery voltage operation a high efficient low parts count DC/DC converter was developed, and used in a satellite for the battery charge and battery discharge function.The converter consists in a bidirectional, non regulating DC/DC converter connected to a discharge regulating Buck converter and a charge regulating Buck converter.The Bidirectional non regulating DC/DC converter performs with relatively high efficiency even at relatively high currents, which here means up to 35Amps.This performance was obtained through the use of power MOSFET's with on- resistances of only a few mille Ohms connected to a special transformer allowing paralleling several transistor stages on the low voltage side of the transformer. The design is patent protected. Synchronous rectification leads to high efficiency at the low battery voltages considered, which was in the range 2,7- 4,3 Volt DC.The converter performs with low switching losses as zero voltage zero current switching is implemented in all switching positions of the converter.Now, the drive power needed, to switch a relatively large number of low Ohm , hence high drive capacitance, power MOSFET's using conventional drive techniques would limit the overall conversion efficiency.Therefore a resonant drive consuming considerable less power than a conventional drive circuit was implemented in the converter.To the originally built and patent protected bidirectional non regulating DC/DC converter, is added the functionality of regulation.Hereby the need for additional converter stages in form of a Charge Buck regulator and a Discharge Buck regulator is eliminated.The bidirectional DC/DC converter can be used in connection with batteries, motors, etc, where the bidirectional feature, simple design and high performance may be useful.

  1. 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

  2. Towards prostate cancer gene therapy: Development of a chlorotoxin-targeted nanovector for toxic (melittin) gene delivery.

    Science.gov (United States)

    Tarokh, Zahra; Naderi-Manesh, Hossein; Nazari, Mahboobeh

    2017-03-01

    Prostate cancer is the second leading cause of death due to cancer in men. Owing to shortcomings in the current treatments, other therapies are being considered. Toxic gene delivery is one of the most effective methods for cancer therapy. Cationic polymers are able to form stable nanoparticles via interaction with nucleic acids electrostatically. Branched polyethylenimine that contains amine groups has notable buffering capacity and the ability to escape from endosome through the proton sponge effect. However, the cytotoxicity of this polymer is high, and modification is one of the applicable strategies to overcome this problem. In this study, PEI was targeted with chlorotoxin (CTX) via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker. CTX can bind specifically to matrix metalloproteinase-2 that is overexpressed in certain cancers. Melittin as the major component of bee venom has been reported to have anti-cancer activity. This was thus selected to deliver to PC3 cell line. Flow cytometry analysis revealed that transfection efficiency of targeted nanoparticles is significantly higher compared to non-targeted nanoparticles. Targeted nanoparticles carrying the melittin gene also decreased cell viability of PC3 cells significantly while no toxic effects were observed on NIH3T3 cell line. Therefore, CTX-targeted nanoparticles carrying the melittin gene could serve as an appropriate gene delivery system for prostate and other MMP-2 positive cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Selective atonal gene delivery improves balance function in a mouse model of vestibular disease.

    Science.gov (United States)

    Schlecker, C; Praetorius, M; Brough, D E; Presler, R G; Hsu, C; Plinkert, P K; Staecker, H

    2011-09-01

    Loss of balance is often due to loss of vestibular hair cells. In mammals, regeneration of functional hair cells in the mature sensory epithelium is limited; therefore, loss of sensory cells can lead to debilitating balance problems. Delivery of the transcription factor atonal (atoh1) after aminoglycoside ototoxicity has previously been shown to induce the transdifferentiation of supporting cells into new hair cells and restore function. A problem with mouse aminoglycoside models is that the partial loss of hair cells seen in human disease is difficult to establish consistently. To more closely mirror human clinical balance dysfunction, we have used systemic application of 3,3'-iminodipropionitrile (IDPN), a vestibulotoxic nitrile compound known to cause vestibular hair cell loss, to induce a consistent partial loss of vestibular hair cells. To determine if balance function could be restored, we delivered atoh1 using a new adenovirus vector, based on Ad28. The Ad28 adenovector is based on a human serotype with a low seroprevalence that appears to target gene delivery to vestibular supporting cells. To further provide cell type selectivity of gene delivery, we expressed atoh1 using the supporting cell-specific glial fibrillary acid protein promoter. Delivery of this vector to IDPN-damaged vestibular organs resulted in a significant recovery of vestibular hair cells and restoration of balance, as measured by time on rotarod compared with untreated controls.

  4. Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases.

    Science.gov (United States)

    Suen, Colin M; Mei, Shirley H J; Kugathasan, Lakshmi; Stewart, Duncan J

    2013-10-01

    Pulmonary arterial hypertension (PAH) is a devastating disease that, despite significant advances in medical therapies over the last several decades, continues to have an extremely poor prognosis. Gene therapy is a method to deliver therapeutic genes to replace defective or mutant genes or supplement existing cellular processes to modify disease. Over the last few decades, several viral and nonviral methods of gene therapy have been developed for preclinical PAH studies with varying degrees of efficacy. However, these gene delivery methods face challenges of immunogenicity, low transduction rates, and nonspecific targeting which have limited their translation to clinical studies. More recently, the emergence of regenerative approaches using stem and progenitor cells such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have offered a new approach to gene therapy. Cell-based gene therapy is an approach that augments the therapeutic potential of EPCs and MSCs and may deliver on the promise of reversal of established PAH. These new regenerative approaches have shown tremendous potential in preclinical studies; however, large, rigorously designed clinical studies will be necessary to evaluate clinical efficacy and safety. © 2013 American Physiological Society. Compr Physiol 3:1749-1779, 2013.

  5. Low cytotoxicity fluorescent PAMAM dendrimer as gene carriers for monitoring the delivery of siRNA

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Lingmei [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Huang, Saipeng [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Chen, Zhao [Xi’an Jiaotong University, School of Science (China); Li, Yanchao [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Liu, Ke [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Liu, Yang, E-mail: yliu@iccas.ac.cn; Du, Libo, E-mail: dulibo@iccas.ac.cn [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China)

    2015-09-15

    Visual detection of gene vectors has attracted a great deal of attention due to the application of these vectors in monitoring and evaluating the effect of gene carriers in living cells. A non-viral vector, the fluorescent PAMAM dendrimer (F-PAMAM), was synthesized through conjugation of PAMAM dendrimers and fluorescein. In vitro and ex vivo experiments show that F-PAMAM exhibits superphotostability, low cytotoxicity and facilitates endocytosis by A549 cells. The vector has a high siRNA binding affinity and it increases the efficiency of cy5-siRNA delivery in A549 cells, in comparison with a cy5-siRNA monomer. Our results provide a new method for simultaneously monitoring the delivery of siRNA and its non-viral carriers in living cells.

  6. The effect of dexamethasone/cell-penetrating peptide nanoparticles on gene delivery for inner ear therapy

    Directory of Open Access Journals (Sweden)

    Yoon JY

    2016-11-01

    Full Text Available Ji Young Yoon,1 Keum-Jin Yang,2 Shi-Nae Park,3 Dong-Kee Kim,3 Jong-Duk Kim1 1Department of Chemical and Biomolecular Engineering, BK 21 Plus Program, Korea Advanced Institute of Science and Technology, Guseong-Dong, Yuseong-Gu, Daejeon, 2Clinical Research Institute, St Mary’s Hospital, Daejeon, 3Department of Otolaryngology – Head and Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Abstract: Dexamethasone (Dex-loaded PHEA-g-C18-Arg8 (PCA nanoparticles (PCA/Dex were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18 core with Dex and an arginine 8 (Arg8 peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA. The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120–140 nm and a zeta potential of 20–25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr than did Dex-free PCA nanoparticles and Lipofectamine® reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30

  7. 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.

  8. Hydrodynamic gene delivery in human skin using a hollow microneedle device.

    Science.gov (United States)

    Dul, M; Stefanidou, M; Porta, P; Serve, J; O'Mahony, C; Malissen, B; Henri, S; Levin, Y; Kochba, E; Wong, F S; Dayan, C; Coulman, S A; Birchall, J C

    2017-11-10

    Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. [The progesterone receptor gene polymorphism as factor of risk for the preterm delivery].

    Science.gov (United States)

    Oliveira, Tenilson Amaral; da Cunha, Danielle Renzoni; Policastro, Adriana; Traina, Évelyn; Gomes, Mariano Tamura; Cordioli, Eduardo

    2011-06-01

    to investigate the association between gene polymorphism of the progesterone receptor (PROGINS) and the risk of premature birth. In this case-control study, 57 women with previous premature delivery (Case Group) and 57 patients with delivery at term in the current pregnancy and no history of preterm delivery (Control Group) were selected. A 10 mL amount of peripheral blood was collected by venipuncture and genomic DNA was extracted followed by the polymerase chain reaction (PCR) under specific conditions for this polymorphism and 2% agarose gel electrophoresis. The bands were visualized with an ultraviolet light transilluminator. Genotype and allele PROGINS frequencies were compared between the two groups by the χ2 test, with the level of significance set at value p < 0.05. The Odds Ratio (OR) was also used, with 95% confidence intervals. PROGINS genotypic frequencies were 75.4% T1/T1, 22.8% T1/T2 and 1.8% T2/T2 in the Group with Preterm Delivery and 80.7% T1/T1, 19.3% T1/T2 and 0% T2/T2 in the term Delivery Group. There were no differences between groups when genotype and allele frequencies were analyzed: p = 0.4 (OR = 0.7) and p = 0.4 (OR = 0.7). the present study suggests that the presence of PROGINS polymorphism in our population does not constitute a risk factor for premature birth.

  10. Hydrodynamic gene delivery in human skin using a hollow microneedle device

    OpenAIRE

    Dul, M.; Stefanidou, M.; Porta, P.; Serve, J.; O'Mahony, Conor; Malissen, B.; Henri, S.; Levin, Y.; Kochba, E.; Wong, F. S.; Dayan, C.; Coulman, S. A.; Birchall, J. C.

    2017-01-01

    Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potenti...

  11. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    Science.gov (United States)

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  12. Polymorphisms in DC-SIGN and L-SIGN genes are associated with HIV-1 vertical transmission in a Northeastern Brazilian population.

    Science.gov (United States)

    da Silva, Ronaldo Celerino; Segat, Ludovica; Zanin, Valentina; Arraes, Luiz Claudio; Crovella, Sergio

    2012-11-01

    DC-SIGN and L-SIGN are receptors expressed on specialized macrophages in decidua, (Hofbauer and placental capillary endothelial cells), known to interact with several pathogens, including HIV-1. To disclose the possible involvement of these molecules in the susceptibility to HIV vertical transmission, we analyzed DC-SIGN and L-SIGN gene single nucleotide polymorphisms (SNPs) in 192 HIV-1 positive children and 58 HIV-1 negative children all born to HIV-1 positive mothers, as well as 96 healthy uninfected children not exposed to HIV-1, all from Northeast Brazil. The frequency of three SNPs in the DC-SIGN promoter (-139G>A, -201G>T and -336A>G) were significantly different when comparing HIV positive children with HIV-1 exposed uninfected children, indicating an association with susceptibility to HIV-1 vertical transmission. This genetic association suggests that DC-SIGN molecule may play a role in susceptibility to HIV-1 infection through vertical transmission. Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  13. Protein antigen delivery by gene gun-mediated epidermal antigen incorporation (EAI).

    Science.gov (United States)

    Scheiblhofer, Sandra; Ritter, Uwe; Thalhamer, Josef; Weiss, Richard

    2013-01-01

    The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this chapter we present a method for delivery of the model antigen ovalbumin into the skin of mice termed epidermal antigen incorporation and describe in detail how antigen-specific proliferation in draining lymph nodes can be followed by flow cytometry.

  14. Polydnaviruses of Parasitic Wasps: Domestication of Viruses To Act as Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Michael R. Strand

    2012-01-01

    Full Text Available Symbiosis is a common phenomenon in which associated organisms can cooperate in ways that increase their ability to survive, reproduce, or utilize hostile environments. Here, we discuss polydnavirus symbionts of parasitic wasps. These viruses are novel in two ways: (1 they have become non-autonomous domesticated entities that cannot replicate outside of wasps; and (2 they function as a delivery vector of genes that ensure successful parasitism of host insects that wasps parasitize. In this review we discuss how these novelties may have arisen, which genes are potentially involved, and what the consequences have been for genome evolution.

  15. Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

    Directory of Open Access Journals (Sweden)

    Zongchao Han

    Full Text Available Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV, makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs, critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.

  16. Efficient and safe gene delivery to human corneal endothelium using magnetic nanoparticles.

    Science.gov (United States)

    Czugala, Marta; Mykhaylyk, Olga; Böhler, Philip; Onderka, Jasmine; Stork, Björn; Wesselborg, Sebastian; Kruse, Friedrich E; Plank, Christian; Singer, Bernhard B; Fuchsluger, Thomas A

    2016-07-01

    To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.

  17. Gene delivery process in a single animal cell after femtosecond laser microinjection

    Science.gov (United States)

    Hosokawa, Yoichiroh; Iguchi, Seriya; Yasukuni, Ryohei; Hiraki, Yuji; Shukunami, Chisa; Masuhara, Hiroshi

    2009-09-01

    Microinjection of extracellular molecules into a single animal cell was performed by an amplified femtosecond laser irradiation. When a single-shot laser pulse was focused on the plasma membrane of a single fibroblast from the mouse cell line NIH3T3 with a high-numerical aperture objective lens, a transient hole with a diameter of 1 μm was formed. The delivery process of extracellular molecules immediately after the hole formation was monitored by a fluorescence staining with fluoresceinisothiocyanate-dextran (FITC-dextran). Then the gene expression was confirmed using a DNA plasmid of an enhanced green fluorescent protein (EGFP). The gene expression was observed when the laser pulse was focused first on the cellular membrane and then on the nuclear membrane, while the gene was not expressed when the laser was focused only on the cellular membrane. On the basis of these results, the efficiency of gene delivery by the femtosecond laser microinjection and the subsequent gene expression were clarified.

  18. Synergistic effects between natural histone mixtures and polyethylenimine in non-viral gene delivery in vitro.

    Science.gov (United States)

    Schneeweiss, Anne; Buyens, Kevin; Giese, Matthias; Sanders, Niek; Ulbert, Sebastian

    2010-11-15

    Nanoparticles made of plasmid DNA (pDNA) and cationic polymers are promising strategies for non-viral gene delivery. However, many cationic polymers are toxic to cells when used in higher concentrations. Positively charged proteins, such as histones, are biodegradable and a good alternative, especially for potential in vivo applications. It has previously been shown that histones are able to complex DNA and mediate transfection of cells. To investigate possible synergistic effects between the different histone types and to avoid the use of recombinant proteins, we analysed whether natural histone mixtures would be functional as gene carriers. Core and linker histones from calf thymus and from chicken erythrocytes were used to transfect different cell lines. The protein mixtures efficiently complexed the pDNA, and the resulting particles entered the cells. However, only marginal expression of the gene encoded by the pDNA was observed. Transfection rates increased drastically when minimal amounts of the basic polymer polyethylenimine (PEI) were added to the particles. Neither PEI nor histones alone mediated any transfection under the conditions where a combination of both worked efficiently, and the combined particles were well tolerated by the cells. These results demonstrate that histone mixtures from natural sources in combination with minimal amounts of PEI can be used as gene carriers. This might have consequences for the development of novel gene delivery strategies, such as DNA vaccines, with minimal side-effects. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Microneedle delivery of plasmid DNA to living human skin: formulation coating, skin insertion and gene expression

    Science.gov (United States)

    Pearton, Marc; Saller, Verena; Coulman, Sion A; Gateley, Chris; Anstey, Alexander V; Zarnitsyn, Vladimir; Birchall, James C

    2012-01-01

    Microneedle delivery of nucleic acids, in particular plasmid DNA (pDNA), to the skin represents a potential new approach for the clinical management of genetic skin diseases and cutaneous cancers, and for intracutaneous genetic immunization. In this study excised human skin explants were used to investigate and optimize key parameters that will determine stable and effective microneedle-facilitated pDNA delivery. These include (i) high dose-loading of pDNA onto microneedle surfaces, (ii) stability and functionality of the coated pDNA, (iii) skin penetration capability of pDNA-coated microneedles, and (iv) efficient gene expression in human skin. Optimization of a dip-coating method enabled significant increases in the loading capacity, up to 100 micrograms of pDNA per 5-microneedle array. Coated microneedles were able to reproducibly perforate human skin at low (coated pDNA was partially compromised on storage, although this was improved through the addition of saccharide excipients without detriment to the biological functionality of pDNA. The pDNA-coated microneedles facilitated reporter gene expression in viable human skin. The efficiency of gene expression from coated microneedles will depend upon suitable DNA loading, efficient and reproducible skin puncture and rapid in situ dissolution of the plasmid at the site of delivery. PMID:22516089

  20. Chitosan/TPP-hyaluronic acid nanoparticles: a new vehicle for gene delivery to the spinal cord.

    Science.gov (United States)

    Gwak, So-Jung; Jung, Jong Kwon; An, Sung Su; Kim, Hyo Jin; Oh, Jin Soo; Pennant, William A; Lee, Hye Yeong; Kong, Min Ho; Kim, Keung Nyun; Yoon, Do Heum; Ha, Yoon

    2012-01-01

    Gene delivery offers therapeutic promise for the treatment of neurological diseases and spinal cord injury. Several studies have offered viral vectors as vehicles to deliver therapeutic agents, yet their toxicity and immunogenicity, along with the cost of their large-scale formulation, limits their clinical use. As such, non-viral vectors are attractive in that they offer improved safety profiles compared to viruses. Poly(ethylene imine) (PEI) is one of the most extensively studied non-viral vectors, but its clinical value is limited y its cytotoxicity. Recently, chitosan/DNA complex nanoparticles have een considered as a vector for gene delivery. Here, we demonstrate that DNA nanoparticles made of hyaluronic acid (HA) and chitosan have low cytotoxicity and induce high transgene expression in neural stem cells and organotypic spinal cord slice tissue. Chitosan-TPP/HA nanoparticles were significantly less cytotoxic than PEI at various concentrations. Additionally, chitosan-TPP/HA nanoparticles with pDNA induced higher transgene expression in vitro for a longer duration than PEI in neural stem cells. These results suggest chitosan-TPP/HA nanoparticles may have the potential to serve as an option for gene delivery to the spinal cord.

  1. Biopolymer-Based Nanoparticles for Drug/Gene Delivery and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Keiji Numata

    2013-01-01

    Full Text Available There has been a great interest in application of nanoparticles as biomaterials for delivery of therapeutic molecules such as drugs and genes, and for tissue engineering. In particular, biopolymers are suitable materials as nanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability and low immunogenicity. Biopolymers are polymers that are produced from living organisms, which are classified in three groups: polysaccharides, proteins and nucleic acids. It is important to control particle size, charge, morphology of surface and release rate of loaded molecules to use biopolymer-based nanoparticles as drug/gene delivery carriers. To obtain a nano-carrier for therapeutic purposes, a variety of materials and preparation process has been attempted. This review focuses on fabrication of biocompatible nanoparticles consisting of biopolymers such as protein (silk, collagen, gelatin, β-casein, zein and albumin, protein-mimicked polypeptides and polysaccharides (chitosan, alginate, pullulan, starch and heparin. The effects of the nature of the materials and the fabrication process on the characteristics of the nanoparticles are described. In addition, their application as delivery carriers of therapeutic drugs and genes and biomaterials for tissue engineering are also reviewed.

  2. Capillary electrophoresis analysis of poly(ethylene glycol) and ligand-modified polylysine gene delivery vectors.

    Science.gov (United States)

    Guo, Yan; Sun, Ye; Gu, Jianren; Xu, Yuhong

    2007-04-15

    Cationic polymers including polylysine (PLL) and polyethylenimine are being widely tested as gene delivery vectors in various gene therapy applications. In many cases, the polymers were further modified by hydrophilic polymer grafting or ligand conjugation, which had been shown to greatly affect the vector stability, delivery efficiency and specificity. The characterization of modified polycation is particularly critical for quality control and vector development. Here several different separation modes using capillary electrophoresis for the analytical characterization of the modified polymers are described. PLL molecules were grafted with poly(ethylene glycol) (PEG) chain or conjugated with epidermal growth factor and analyzed under various analytical conditions. Poly(N,N'-dimethylacrylamide)-coated capillary was used to analyze the modified PLL to reduce the interaction between the samples and the capillary wall. PLLs containing different numbers of conjugated ligands were well separated with the coating method but, for PLL-g-PEG, the separation was poor under the same conditions. A method using low buffer pH and hydroxypropylmethyl cellulose additive was developed. These methods are useful to characterize various polycations and important for the quality control and application of potential gene delivery vectors.

  3. Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice

    Directory of Open Access Journals (Sweden)

    Mitsuru Ando

    2014-01-01

    Full Text Available Sustained gene delivery of interferon (IFN γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

  4. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    Science.gov (United States)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  5. Radiation-Tolerant DC-DC Converters

    Science.gov (United States)

    Skutt, Glenn; Sable, Dan; Leslie, Leonard; Graham, Shawn

    2012-01-01

    A document discusses power converters suitable for space use that meet the DSCC MIL-PRF-38534 Appendix G radiation hardness level P classification. A method for qualifying commercially produced electronic parts for DC-DC converters per the Defense Supply Center Columbus (DSCC) radiation hardened assurance requirements was developed. Development and compliance testing of standard hybrid converters suitable for space use were completed for missions with total dose radiation requirements of up to 30 kRad. This innovation provides the same overall performance as standard hybrid converters, but includes assurance of radiation- tolerant design through components and design compliance testing. This availability of design-certified radiation-tolerant converters can significantly reduce total cost and delivery time for power converters for space applications that fit the appropriate DSCC classification (30 kRad).

  6. Co-silencing of tomato S-adenosylhomocysteine hydrolase genes confers increased immunity against Pseudomonas syringae pv. tomato DC3000 and enhanced tolerance to drought stress

    Directory of Open Access Journals (Sweden)

    Li Xiao Hui

    2015-09-01

    Full Text Available S-adenosylhomocysteine hydrolase (SAHH, catalyzing the reversible hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.

  7. Milliwatt dc/dc Inverter

    Science.gov (United States)

    Mclyman, C. W.

    1983-01-01

    Compact dc/dc inverter uses single integrated-circuit package containing six inverter gates that generate and amplify 100-kHz square-wave switching signal. Square-wave switching inverts 10-volt local power to isolated voltage at another desired level. Relatively high operating frequency reduces size of filter capacitors required, resulting in small package unit.

  8. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

    Directory of Open Access Journals (Sweden)

    Yarong Liu

    2014-01-01

    Full Text Available Adeno-associated virus type 2 (AAV2 is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs. We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.

  9. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    Science.gov (United States)

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  10. Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

    Science.gov (United States)

    Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

    2012-01-01

    The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

  11. Chitosan tripolyphosphate (CS/TPP) nanoparticles: preparation, characterization and application for gene delivery in shrimp.

    Science.gov (United States)

    Vimal, S; Abdul Majeed, S; Taju, G; Nambi, K S N; Sundar Raj, N; Madan, N; Farook, M A; Rajkumar, T; Gopinath, D; Sahul Hameed, A S

    2013-12-01

    The present study examines the use of CS/TPP nanoparticles for gene delivery in different tissues of shrimp through oral route. The viral gene of WSSV was used to construct DNA vaccines using pcDNA 3.1, a eukaryotic expression vector and the constructs were named as pVP28. The CS/TPP nanoparticles were synthesized by ionic gelation process and these particles were characterized. The structure and morphology of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by Immuno-dot blot, ELISA and RT-PCR analyses. The results indicate that DNA can be easily delivered into shrimp by feeding with CS/TPP nanoparticles. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Comparison of different cationized proteins as biomaterials for nanoparticle-based ocular gene delivery.

    Science.gov (United States)

    Zorzi, Giovanni K; Párraga, Jenny E; Seijo, Begoña; Sanchez, Alejandro

    2015-11-01

    Cationized polymers have been proposed as transfection agents for gene therapy. The present work aims to improve the understanding of the potential use of different cationized proteins (atelocollagen, albumin and gelatin) as nanoparticle components and to investigate the possibility of modulating the physicochemical properties of the resulting nanoparticle carriers by selecting specific protein characteristics in an attempt to improve current ocular gene-delivery approaches. The toxicity profiles, as well as internalization and transfection efficiency, of the developed nanoparticles can be modulated by modifying the molecular weight of the selected protein and the amine used for cationization. The most promising systems are nanoparticles based on intermediate molecular weight gelatin cationized with the endogenous amine spermine, which exhibit an adequate toxicological profile, as well as effective association and protection of pDNA or siRNA molecules, thereby resulting in higher transfection efficiency and gene silencing than the other studied formulations. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Ultrasound and microbubble-targeted delivery of therapeutic compounds : ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles

    NARCIS (Netherlands)

    Juffermans, L J M; Meijering, D B M; van Wamel, A; Henning, R H; Kooiman, K; Emmer, M; de Jong, N; van Gilst, W H; Musters, R; Paulus, W J; van Rossum, A C; Deelman, L E; Kamp, O

    The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene

  14. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

    2015-01-01

    Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...... with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle....

  15. Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

    Science.gov (United States)

    Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-11-01

    The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

  16. Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Chen; Jie, Leng; Yongqi, Wang [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Weiming, Xiao [Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Juqun, Xi [Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, 225009 (China); Yanbing, Ding [Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Li, Qian [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Xingyuan, Pan [Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009 (China); Mingchun, Ji [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Weijuan, Gong, E-mail: wjgong@yzu.edu.cn [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, 225009 (China); Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009 (China); Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 (China)

    2015-07-31

    Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8{sup +} T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy. - Highlights: • Generation of a nanoparticle for delivery of dsNKG2D-IL-15 gene. • Characterization of the gene nanoparticle. • Antitumor activity mediated by the gene nanoparticle.

  17. Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques.

    Science.gov (United States)

    Gombash, S E; Cowley, C J; Fitzgerald, J A; Lepak, C A; Neides, M G; Hook, K; Todd, L J; Wang, G-D; Mueller, C; Kaspar, B K; Bielefeld, E C; Fischer, A J; Wood, J D; Foust, K D

    2017-10-01

    Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or phosphate-buffered saline. Piglets were euthanized three weeks post injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP-positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9-treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders.

  18. Cell-type-specific gene delivery into neuronal cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Parveen, Zahida; Mukhtar, Muhammad; Rafi, Mohammed; Wenger, David A.; Siddiqui, Khwaja M.; Siler, Catherine A.; Dietzschold, Bernhard; Pomerantz, Roger J.; Schnell, Matthias J.; Dornburg, Ralph

    2003-01-01

    The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain

  19. 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.

  20. Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium.

    Directory of Open Access Journals (Sweden)

    Maria D I Manunta

    Full Text Available Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy.The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI. We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo.RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6 compatible with deposition in the central and lower airways.RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.

  1. Dehalogenimonas lykanthroporepellens BL-DC-9T simultaneously transcribes many rdhA genes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors.

    Science.gov (United States)

    Mukherjee, Kalpataru; Bowman, Kimberly S; Rainey, Fred A; Siddaramappa, Shivakumara; Challacombe, Jean F; Moe, William M

    2014-05-01

    The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  2. Non-viral gene delivery strategies for gene therapy: a “ménage à trois” among nucleic acids, materials, and the biological environment

    International Nuclear Information System (INIS)

    Pezzoli, Daniele; Candiani, Gabriele

    2013-01-01

    Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription–translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

  3. Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.

    2002-01-01

    The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

  4. Amphiphilic block co-polymers: preparation and application in nanodrug and gene delivery.

    Science.gov (United States)

    Xiong, Xiao-Bing; Binkhathlan, Ziyad; Molavi, Ommoleila; Lavasanifar, Afsaneh

    2012-07-01

    Self-assembly of amphiphilic block co-polymers composed of poly(ethylene oxide) (PEO) as the hydrophilic block and poly(ether)s, poly(amino acid)s, poly(ester)s and polypropyleneoxide (PPO) as the hydrophobic block can lead to the formation of nanoscopic structures of different morphologies. These structures have been the subject of extensive research in the past decade as artificial mimics of lipoproteins and viral vectors for drug and gene delivery. The aim of this review is to provide an overview of the synthesis of commonly used amphiphilic block co-polymers. It will also briefly go over some pharmaceutical applications of amphiphilic block co-polymers as "nanodelivery systems" for small molecules and gene therapeutics. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  5. Integration of Drug, Protein, and Gene Delivery Systems with Regenerative Medicine

    Science.gov (United States)

    Lorden, Elizabeth R.; Levinson, Howard M.; Leong, Kam W.

    2013-01-01

    Regenerative medicine has the potential to drastically change the field of health care from reactive to preventative and restorative. Exciting advances in stem cell biology and cellular reprogramming have fueled the progress of this field. Biochemical cues in the form of small molecule drugs, growth factors, zinc finger protein transcription factors and nucleases, transcription activator-like effector nucleases, monoclonal antibodies, plasmid DNA, aptamers, or RNA interference agents can play an important role to influence stem cell differentiation and the outcome of tissue regeneration. Many of these biochemical factors are fragile and must act intracellularly at the molecular level. They require an effective delivery system, which can take the form of a scaffold (e.g. hydrogels and electrospun fibers), carrier (viral and nonviral), nano- and micro-particle, or genetically modified cell. In this review, we will discuss the history and current technologies of drug, protein and gene delivery in the context of regenerative medicine. Next we will present case examples of how delivery technologies are being applied to promote angiogenesis in non-healing wounds or prevent angiogenesis in age related macular degeneration. Finally, we will conclude with a brief discussion of the regulatory pathway from bench-to-bedside for the clinical translation of these novel therapeutics. PMID:25787742

  6. Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

    Science.gov (United States)

    Kakinohana, Osamu; Hefferan, Michael P.; Miyanohara, Atsushi; Nejime, Tetsuya; Marsala, Silvia; Juhas, Stefan; Juhasova, Jana; Motlik, Jan; Kucharova, Karolina; Strnadel, Jan; Platoshyn, Oleksandr; Lazar, Peter; Galik, Jan; Vinay, Laurent; Marsala, Martin

    2012-01-01

    Background Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABAB receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. Methods/Principal Findings Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. Conclusions/Significance These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel

  7. Fourth Generation Phosphorus-Containing Dendrimers: Prospective Drug and Gene Delivery Carrier

    Science.gov (United States)

    Shcharbin, D.; Dzmitruk, V.; Shakhbazau, A.; Goncharova, N.; Seviaryn, I.; Kosmacheva, S.; Potapnev, M.; Pedziwiatr-Werbicka, E.; Bryszewska, M.; Talabaev, M.; Chernov, A.; Kulchitsky, V.; Caminade, A.-M.; Majoral, J.-P.

    2011-01-01

    Research concerning new targeting delivery systems for pharmacologically active molecules and genetic material is of great importance. The aim of the present study was to investigate the potential of fourth generation (P4) cationic phosphorus-containing dendrimers to bind fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS), anti-neoplastic drug cisplatin, anti-HIV siRNA siP24 and its capability to deliver green fluorescent protein gene (pGFP) into cells. The interaction between P4 and ANS (as the model drug) was investigated. The binding constant and the number of binding centers per one molecule of P4 were determined. In addition, the dendriplex between P4 and anti-HIV siRNA siP24 was characterized using circular dichroism, fluorescence polarization and zeta-potential methods; the average hydrodynamic diameter of the dendriplex was calculated using zeta-size measurements. The efficiency of transfection of pGFP using P4 was determined in HEK293 cells and human mesenchymal stem cells, and the cytotoxicity of the P4-pGFP dendriplex was studied. Furthermore, enhancement of the toxic action of the anti-neoplastic drug cisplatin by P4 dendrimers was estimated. Based on the results, the fourth generation cationic phosphorus-containing dendrimers seem to be a good drug and gene delivery carrier candidate. PMID:24310590

  8. Mapping the AAV capsid host antibody response towards the development of second generation gene delivery vectors

    Directory of Open Access Journals (Sweden)

    Yu-Shan eTseng

    2014-01-01

    Full Text Available The recombinant Adeno-associated virus (rAAV gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2. Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from monoclonal antibodies, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.

  9. A Simple Zn2+ Complex-Based Composite System for Efficient Gene Delivery.

    Directory of Open Access Journals (Sweden)

    Zhe Zhang

    Full Text Available Metal complexes might become a new type of promising gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, and appropriate density and distribution of positive charges. In this study, Zn2+ complexes (1-10 formed with a series of ligands contained benzimidazole(bzimwere prepared and characterized. They were observed to have different affinities for DNA, dependent on their numbers of positive charges, bzim groups, and coordination structures around Zn2+. The binding induced DNA to condensate into spherical nanoparticles with ~ 50 nm in diameter. The cell transfection efficiency of the DNA nanoparticles was poor, although they were low toxic. The sequential addition of the cell-penetrating peptide (CPP TAT(48-60 and polyethylene glycol (PEG resulted in the large DNA condensates (~ 100 nm in diameter and the increased cellular uptake. The clathrin-mediated endocytosis was found to be a key cellular uptake pathway of the nanoparticles formed with or without TAT(48-60 or/and PEG. The DNA nanoparticles with TAT(48-60 and PEG was found to have the cell transfection efficiency up to 20% of the commercial carrier Lipofect. These results indicated that a simple Zn2+-bzim complex-based composite system can be developed for efficient and low toxic gene delivery through the combination with PEG and CPPs such as TAT.

  10. Genomic signature and toxicogenomics comparison of polycationic gene delivery nanosystems in human alveolar epithelial A549 cells

    Directory of Open Access Journals (Sweden)

    J Barar

    2009-10-01

    Full Text Available "nBackground and the purpose of the study: Of the gene delivery systems, non-viral polycationic gene delivery nanosystems have been alternatively exploited as a relatively safe delivery reagents compared to viral vectors. However, little is known about the genomic impacts of these delivery systems in target cells/tissues. In this study, the toxicogenomics and genotoxicity potential of some selected polycationic lipid/polymer based nanostructures (i.e., Oligofectamine® (OF, starburst polyamidoamine Polyfect® (PF and diaminobutane (DAB dendrimers were investigated in human alveolar epithelial A549 cells. "nMethods: To study the nature and the ontology of the gene expression changes in A549 cells upon treatment with polycationic nanostructures, MTT assay and microarray gene expression profiling methodology were employed. For microarray analysis, cyanine (Cy3/Cy5 labeled cDNA samples from treated and untreated cells were hybridized on target arrays housing 200 genes. "nResults and major conclusions: The polycationic nanosystems induced significant gene expression changes belonging to different genomic ontologies such as cell defence and apoptosis pathways. These data suggest that polycationic nanosystems can elicit multiple gene expression changes in A549 cells upon their chemical structures and interactions with cellular/subcellular components. Such impacts may interfere with the main goals of the desired genemedicine.

  11. Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

    OpenAIRE

    Zanta, Maria Antonietta; Belguise-Valladier, Pascale; Behr, Jean-Paul

    1999-01-01

    Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of ...

  12. A Review of Gene Delivery and Stem Cell Based Therapies for Regenerating Inner Ear Hair Cells

    Directory of Open Access Journals (Sweden)

    Michael S. Detamore

    2011-09-01

    Full Text Available Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration.

  13. Bioreducible liposomes for gene delivery: from the formulation to the mechanism of action.

    Directory of Open Access Journals (Sweden)

    Gabriele Candiani

    Full Text Available BACKGROUND: A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes. On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH, these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25:50:25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro. CONCLUSIONS/SIGNIFICANCE: The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies

  14. Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

    Science.gov (United States)

    Lv, Lulu; Zhao, Huiqing

    2014-01-01

    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. PMID:24392152

  15. Virus-Induced Gene Silencing-Based Functional Analyses Revealed the Involvement of Several Putative Trehalose-6-Phosphate Synthase/Phosphatase Genes in Disease Resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in Tomato.

    Science.gov (United States)

    Zhang, Huijuan; Hong, Yongbo; Huang, Lei; Liu, Shixia; Tian, Limei; Dai, Yi; Cao, Zhongye; Huang, Lihong; Li, Dayong; Song, Fengming

    2016-01-01

    Trehalose and its metabolism have been demonstrated to play important roles in control of plant growth, development, and stress responses. However, direct genetic evidence supporting the functions of trehalose and its metabolism in defense response against pathogens is lacking. In the present study, genome-wide characterization of putative trehalose-related genes identified 11 SlTPSs for trehalose-6-phosphate synthase, 8 SlTPPs for trehalose-6-phosphate phosphatase and one SlTRE1 for trehalase in tomato genome. Nine SlTPSs, 4 SlTPPs, and SlTRE1 were selected for functional analyses to explore their involvement in tomato disease resistance. Some selected SlTPSs, SlTPPs, and SlTRE1 responded with distinct expression induction patterns to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 as well as to defense signaling hormones (e.g., salicylic acid, jasmonic acid, and a precursor of ethylene). Virus-induced gene silencing-mediated silencing of SlTPS3, SlTPS4, or SlTPS7 led to deregulation of ROS accumulation and attenuated the expression of defense-related genes upon pathogen infection and thus deteriorated the resistance against B. cinerea or Pst DC3000. By contrast, silencing of SlTPS5 or SlTPP2 led to an increased expression of the defense-related genes upon pathogen infection and conferred an increased resistance against Pst DC3000. Silencing of SlTPS3, SlTPS4, SlTPS5, SlTPS7, or SlTPP2 affected trehalose level in tomato plants with or without infection of B. cinerea or Pst DC3000. These results demonstrate that SlTPS3, SlTPS4, SlTPS5, SlTPS7, and SlTPP2 play roles in resistance against B. cinerea and Pst DC3000, implying the importance of trehalose and tis metabolism in regulation of defense response against pathogens in tomato.

  16. Virus-induced Gene Silencing-based Functional Analyses Revealed the Involvement of Several Putative Trehalose-6-Phosphate Synthase/Phosphatase Genes in Disease Resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in Tomato

    Directory of Open Access Journals (Sweden)

    Huijuan Zhang

    2016-08-01

    Full Text Available Trehalose and its metabolism have been demonstrated to play important roles in control of plant growth, development and stress responses. However, direct genetic evidence supporting the functions of trehalose and its metabolism in defense response against pathogens is lacking. In the present study, genome-wide characterization of putative trehalose-related genes identified 11 SlTPSs for trehalose-6-phosphate synthase, 8 SlTPPs for trehalose-6-phosphate phosphatase and one SlTRE1 for trehalase in tomato genome. Nine SlTPSs, 4 SlTPPs and SlTRE1 were selected for functional analyses to explore their involvement in tomato disease resistance. Some selected SlTPSs, SlTPPs and SlTRE1 responded with distinct expression induction patterns to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst DC3000 as well as to defense signaling hormones (e.g. salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-mediated silencing of SlTPS3, SlTPS4 or SlTPS7 led to deregulation of ROS accumulation and attenuated the expression of defense-related genes upon pathogen infection and thus deteriorated the resistance against B. cinerea or Pst DC3000. By contrast, silencing of SlTPS5 or SlTPP2 led to an increased expression of the defense-related genes upon pathogen infection and conferred an increased resistance against Pst DC3000. Silencing of SlTPS3, SlTPS4, SlTPS5, SlTPS7 or SlTPP2 affected trehalose level in tomato plants with or without infection of B. cinerea or Pst DC3000. These results demonstrate that SlTPS3, SlTPS4, SlTPS5, SlTPS7 and SlTPP2 play roles in resistance against B. cinerea and Pst DC3000, implying the importance of trehalose and tis metabolism in regulation of defense response against pathogens in tomato.

  17. Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice.

    Science.gov (United States)

    Park, Hongsuk; Cho, Sungpil; Janat-Amsbury, Margit M; Bae, You Han

    2015-12-01

    Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Quaternized chitosan/rectorite intercalative materials for a gene delivery system

    International Nuclear Information System (INIS)

    Wang Xiaoying; Du Yumin; Pei Xiaofeng; Li Yan

    2008-01-01

    Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96 h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120 h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier

  19. Polyamidoamine (PAMAM Dendrimers Modified with Cathepsin-B Cleavable Oligopeptides for Enhanced Gene Delivery

    Directory of Open Access Journals (Sweden)

    Seulgi Lee

    2017-06-01

    Full Text Available Because of the complex mechanisms mediating cancer onset, prognosis, and metastatic behavior, different therapeutic approaches targeting these mechanisms have been investigated. Recent advancements in nanocarrier-based drug and gene delivery methods have encouraged scientific groups to investigate various novel therapeutic techniques. In this study, a poly(amidoamine (PAMAM polymer-based gene carrier containing the cathepsin B-enzyme sensitive sequence (glycine-phenylalanine-leucine-glycine, GFLG was evaluated to determine transfection efficiency. Following the GFLG sequence, the surface of PAMAM generation 4 (G4 was conjugated with histidine (H and arginine (R for improved endosomal escape and cellular uptake, respectively. The successful synthesis of G4-GLFG-H-R was confirmed by 1H-nuclear magnetic resonance spectroscopy. The polyplex composed of G4-GLFG-H-R and pDNA was simulated by the enzyme cathepsin B and induced endosomal escape of pDNA, which was confirmed by gel electrophoresis. Compared with the G4 control, enzyme-sensitive G4-GLFG-H-R showed higher transfection efficiency and lower cytotoxicity in HeLa cells. These results demonstrated that G4-GLFG-H-R may be a highly potent and efficient carrier for gene therapy applications.

  20. Targeted gene delivery in the cricket brain, using in vivo electroporation.

    Science.gov (United States)

    Matsumoto, Chihiro Sato; Shidara, Hisashi; Matsuda, Koji; Nakamura, Taro; Mito, Taro; Matsumoto, Yukihisa; Oka, Kotaro; Ogawa, Hiroto

    2013-12-01

    The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb'-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Quaternized chitosan/rectorite intercalative materials for a gene delivery system

    Science.gov (United States)

    Wang, Xiaoying; Pei, Xiaofeng; Du, Yumin; Li, Yan

    2008-09-01

    Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96 h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120 h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier.

  2. Novel pH-Sensitive Cationic Lipids with Linear Ortho Ester Linkers for Gene Delivery

    Science.gov (United States)

    Chen, Haigang; Zhang, Huizhen; Thor, Der; Rahimian, Roshanak; Guo, Xin

    2012-01-01

    In an effort to develop pH-sensitive lipoplexes for efficient gene delivery, we report three novel cationic lipids containing a linear ortho ester linker that conjugates either the headgroup (Type I) or one hydrocarbon chain (Type II) with the rest of the lipid molecule. The cationic lipids carry either an iodide or a chloride counterion. Compared to our previously reported cyclic ortho ester linker, the linear ortho ester linker facilitated the construction of cationic liposomes and lipoplexes with different helper lipids. The chloride counterion not only facilitated the hydration of the lipid films during liposome construction, but also enhanced the hydrolysis of the ortho ester linker in the lipoplexes. After incubation at endosomal pH 5.5, the Type I lipoplexes aggregated and destabilized the endosome-mimicking model liposomes, but not the Type II lipoplexes. The helper lipids (DOPE or cholesterol) of the lipoplexes enhanced the pH-sensitivity of the Type I lipoplexes. In CV-1 cells (monkey kidney fibroblast), the Type I ortho ester-based lipoplexes, especially those with the chloride counterion, significantly improved the gene transfection efficiency, in some cases by more than 100 fold, compared to their pH-insensitive counterparts consisting of DOTAP. The gene transfection efficiency of the ortho ester-based lipoplexes was well correlated with their rate of aggregation and membrane destabilization in response to the endosomal pH 5.5. PMID:22480493

  3. Efficient gene delivery to human umbilical cord mesenchymal stem cells by cationized Porphyra yezoensis polysaccharide nanoparticles.

    Science.gov (United States)

    Yu, Qingtong; Cao, Jin; Chen, Baoding; Deng, Wenwen; Cao, Xia; Chen, Jingjing; Wang, Yan; Wang, Shicheng; Yu, Jiangnan; Xu, Ximing; Gao, Xiangdong

    2015-01-01

    This study centered on an innovative application of Porphyra yezoensis polysaccharide (PPS) with cationic modification as a safe and efficient nonviral gene vector to deliver a plasmid encoding human Wnt3a (pWnt3a) into human umbilical cord mesenchymal stem cells (HUMSCs). After modification with branched low-molecular-weight (1,200 Da) polyethylenimine, the cationized PPS (CPPS) was combined with pWnt3a to form spherical nanoscale particles (CPPS-pWnt3a nanoparticles). Particle size and distribution indicated that the CPPS-pWnt3a nanoparticles at a CPPS:pWnt3a weight ratio of 40:1 might be a potential candidate for DNA plasmid transfection. A cytotoxicity assay demonstrated that the nanoparticles prepared at a CPPS:pWnt3a weight ratio of 40:1 were nontoxic to HUMSCs compared to those of Lipofectamine 2000 and polyethylenimine (25 kDa). These nanoparticles were further transfected to HUMSCs. Western blotting demonstrated that the nanoparticles (CPPS:pWnt3a weight ratio 40:1) had the greatest transfection efficiency in HUMSCs, which was significantly higher than that of Lipofectamine 2000; however, when the CPPS:pWnt3a weight ratio was increased to 80:1, the nanoparticle-treated group showed no obvious improvement in translation efficiency over Lipofectamine 2000. Therefore, CPPS, a novel cationic polysaccharide derived from P. yezoensis, could be developed into a safe, efficient, nonviral gene vector in a gene-delivery system.

  4. AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons: Implications for Gene Therapy and Disease Models

    Directory of Open Access Journals (Sweden)

    Katrina Albert

    2017-02-01

    Full Text Available Gene delivery using adeno-associated virus (AAV vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson′s disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson′s disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson′s disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson′s disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV-a-synuclein (a-syn to target substantia nigra dopamine neurons to produce an α-syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP, which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in

  5. Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Wen Jen, E-mail: wjlin@ntu.edu.tw; Chien, Wei Hsuan [National Taiwan University, School of Pharmacy, Graduate Institute of Pharmaceutical Sciences (China)

    2015-09-15

    The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly(d,l-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

  6. Nonviral Gene Delivery from Nonwoven Fibrous Scaffolds Fabricated by Interfacial Complexation of Polyelectrolytes

    Science.gov (United States)

    Lim, Shawn H.; Liao, I-Chien; Leong, Kam W.

    2008-01-01

    We investigated a novel nonwoven fibrous scaffold as a vehicle for delivery of DNA. Fibers were formed by polyelectrolyte complexation of water-soluble chitin and alginate, and PEI–DNA nanoparticles were encapsulated during the fiber drawing process. Nanoparticles released from the fibers over time retained their bioactivity and successfully transfected cells seeded on the scaffold in a sustained manner. Transgene expression in HEK293 cells and human dermal fibroblasts seeded on the transfecting scaffolds was significant even after 2 weeks of culture compared to 3-day expression in two-dimensional controls. Fibroblasts seeded on scaffolds containing DNA encoding basic fibroblast growth factor (bFGF) demonstrated prolonged secretion of bFGF at levels significantly higher than baseline. This work establishes the potential of this fibrous scaffold as a matrix capable of delivering genes to direct and support cellular development in tissue engineering. PMID:16497560

  7. Nano-niosomes in drug, vaccine and gene delivery: a rapid overview

    Directory of Open Access Journals (Sweden)

    Abbas Pardakhty

    2013-10-01

    Full Text Available   Abstract Niosomes, non-ionic surfactant vesicles (NSVs, are the hydrated lipids composed mainly of different classes of non-ionic surfactants, introduced in the seventies as a cosmetic vehicle. Nowadays, niosomes are used as important new drug delivery systems by many research groups and also they are effective immunoadjuvants which some commercial forms are available in the market. These vesicles recently used as gene transfer vectors as well. This review article presents a brief report about the achievements in the field of nanoscience related to NSVs. Different polar head groups from a vast list of various surfactants with one, two or three lipophilic alkyl, perfluoroalkyl and steroidal moieties may be utilized to form the proper vesicular structures for encapsulating both hydrophilic and hydrophobic compounds. The methods of niosome preparation, the vesicle stability related aspects and many examples of pharmaceutical applications of NSVs will be presented. The routes of administration of these amphiphilic assemblies are also discussed. 

  8. Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

    Science.gov (United States)

    Hong, Tu; Guo, Honglian; Xu, Yaqiong

    2011-10-01

    Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

  9. Graphene oxide-cationic polymer conjugates: Synthesis and application as gene delivery vectors.

    Science.gov (United States)

    Teimouri, Mohsen; Nia, Azadeh Hashem; Abnous, Khalil; Eshghi, Hossein; Ramezani, Mohammad

    2016-01-01

    Nanomedicine as the interface between nanotechnology and medical sciences is a new area that has attracted the attention of vast groups of researchers. Carbon nanomaterials are common platform for synthesis of nanoparticles for biomedical applications due to their low cytotoxicity and feasible internalization into mammalian cell lines (Yang et al., 2007; Arora et al., 2014; Oh and Park, 2014). Synthesis of vectors based on various cationic polymers polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM) and their derivatives were considered as a strategy for transferring plasmid DNA and treatment of genetic diseases. Considering the low cytotoxicity of graphene, chemical modification of its surface has led to fabrication of novel gene delivery systems based on graphene and graphene oxide. Herein we report the synthesis of three groups of vectors based on conjugation of graphene oxide (GO) with alkylated derivatives of three different cationic polymers (polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM)) through different linkers including surface carboxyl group, glycine and spermidine. Two main challenges in design of gene delivery vectors is decreasing cytotoxicity while improving the transfection efficiency. All synthesized vectors showed significantly lower cellular toxicity compared to bare polymer. A plasmid encoding green fluorescent protein (GFP) was used to evaluate the transfection efficiency of nanoparticles both qualitatively using live cell fluorescent imaging and quantitatively using flow cytometry and each vector was compared to its polymer base. Most successful conjugation strategy was observed in the case of PEI conjugates among which most efficient vector was PEI-GO conjugate bearing glycine linker. This vector was 9 fold more effective in terms of the percent of EGFP transfected cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Nonviral gene-delivery by highly fluorinated gemini bispyridinium surfactant-based DNA nanoparticles.

    Science.gov (United States)

    Fisicaro, Emilia; Compari, Carlotta; Bacciottini, Franco; Contardi, Laura; Pongiluppi, Erika; Barbero, Nadia; Viscardi, Guido; Quagliotto, Pierluigi; Donofrio, Gaetano; Krafft, Marie Pierre

    2017-02-01

    Biological and thermodynamic properties of a new homologous series of highly fluorinated bispyridinium cationic gemini surfactants, differing in the length of the spacer bridging the pyridinium polar heads in 1,1' position, are reported for the first time. Interestingly, gene delivery ability is closely associated with the spacer length due to a structural change of the molecule in solution. This conformation change is allowed when the spacer reaches the right length, and it is suggested by the trends of the apparent and partial molar enthalpies vs molality. To assess the compounds' biological activity, they were tested with an agarose gel electrophoresis mobility shift assay (EMSA), MTT proliferation assay and Transient Transfection assays on a human rhabdomyosarcoma cell line. Data from atomic force microscopy (AFM) allow for morphological characterization of DNA nanoparticles. Dilution enthalpies, measured at 298K, enabled the determination of apparent and partial molar enthalpies vs molality. All tested compounds (except that with the longest spacer), at different levels, can deliver the plasmid when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). The compound with a spacer formed by eight carbon atoms gives rise to a gene delivery ability that is comparable to that of the commercial reagent. The compound with the longest spacer compacts DNA in loosely condensed structures by forming bows, which are not suitable for transfection. Regarding the compounds' hydrogenated counterparts, the tight relationship between the solution thermodynamics data and their biological performance is amazing, making "old" methods the foundation to deeply understanding "new" applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Mannosylated Chitosan Nanoparticles Based Macrophage-Targeting Gene Delivery System Enhanced Cellular Uptake and Improved Transfection Efficiency.

    Science.gov (United States)

    Peng, Yixing; Yao, Wenjun; Wang, Bo; Zong, Li

    2015-04-01

    Gene transfer mediated by mannosylated chitosan (MCS) is a safe and promising approach for gene and vaccine delivery. MCS nanoparticles based gene delivery system showed high in vivo delivery efficiency and elicited strong immune responses in mice. However, little knowledge about the cell binding, transfection efficiency and intracellular trafficking of MCS nanoparticles had been acquired. In this study, using gastrin-releasing peptide as a model plasmid (pGRP), the binding of MCS/pGRP nanoparticles to macrophages and the intracellular trafficking of MCS/pGRP nanoparticles in macrophages were investigated. MCS-mediated transfection efficiency in macrophages was also evaluated using pGL-3 as a reporter gene. The results showed that the binding and transfection efficiency of MCS nanoparticles in macrophages was higher than that of CS, which was attributed to the interaction between mannose ligands in MCS and mannose receptors on the surface of macrophages. Observation with a confocal laser scanning microscope indicated the cellular uptake of MCS/pGRP nanoparticles were more than that of CS/pGRP nanoparticles in macrophages. MCS/pGRP nanoparticles were taken up by macrophages and most of them were entrapped in endosomal/lysosomal compartments. After the nanoparticles escaping from endosomal/lysosomal compartments, naked pGRP entered the nucleus, and a few MCS might enter the nucleus in terms of nanoparticles. Overall, MCS has the potential to be an excellent macrophage-targeting gene delivery carrier.

  12. Efficient gene delivery to human umbilical cord mesenchymal stem cells by cationized Porphyra yezoensis polysaccharide nanoparticles

    Directory of Open Access Journals (Sweden)

    Yu Q

    2015-11-01

    Full Text Available Qingtong Yu,1,2 Jin Cao,2 Baoding Chen,3 Wenwen Deng,2 Xia Cao,2 Jingjing Chen,2 Yan Wang,2 Shicheng Wang,2 Jiangnan Yu,2 Ximing Xu,2 Xiangdong Gao1 1School of Life Science and Technology, China Pharmaceutical University, Nanjing, 2Department of Pharmaceutics, School of Pharmacy and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University, 3Department of Medical Ultrasound, Affiliated Hospital of Jiangsu University, Zhenjiang, People’s Republic of China Abstract: This study centered on an innovative application of Porphyra yezoensis polysaccharide (PPS with cationic modification as a safe and efficient nonviral gene vector to deliver a plasmid encoding human Wnt3a (pWnt3a into human umbilical cord mesenchymal stem cells (HUMSCs. After modification with branched low-molecular-weight (1,200 Da polyethylenimine, the cationized PPS (CPPS was combined with pWnt3a to form spherical nanoscale particles (CPPS-pWnt3a nanoparticles. Particle size and distribution indicated that the CPPS-pWnt3a nanoparticles at a CPPS:pWnt3a weight ratio of 40:1 might be a potential candidate for DNA plasmid transfection. A cytotoxicity assay demonstrated that the nanoparticles prepared at a CPPS:pWnt3a weight ratio of 40:1 were nontoxic to HUMSCs compared to those of Lipofectamine 2000 and polyethylenimine (25 kDa. These nanoparticles were further transfected to HUMSCs. Western blotting demonstrated that the nanoparticles (CPPS:pWnt3a weight ratio 40:1 had the greatest transfection efficiency in HUMSCs, which was significantly higher than that of Lipofectamine 2000; however, when the CPPS:pWnt3a weight ratio was increased to 80:1, the nanoparticle-treated group showed no obvious improvement in translation efficiency over Lipofectamine 2000. Therefore, CPPS, a novel cationic polysaccharide derived from P. yezoensis, could be developed into a safe, efficient, nonviral gene vector in a gene-delivery system. Keywords: Porphyra yezoensis, nanoparticle

  13. Mesoporous Silica Nanomaterials for Applications in Catalysis, Sensing, Drug Delivery and Gene Transfection

    Energy Technology Data Exchange (ETDEWEB)

    Radu, Daniela Rodica [Iowa State Univ., Ames, IA (United States)

    2004-01-01

    The central theme of this dissertation is represented by the versatility of mesoporous silica nanomaterials in various applications such as catalysis and bio-applications, with main focus on biological applications of Mesoporous Silica Nanospheres (MSN). The metamorphosis that we impose to these materials from catalysis to sensing and to drug and gene delivery is detailed in this dissertation. First, we developed a synthetic method that can fine tune the amount of chemically accessible organic functional groups on the pores surface of MSN by exploiting electrostatic and size matching between the cationic alkylammonium head group of the cetyltrimethylammonium bromide (CTAB) surfactant and various anionic organoalkoxysilane precursors at the micelle-water interface in a base-catalyzed condensation reaction of silicate. Aiming nature imitation, we demonstrated the catalytic abilities of the MSNs, We utilized an ethylenediamine functional group for chelating Cu2+ as a catalytic functional group anchored inside the mesopores. Thus, a polyalkynylene-based conducting polymer (molecular wire) was synthesized within the Cu-functionalized MSNs silica catalyst. For sensing applications, we have synthesized a poly(lactic acid) coated mesoporous silica nanosphere (PLA-MSN) material that serves as a fluorescence sensor system for detection of amino-containing neurotransmitters in neutral aqueous buffer. We exploited the mesoporosity of MSNs for encapsulating pharmaceutical drugs. We examined bio-friendly capping molecules such as polyamidoamine dendrimers of generations G2 to G4, to prevent the drug leaching. Next, the drug delivery system employed MSNs loaded with Doxorubicin, an anticancer drug. The results demonstrated that these nano-Trojan horses have ability to deliver Doxorubicin to cancer cells and induce their death. Finally, to demonstrate the potential of MSN as an universal cellular transmembrane nanovehicle, we anchored positively charged dendrimers on

  14. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

    Science.gov (United States)

    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  15. Amino acid-substituted gemini surfactant-based nanoparticles as safe and versatile gene delivery agents.

    Science.gov (United States)

    Singh, Jagbir; Yang, Peng; Michel, Deborah; Verrall, Ronald E; Foldvari, Marianna; Badea, Ildiko

    2011-05-01

    Gene based therapy represents an important advance in the treatment of diseases that heretofore have had either no treatment or cure. To capitalize on the true potential of gene therapy, there is a need to develop better delivery systems that can protect these therapeutic biomolecules and deliver them safely to the target sites. Recently, we have designed and developed a series of novel amino acid-substituted gemini surfactants with the general chemical formula C(12)H(25) (CH(3))(2)N(+)-(CH(2))(3)-N(AA)-(CH(2))(3)-N(+) (CH(3))(2)-C(12)H(25) (AA= glycine, lysine, glycyl-lysine and, lysyl-lysine). These compounds were synthesized and tested in rabbit epithelial cells using a model plasmid and a helper lipid. Plasmid/gemini/lipid (P/G/L) nanoparticles formulated using these novel compounds achieved higher gene expression than the nanoparticles containing the parent unsubstituted compound. In this study, we evaluated the cytotoxicity of P/G/L nanoparticles and explored the relationship between transfection efficiency/toxicity and their physicochemical characteristics (such as size, binding properties, etc.). An overall low toxicity is observed for all complexes with no significant difference among substituted and unsubstituted compounds. An interesting result revealed by the dye exclusion assay suggests a more balanced protection of the DNA by the glycine and glycyl-lysine substituted compounds. Thus, the higher transfection efficiency is attributed to the greater biocompatibility and flexibility of the amino acid/peptide-substituted gemini surfactants and demonstrates the feasibility of using amino acid-substituted gemini surfactants as gene carriers for the treatment of diseases affecting epithelial tissue.

  16. 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

  17. Deriving isolates of powdery mildew (Blumeria graminis DC. f.sp. avenae Em. Marchal. in common oat (Avena sativa L. and using them to identify selected resistance genes

    Directory of Open Access Journals (Sweden)

    Sylwia Okoń

    2012-12-01

    Full Text Available Powdery mildew in common oat is caused by Blumeria graminis DC. f.sp. avenae Em. Marchal. Host-pathogen tests are commonly used to identify and locate resistance genes to powdery mildew in cereals. The aim of the study was to determine the virulence of powdery mildew isolates obtained from powdery mildew populations harvested in Poland and to identify OMR1, OMR2 and OMR3 resistance genes to powdery mildew in F2 populations of inter-cultivar hybrids of common oat: Bruno × Fuchs, Jumbo × Fuchs and Mostyn × Fuchs. On the basis of the analysis conducted, isolates enabling division of the studied populations into groups of resistant and susceptible plants were selected. M10 and M14 isolates were chosen for the population which was obtained from crossbreeding of ‘Bruno’ with ‘Fuchs’; these isolates demonstrated avirulence to Bruno cultivar containing OMR1 gene. In order to divide population obtained from crossbreeding of ‘Jumbo’ with ‘Fuchs’, M13 and M16 isolates were chosen; they demonstrated avirulence to the cultivar Jumbo containing the OMR2 gene. On the basis of the tests conducted, it was impossible to select isolates characterised by avirulence to the OMR3 gene. In the F2 population of Bruno × Fuchs and Jumbo × Fuchs hybrids, a division was made into resistant and susceptible plants. The obtained results were verified by the 2 test; the proportion in the dispersion matching model was found to be 3 resistant plants: 1 sensitive plant both in the Bruno × Fuchs and Jumbo × Fuchs populations. Such dispersion indicated that the resistance to powdery mildew in the studied cultivars Bruno and Jumbo was conditioned by single dominant genes.

  18. A rapid pathway toward a superb gene delivery system: programming structural and functional diversity into a supramolecular nanoparticle library.

    Science.gov (United States)

    Wang, Hao; Liu, Kan; Chen, Kuan-Ju; Lu, Yujie; Wang, Shutao; Lin, Wei-Yu; Guo, Feng; Kamei, Ken-ichiro; Chen, Yi-Chun; Ohashi, Minori; Wang, Mingwei; Garcia, Mitch André; Zhao, Xing-Zhong; Shen, Clifton K-F; Tseng, Hsian-Rong

    2010-10-26

    Nanoparticles are regarded as promising transfection reagents for effective and safe delivery of nucleic acids into a specific type of cells or tissues providing an alternative manipulation/therapy strategy to viral gene delivery. However, the current process of searching novel delivery materials is limited due to conventional low-throughput and time-consuming multistep synthetic approaches. Additionally, conventional approaches are frequently accompanied with unpredictability and continual optimization refinements, impeding flexible generation of material diversity creating a major obstacle to achieving high transfection performance. Here we have demonstrated a rapid developmental pathway toward highly efficient gene delivery systems by leveraging the powers of a supramolecular synthetic approach and a custom-designed digital microreactor. Using the digital microreactor, broad structural/functional diversity can be programmed into a library of DNA-encapsulated supramolecular nanoparticles (DNA⊂SNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA⊂SNPs library identified the DNA⊂SNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNA⊂SNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads.

  19. Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.

    Science.gov (United States)

    Lamichhane, Tek N; Raiker, Rahul S; Jay, Steven M

    2015-10-05

    Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

  20. Amphiphilic polymers formed from ring-opening polymerization: a strategy for the enhancement of gene delivery.

    Science.gov (United States)

    Zhang, Yi-Mei; Huang, Zheng; Zhang, Ji; Wu, Wan-Xia; Liu, Yan-Hong; Yu, Xiao-Qi

    2017-03-28

    Cationic liposomes and polymers are both important candidates for use as non-viral gene vectors. However, both of them have special shortcomings and application limits. This work is devoted to the combination of advantages of liposomes and polymers. The ring-opening polymerization strategy was used for the preparation of amphiphilic polymers from cyclen-based cationic small lipids. The non-hydrophobic polymer and the corresponding lipids were also prepared for performing structure-activity relationship studies. Gel electrophoresis results reveal that both the lipopolymers and liposomes could effectively condense DNA into nanoparticles and protect DNA from degradation. Compared to polymers, the DNA binding ability of liposomes is more affected by hydrophobic tails. Under the same dosage, the synthetic polymers have stronger DNA binding ability than the liposomes. In vitro transfection experiments show that the polymers could give better transfection efficiency, which was much higher than those of the corresponding liposomes and non-hydrophobic polymer. The oleyl moiety is suitable for lipidic vectors, but things were different for polymers. Under optimized conditions, up to 14.2 times higher transfection efficiency than that for 25 kDa bPEI could be obtained. More importantly, the lipopolymers showed much better serum tolerance, which was further confirmed by protein adsorption, gel electrophoresis, flow cytometry, and CLSM assays. The results indicate that ring-opening polymerization is a promising strategy for the enhancement of the gene delivery efficiency and biocompatibility of cationic lipids.

  1. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.

    Science.gov (United States)

    Liu, Chang; Zhang, Li; Liu, Hao; Cheng, Kun

    2017-11-28

    The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Perfluorochemical Liquid-Adenovirus Suspensions Enhance Gene Delivery to the Distal Lung

    Directory of Open Access Journals (Sweden)

    Jeffrey A. Kazzaz

    2011-01-01

    Full Text Available We compared lung delivery methods of recombinant adenovirus (rAd: (1 rAd suspended in saline, (2 rAd suspended in saline followed by a pulse-chase of a perfluorochemical (PFC liquid mixture, and (3 a PFC-rAd suspension. Cell uptake, distribution, and temporal expression of rAd were examined using A549 cells, a murine model using luciferase bioluminescence, and histological analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2–4 h. rAd transgene expression was improved in alveolar epithelial cells, and the level and distribution of luciferase expression when delivered in PFC-rAd suspensions consistently peaked at 24 h. These results demonstrate that PFC-rAd suspensions improve distribution and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.

  3. Polyglycerol-functionalized nanodiamond as a platform for gene delivery: Derivatization, characterization, and hybridization with DNA

    Directory of Open Access Journals (Sweden)

    Li Zhao

    2014-03-01

    Full Text Available A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (–OH → –OTs (tosylate → –N3 in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8 terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA through electrostatic attraction, making them promising as nonviral vectors for gene delivery.

  4. Synthetic polyspermine imidazole-4, 5-amide as an efficient and cytotoxicity-free gene delivery system

    Directory of Open Access Journals (Sweden)

    Duan S

    2012-07-01

    Full Text Available Shi-Yue Duan, Xue-Mei Ge, Nan Lu, Fei Wu, Weien Yuan, Tuo JinSchool of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, People's Republic of ChinaAbstract: A chemically dynamic spermine-based polymer: polyspermine imidazole-4, 5-amide (PSIA, Mw > 7 kDa was designed, synthesized, and evaluated in terms of its ability to deliver nucleic acids. This polymer was made from an endogenous monomer professionally condensing genes in sperms, spermine, and a known safety drug metabolite, imidazole-4, 5-dicarboxylic acid, through a bis-amide bond conjugated with the imidazole ring. This polymer can condense pDNA at a W/W ratio above 10 to form polyplexes (100–200 nm in diameter, which is consistent with the observation by transmission electron microscopy (TEM, and the zeta potential was in the range of 10–20 mV. The pDNA packaged polymer was stable in phosphate buffer solution (PBS at pH 7.4 (simulated body fluid while the polyplexes were releasing pDNA into the solution at pH 5.8 (simulated endo-lysosomes due to the degradation of the bis-amide linkages in response to changes in pH values. PSIA-polyplexes were able to achieve efficient cellular uptake and luciferase gene silencing by co-transfection of pDNA and siRNA in COS-7 cells and HepG2 cells with negligible cytotoxicity. Biodistribution of Rhodamine B-labeled PSIA-polyplexes after being systemically injected in BALB/c nude-mice showed that the polyplexes circulated throughout the body, accumulated mainly in the kidney at 4 hours of sample administration, and moved to the liver and spleen after 24 hours. All the results suggested that PSIA offered a promising example to balance the transfection efficiency and toxicity of a synthetic carrier system for the delivery of therapeutic nucleic acids.Keywords: gene delivery, polyspermine, cytotoxicity, transfection efficiency, biodistribution

  5. An efficient nonviral gene-delivery vector based on hyperbranched cationic glycogen derivatives

    Directory of Open Access Journals (Sweden)

    Liang X

    2014-01-01

    Full Text Available Xuan Liang,1,* Xianyue Ren,2,* Zhenzhen Liu,1 Yingliang Liu,1 Jue Wang,2 Jingnan Wang,2 Li-Ming Zhang,1 David YB Deng,2 Daping Quan,1 Liqun Yang1 1Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-Sen University, Guangzhou, People's Republic of China; 2Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China *Both these authors contributed equally to this work Background: The purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector. Methods: A series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino-1-propylamine (DMAPA-Glyp and 1-(2-aminoethyl piperazine (AEPZ-Glyp residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid–base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney and the CNE2 (human nasopharyngeal carcinoma cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley

  6. Poly(amido amine)s as gene delivery vectors: effects of quaternary nicotinamide moieties in the side chains

    NARCIS (Netherlands)

    Mateos timoneda, Miguel; Lok, Martin C.; Hennink, Wim E.; Feijen, Jan; Engbersen, Johannes F.J.

    2008-01-01

    To evaluate the effect of quaternary nicotinamide pendant groups on gene delivery properties, a series of poly(amido amine) (co)polymers were synthesized by Michael addition polymerization of N, N-cystaminebisacrylamide with variable ratios of 1-(4-aminobutyl)-3-carbamoylpyridinium (Nic-BuNH2), and

  7. Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

    Science.gov (United States)

    Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A

    1998-06-01

    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

  8. Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine

    Directory of Open Access Journals (Sweden)

    Cody S. Lee

    2017-06-01

    Full Text Available With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.

  9. Coating barium titanate nanoparticles with polyethylenimine improves cellular uptake and allows for coupled imaging and gene delivery.

    Science.gov (United States)

    Dempsey, Christopher; Lee, Isac; Cowan, Katie R; Suh, Junghae

    2013-12-01

    Barium titanate nanoparticles (BT NP) belong to a class of second harmonic generating (SHG) nanoprobes that have recently demonstrated promise in biological imaging. Unfortunately, BT NPs display low cellular uptake efficiencies, which may be a problem if cellular internalization is desired or required for a particular application. To overcome this issue, while concomitantly developing a particle platform that can also deliver nucleic acids into cells, we coated the BT NPs with the cationic polymer polyethylenimine (PEI)-one of the most effective nonviral gene delivery agents. Coating of BT with PEI yielded complexes with positive zeta potentials and resulted in an 8-fold increase in cellular uptake of the BT NPs. Importantly, we were able to achieve high levels of gene delivery with the BT-PEI/DNA complexes, supporting further efforts to generate BT platforms for coupled imaging and gene therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

    Science.gov (United States)

    Lopes, Cátia DF; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  11. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Andrew H. Baker

    2010-10-01

    Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

  12. The force analysis for superparamagnetic nanoparticles-based gene delivery in an oscillating magnetic field

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jiajia [State Key Laboratory of Electrical Insulation and Power Equipment, Xi’an Jiaotong University, No. 28 Xianning West Road, Xi’an, Shaanxi Province 710049 (China); Shi, Zongqian, E-mail: zqshi@mail.xjtu.edu.cn [State Key Laboratory of Electrical Insulation and Power Equipment, Xi’an Jiaotong University, No. 28 Xianning West Road, Xi’an, Shaanxi Province 710049 (China); Jia, Shenli [State Key Laboratory of Electrical Insulation and Power Equipment, Xi’an Jiaotong University, No. 28 Xianning West Road, Xi’an, Shaanxi Province 710049 (China); Zhang, Pengbo [Department of Anesthesiology, Second Affiliated Hospital of Xi’an Jiaotong University School of Medicine, No.157 West 5 Road, Xi’an, Shaanxi Province 710004 (China)

    2017-04-01

    Due to the peculiar magnetic properties and the ability to function in cell-level biological interaction, superparamagnetic nanoparticles (SMNP) have been being the attractive carrier for gene delivery. The superparamagnetic nanoparticles with surface-bound gene vector can be attracted to the surface of cells by the Kelvin force provided by external magnetic field. In this article, the influence of the oscillating magnetic field on the characteristics of magnetofection is studied in terms of the magnetophoretic velocity. The magnetic field of a cylindrical permanent magnet is calculated by equivalent current source (ECS) method, and the Kelvin force is derived by using the effective moment method. The results show that the static magnetic field accelerates the sedimentation of the particles, and drives the particles inward towards the axis of the magnet. Based on the investigation of the magnetophoretic velocity of the particle under horizontally oscillating magnetic field, an oscillating velocity within the amplitude of the magnet oscillation is observed. Furthermore, simulation results indicate that the oscillating amplitude plays an important role in regulating the active region, where the particles may present oscillating motion. The analysis of the magnetophoretic velocity gives us an insight into the physical mechanism of the magnetofection. It's also helpful to the optimal design of the magnetofection system. - Highlights: • We compare the results of the ECS method and FEA method with the commercial software, Ansys. • We analyze the physic mechanism of the oscillating motion of the particles in the presence of an oscillating magnet. • We discuss the influence of the oscillating amplitude of the magnet on the behavior of the particle.

  13. Optimizing cationic and neutral lipids for efficient gene delivery at high serum content.

    Science.gov (United States)

    Chan, Chia-Ling; Ewert, Kai K; Majzoub, Ramsey N; Hwu, Yeu-Kuang; Liang, Keng S; Leal, Cecília; Safinya, Cyrus R

    2014-01-01

    Cationic liposome (CL)-DNA complexes are promising gene delivery vectors with potential application in gene therapy. A key challenge in creating CL-DNA complexes for application is that their transfection efficiency (TE) is adversely affected by serum. In particular, little is known about the effects of a high serum content on TE, even though this may provide design guidelines for application in vivo. We prepared CL-DNA complexes in which we varied the neutral lipid [1,2-dioleoyl-sn-glycerophosphatidylcholine, glycerol-monooleate (GMO), cholesterol], the headgroup charge and chemical structure of the cationic lipid, and the ratio of neutral to cationic lipid; we then measured the TE of these complexes as a function of serum content and assessed their cytotoxicity. We tested selected formulations in two human cancer cell lines (M21/melanoma and PC-3/prostate cancer). In the absence of serum, all CL-DNA complexes of custom-synthesized multivalent lipids show high TE. Certain combinations of multivalent lipids and neutral lipids, such as MVL5(5+)/GMO-DNA complexes or complexes based on the dendritic-headgroup lipid TMVLG3(8+) exhibited high TE both in the absence and presence of serum. Although their TE still dropped to a small extent in the presence of serum, it reached or surpassed that of benchmark commercial transfection reagents, particularly at a high serum content. Two-component vectors (one multivalent cationic lipid and one neutral lipid) can rival or surpass benchmark reagents at low and high serum contents (up to 50%, v/v). We propose guidelines for optimizing the serum resistance of CL-DNA complexes based on a given cationic lipid. Copyright © 2014 John Wiley & Sons, Ltd.

  14. The force analysis for superparamagnetic nanoparticles-based gene delivery in an oscillating magnetic field

    International Nuclear Information System (INIS)

    Sun, Jiajia; Shi, Zongqian; Jia, Shenli; Zhang, Pengbo

    2017-01-01

    Due to the peculiar magnetic properties and the ability to function in cell-level biological interaction, superparamagnetic nanoparticles (SMNP) have been being the attractive carrier for gene delivery. The superparamagnetic nanoparticles with surface-bound gene vector can be attracted to the surface of cells by the Kelvin force provided by external magnetic field. In this article, the influence of the oscillating magnetic field on the characteristics of magnetofection is studied in terms of the magnetophoretic velocity. The magnetic field of a cylindrical permanent magnet is calculated by equivalent current source (ECS) method, and the Kelvin force is derived by using the effective moment method. The results show that the static magnetic field accelerates the sedimentation of the particles, and drives the particles inward towards the axis of the magnet. Based on the investigation of the magnetophoretic velocity of the particle under horizontally oscillating magnetic field, an oscillating velocity within the amplitude of the magnet oscillation is observed. Furthermore, simulation results indicate that the oscillating amplitude plays an important role in regulating the active region, where the particles may present oscillating motion. The analysis of the magnetophoretic velocity gives us an insight into the physical mechanism of the magnetofection. It's also helpful to the optimal design of the magnetofection system. - Highlights: • We compare the results of the ECS method and FEA method with the commercial software, Ansys. • We analyze the physic mechanism of the oscillating motion of the particles in the presence of an oscillating magnet. • We discuss the influence of the oscillating amplitude of the magnet on the behavior of the particle.

  15. Interaction of DNA/nuclear protein/polycation and the terplexes for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Shen Yuan; Pan Shirong; Feng Min; Wen Yuting; Deng Jingjing; Luo Xin; Wu Chuanbin [School of Pharmaceutical Sciences, Sun Yat-sen University, Zhongshan II Road 74, Guangzhou 510080 (China); Peng Hui, E-mail: fengmin@mail.sysu.edu.cn [School of Zhongshan Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080 (China)

    2010-01-29

    Nuclear transport of exogenous DNA is a major barrier to nonviral gene delivery that needs to be addressed in the design of new vectors. In this study, we prepared pDNA/HMGB1/PEG-PEI terplexes to promote nuclear import. HMGB1 in the terplexes was used to assist the transportation of pDNA into the nucleus of cells, since it contained nuclear localization signal (NLS); PEG chains were introduced to stabilize pDNA/vector terplexes and reduce the cytotoxicity. HMGB1/PEG-PEI combined vectors have been investigated specifically for their structure interaction by atomic force microscopy and circular dichroic spectroscopy. The results demonstrated that the HMGB1 molecule could bind with the pDNA chains, but not condense pDNA well. The PEG-PEI further compacted pDNA/HMGB1 complexes into nanosized spherical terplexes. The pDNA delivered by HMGB1/PEG-PEI combined vectors was significantly accumulated in the nucleus of cells, as observed by confocal laser scanning microscopy. The percentage of GFP-transfected cells and VEGF protein expression level induced by HMGB1/PEG-PEI were 2.6-4.9-fold and 1.4-2.8-fold higher, respectively, than that of a common cationic polymer PEI 25 kDa. Therefore, the HMGB1/PEG-PEI combined vector could be used as a versatile vector for promoting exogenous DNA nuclear localization, thereby enhancing its expression.

  16. Dendritic cationic lipids with highly charged headgroups for efficient gene delivery.

    Science.gov (United States)

    Ewert, Kai K; Evans, Heather M; Bouxsein, Nathan F; Safinya, Cyrus R

    2006-01-01

    Gene therapy is expected to lead to powerful new approaches for curing many diseases, a potential that is currently explored in worldwide clinical trials. Nonviral DNA delivery systems are desirable to overcome the inherent problems of viral vectors, but their current efficiency requires improvement and the understanding of their mechanism of action is incomplete. We have synthesized new multivalent cationic lipids with highly charged dendritic headgroups to probe the structure-transfection efficiency relationships of cationic liposome (CL)-DNA complexes, a prevalent nonviral vector. The lipid headgroups are constructed from ornithine cores and ornithine or carboxyspermine endgroups. The dendritic lipids were prepared on a gram scale, using a synthetic scheme that permits facile variation of the lipid building blocks headgroup, spacer, and hydrophobic moiety. They carry four to sixteen positive charges in their headgroups. Complexes of DNA with mixtures of the dendritic lipids and neutral 1,2-dioleoyl-sn-glycero phosphatidylcholine (DOPC) exhibit novel structures at high contents of the highly charged lipids, while the well-known lamellar phase is formed at high contents of DOPC. DNA complexes of the new dendritic lipids efficiently transfect mammalian cells in culture without cytotoxicity and, in contrast to lamellar complexes, maintain high transfection efficiency over a broad range of composition.

  17. Synthesis of water-based cationic polyurethane for antibacterial and gene delivery applications.

    Science.gov (United States)

    Wu, Geng-Hsi; Hsu, Shan-Hui

    2016-10-01

    Cationic polymers are often used as antimicrobial materials and transfection reagents. Water-based process could reduce environmental pollution and prevent the risk of solvent residue in the final product. In this study, waterborne biodegradable cationic polyurethane (WCPU) was synthesized by reacting polycaprolactone (PCL diol), isophorone diisocyanate (IPDI), and N-methyldiethanolamine (N-MDEA) under 75°C. An aqueous dispersion of WCPU nanoparticles (NPs) could be acquired by vigorous stirring under acidic condition. The particles in the dispersion had an average size of ∼80nm and a zeta potential of ∼60mV. When cast into films, the contact angle of the film was ∼67° and the zeta potential was ∼16mV. WCPU NPs demonstrated excellent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (100% inhibition with a contact time of 3h). Meanwhile, the antibacterial ratio of WCPU films to E. coli and S. aureus reached 100% after 24h of contact. Moreover, WCPU NPs could be used as a transfection reagent without significant toxicity for concentrations less than 1000μg/mL and showed the ability to condensate plasmid DNA. The transfection efficiency for HEK293T cells and hBMSCs was ∼60% and ∼30% at 48h, respectively, after the transfection. Therefore, the WCPU synthesized in this study has potential antibacterial and gene delivery applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Sugar-based gene delivery systems: Current knowledge and new perspectives.

    Science.gov (United States)

    Hong, Seo Jin; Ahn, Min Hye; Sangshetti, Jaiprakash; Choung, Pill Hoon; Arote, Rohidas B

    2018-02-01

    Carbohydrates, one of the most abundant natural compounds and key participants in many biological processes, are relevant in medical and industrial fields. In comparison with synthetic polymers, carbohydrates are biocompatible and have intrinsic targeting properties, enabling them to interact with cell-surface receptors. Among the different carbohydrates, polysaccharides are naturally occurring biological molecules with tremendous potential for biomedical applications. The physicochemical properties of these polysaccharide based nanoparticles, such as excellent biocompatibility, surface charge to interact with nucleic acids, low toxicity and cost effectiveness make them superior carriers for nanomedicine. In addition to variety of physicochemical properties, polysaccharides allow the great ease of chemical modification which enables the preparation of wide range of nanoparticles. In this review, we present the state-of-the-art information on the potential of polysaccharides-based polymers as non-viral gene delivery vectors in treating various diseases. Then, we discuss the chemical modification and structure/property relationship of carbohydrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Comparisons of three polyethyleneimine-derived nanoparticles as a gene therapy delivery system for renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Wei Yuquan

    2011-04-01

    Full Text Available Abstract Background Polyethyleneimine (PEI, which can interact with negatively charged DNA through electrostatic interaction to form nanocomplexes, has been widely attempted to use as a gene delivery system. However, PEI has some defects that are not fit for keeping on gene expression. Therefore, some modifications against PEI properties have been done to improve their application value in gene delivery. In this study, three modified PEI derivatives, including poly(ε-caprolactone-pluronic-poly(ε-caprolactone grafted PEI (PCFC-g-PEI, folic acid-PCFC-isophorone diidocyanate-PEI (FA-PEAs and heparin-PEI (HPEI, were evaluated in terms of their cytotoxicity and transfection efficiency in vitro and in vivo in order to ascertain their potential application in gene therapy. Methods MTT assay and a marker GFP gene, encoding green fluorescent protein, were used to evaluate cell toxicity and transfection activity of the three modified PEI in vitro. Renal cell carcinoma (RCC models were established in BALB/c nude mice inoculated with OS-RC-2 cells to detect the gene therapy effects using the three PEI-derived nanoparticles as gene delivery vehicles. The expression status of a target gene Von Hippel-Lindau (VHL in treated tumor tissues was analyzed by semiquantitative RT-PCR and immunohistochemistry. Results Each of three modified PEI-derived biomaterials had an increased transfection efficiency and a lower cytotoxicity compared with its precursor PEI with 25-kD or 2-kD molecule weight in vitro. And the mean tumor volume was obviously decreased 30% by using FA-PEAs to transfer VHL plasmids to treat mice RCC models. The VHL gene expression was greatly improved in the VHL-treated group. While there was no obvious tumor inhibition treated by PCFC-g-PEI:VHL and HPEI:VHL complexes. Conclusions The three modified PEI-derived biomaterials, including PCFC-g-PEI, FA-PEAs and HPEI, had an increased transfection efficiency in vitro and obviously lower toxicities

  20. Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna

    Directory of Open Access Journals (Sweden)

    Christian Hinderer

    2014-01-01

    Full Text Available Adeno-associated virus serotype 9 (AAV9 vectors have recently been shown to transduce cells throughout the central nervous system of nonhuman primates when injected into the cerebrospinal fluid (CSF, a finding which could lead to a minimally invasive approach to treat genetic and acquired diseases affecting the entire CNS. We characterized the transduction efficiency of two routes of vector administration into the CSF of cynomolgus macaques—lumbar puncture, which is typically used in clinical practice, and suboccipital puncture, which is more commonly used in veterinary medicine. We found that delivery of vector into the cisterna magna via suboccipital puncture is up to 100-fold more efficient for achieving gene transfer to the brain. In addition, we evaluated the inflammatory response to AAV9-mediated GFP expression in the nonhuman primate CNS. We found that while CSF lymphocyte counts increased following gene transfer, there were no clinical or histological signs of immune toxicity. Together these data indicate that delivery of AAV9 into the cisterna magna is an effective method for achieving gene transfer in the CNS, and suggest that adapting this uncommon injection method for human trials could vastly increase the efficiency of gene delivery.

  1. Effective Targeted Gene Knockdown in Mammalian Cells Using the piggyBac Transposase-based Delivery System

    Directory of Open Access Journals (Sweden)

    Jesse B Owens

    2013-01-01

    Full Text Available Nonviral gene delivery systems are rapidly becoming a desirable and applicable method to overexpress genes in various types of cells. We have recently developed a piggyBac transposase-based, helper-independent and self-inactivating delivery system (pmGENIE-3 capable of high-efficiency transfection of mammalian cells including human cells. In the following study, we have assessed the potential of this delivery system to drive the expression of short hairpin RNAs to knock down genes in human cells. Two independent pmGENIE-3 vectors were developed to specifically target knockdown of an endogenous gene, telomerase reverse transcriptase (TERT, in telomerase-positive human immortalized cell lines. As compared with a transposase-deficient vector, pmGENIE-3 showed significantly improved short-term transfection efficiency (~4-fold enhancement, 48 hours posttransfection and long-term integration efficiency (~5-fold enhancement following antibiotic selection. We detected a significant reduction of both TERT expression and telomerase activity in both HEK293 and MCF-7 breast carcinoma cells transfected with two pmGENIE-3 construct targeting distinct regions of TERT. Importantly, this knockdown of expression was sufficient to abrogate telomerase function since telomeres were significantly shortened (3–4 Kb, P < 0.001 in both TERT-targeted cell lines following antibiotic selection of stable integrants. Together, these data show the capacity of the piggyBac nonviral delivery system to stably knockdown gene expression in mammalian cells and indicate the potential to develop novel tumor-targeting therapies.

  2. DC Home Appliances for DC Distribution System

    Directory of Open Access Journals (Sweden)

    MUHAMMAD KAMRAN

    2017-10-01

    Full Text Available This paper strengthens the idea of DC distribution system for DC microgrid consisting of a building of 50 apartments. Since the war of currents AC system has been dominant because of the paucity of research in the protection of the DC system. Now with the advance research in power electronics material and components, generation of electricity is inherently DC as by solar PV, fuel cell and thermoelectric generator that eliminates the rectification process. Transformers are replaced by the power electronics buck-boost converters. DC circuit breakers have solved the protection problems for both DC transmission and distribution system. In this paper 308V DC microgrid is proposed and home appliances (DC internal are modified to operate on 48V DC from DC distribution line. Instead of using universal and induction motors in rotary appliances, BLDC (Brushless DC motors are proposed that are highly efficient with minimum electro-mechanical and no commutation losses. Proposed DC system reduces the power conversion stages, hence diminishes the associated power losses and standby losses that boost the overall system efficiency. So in view of all this a conventional AC system can be replaced by a DC system that has many advantages by cost as well as by performance

  3. Synthesis and evaluation of cationic nanomicelles for in vitro and in vivo gene delivery

    Science.gov (United States)

    Mandke, Rhishikesh Subhash

    The goal of proposed study was to contribute towards the development of a nano size, high efficiency and low toxicity non-viral polymeric vector for gene delivery in vitro and in vivo. A series of fatty acid grafted low-molecular-weight chitosan (N-acyl LMWCs) were synthesized, purified and characterized for their physicochemical properties using various analytical techniques such as infrared spectroscopy, elemental analysis and dynamic light scattering. The formulation parameters including pH, sonication duration, and filtration altered the physicochemical characteristics of N-acyl LMWC nanomicelles. The acyl chain length and degree of unsaturation in fatty acids also had an impact on the physicochemical properties and the transfection efficiency of nanomicelles. N-acyl LMWC nanomicelles showed efficient in vitro transfection as visualized and quantified using a reporter plasmid (encoding green fluorescent protein), and therapeutic plasmids (encoding for interleukin-4 and interleukin-10), respectively. The in vitro transfection efficiencies of N-acyl LMWCs with 18:1 and 18:2 grafts (oleic and linoleic acids) were comparable with FuGENERTM HD (marketed non-viral vector) but were ˜8-fold and 35-fold higher as compared to LMWC and naked DNA, respectively. The in vivo transfection efficiency of N-acyl LMWC to deliver plasmids individually encoding IL-4 and IL-10 as well as a bicistronic plasmid encoding both IL-4 and IL-10 was studied in a multiple, low-dose streptozotocin induced diabetic mouse model. The transfection efficiency of pDNA/N-acyl LMWC polyplexes injected via intramuscular route showed significant improvement (pexpression of IL-4 and IL-10 was observed, accompanied by a reduction in interferon-gamma (INF-gamma), and tumor necrosis factor-alpha (TNF-alpha) levels. The pancreas of pDNA/N-acyl LMWC polyplex treated animals exhibited protection from streptozotocin-induced insulitis and the delivery systems were biocompatible. Histological studies revealed

  4. "Forback" Dc-To-Dc Converters

    Science.gov (United States)

    Lukemire, Alan T.

    1992-01-01

    Dc-to-dc power-converter circuits called "forback" resemble circuits of standard configurations called "forward", "flyback", and "Cuk". Circuit employs minor modifications to existing topologies, combines advantages, while eliminating disadvantages, of older circuits.

  5. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells.

    Science.gov (United States)

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-10-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. © 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  6. Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

    Science.gov (United States)

    Tolmachov, Oleg E

    2015-01-01

    Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

  7. Optimization of transfection parameters for ultrasound/SonoVue microbubble-mediated hAng-1 gene delivery in vitro.

    Science.gov (United States)

    Zhou, Qing; Chen, Jin-Ling; Chen, Qian; Wang, Xiao; Deng, Qing; Hu, Bo; Guo, Rui-Qiang

    2012-12-01

    This study aimed to explore the effects of microbubble concentration, gene dosage, cell-microbubble mixing mode and fetal bovine serum (FBS) on gene delivery. 293T cells were transfected with Sonovue microbubbles carrying the hAng-1 gene via ultrasound irradiation. Various ultrasound exposure parameters and microbubble and DNA concentrations were investigated. In addition, FBS and the cell suspension or adherent mode was explored. Transfection efficiency and cell viability were used to determine the optimal transfection parameters. hAng-1 gene transfection efficiency gradually increased with elongation of ultrasound exposure and increasing microbubble concentration. However, if ultrasound irradiation exceeded 1.5 W/cm² and 30 sec or the microbubble concentration was over 20%, hAng-1 gene expression was significantly decreased, coupled with extensive cell death. Gene transfection levels were low under DNA concentrations less than 15 µg/ml. Furthermore, the gene transfer rate was significantly increased under cell suspension mode; FBS had no effect on hAng-1 gene transfection. The integrity of hAng-1 DNA was not affected by ultrasonic irradiation under optimal conditions. The optimal transfection parameters for the hAng-1 gene and Sonovue microbubble were ultrasound exposure of 1.5 W/cm² and 30 sec, 20% microbubbles, 15 µg/ml of DNA and under cell suspension mode.

  8. Mutagenic Analysis of an Adeno-Associated Virus Variant Capable of Simultaneously Promoting Immune Resistance and Robust Gene Delivery.

    Science.gov (United States)

    Kim, Yoojin; Kim, Eunmi; Oh, Seokmin; Yoon, Ye-Eun; Jang, Jae-Hyung

    2018-01-01

    In addition to the ability to boost gene delivery efficiency in many therapeutically relevant cells, the capability of circumventing neutralizing antibody (NAb) inactivation is a key prerequisite that gene carriers must fulfill for their extensive applications as therapeutic agents in many gene therapy trials, especially for cancer treatments. This study revealed that a genetically engineered adeno-associated virus (AAV) variant, AAVr3.45, inherently possesses dual beneficial properties as a gene carrier: (i) efficiently delivering therapeutic genes to many clinically valuable cells (e.g., stem or cancer cells) and (ii) effectively bypassing immunoglobulin (IgG) neutralization. Detailed interpretation of the structural features of AAVr3.45, which was previously engineered from AAV2, demonstrated that the LATQVGQKTA peptide at the heparan sulfate proteoglycan binding domain, especially the presence of cationic lysine on the peptide, served as a key motif for dramatically enhancing its gene delivery capabilities, ultimately broadening its tropisms for many cancer cell lines. Furthermore, the substitution of valine on the AAV2 capsid at the amino acid 719 site to methionine functioned as a coordinator for promoting viral resistance against IgG inactivation. The NAb-resistant characteristics of AAVr3.45 were possibly associated with the LATQVGQKTA sequence itself, indicating that its synergistic cooperation with the point mutation (V719M) is required for maximizing its ability to evade NAb inactivation. The potential of AAVr3.45 as a cancer gene therapy agent was confirmed by provoking apoptosis in breast adenocarcinoma by efficiently delivering a pro-apoptotic gene, BIM (Bcl-2-like protein 11), under high titers of human IgG. Thus, the superior aspects of the NAb-resistant AAVr3.45 as a potential therapeutic agent for systemic injection approaches, especially for cancer gene therapy, were highlighted in this study.

  9. Gene Silencing in Skin After Deposition of Self-Delivery siRNA With a Motorized Microneedle Array Device

    Directory of Open Access Journals (Sweden)

    Robyn P Hickerson

    2013-01-01

    Full Text Available Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.

  10. Adiabatic differential scanning calorimetric study of divalent cation induced DNA - DPPC liposome formulation compacted for gene delivery

    Directory of Open Access Journals (Sweden)

    Erhan Süleymanoglu

    2004-11-01

    Full Text Available Complexes between nucleic acids and phospholipid vesicles have been developed as stable non-viral gene delivery vehicles. Currently employed approach uses positively charged lipid species and a helper zwitterionic lipid, the latter being applied for the stabilization of the whole complex. However, besides problematic steps during their preparation, cationic lipids are toxic for cells. The present work describes some energetic issues pertinent to preparation and use of neutral lipid-DNA self-assemblies, thus avoiding toxicity of lipoplexes. Differential scanning calorimetry data showed stabilization of polynucleotide helix upon its interaction with liposomes in the presence of divalent metal cations. It is thus possible to suggest this self-assembly as an improved formulation for use in gene delivery.

  11. Direct reprogramming of mouse fibroblasts into neural cells via Porphyra yezoensis polysaccharide based high efficient gene co-delivery

    Directory of Open Access Journals (Sweden)

    Qingtong Yu

    2017-11-01

    Full Text Available Abstract Background The cell source for transplantation therapy is always a prerequisite question to be solved in clinical applications. Neural cells are considered non-regenerable, which highly restrict their application in the treatment for nerve injury. Therefore, neural trans-differentiation based on gene transfection provides a new solution to this issue. Compared to viral strategy, non-viral gene delivery systems are considered as a more promising way to achieve this aim. This study centers on a novel application of Porphyra yezoensis polysaccharide as a non-viral gene carrier for the neural trans-differentiation of mouse fibroblasts. Results Ethanediamine modified P. yezoensis polysaccharide (Ed-PYP served as a gene carrier and a group of plasmids that encode Ascl1, Brn4, and Tcf3 (pABT self-assembled into nanoparticles. Results demonstrated that Ed-PYP–pABT nanoparticles at Ed-PYP: pABT weight ratio of 40:1 was the optimal candidate for gene delivery. ELISA assay revealed the highest expression levels of NGF, BDNF and SHH at 14 days after last transfection. Immunofluorescence and western blot assays also showed robust expression of neural markers including Nestin, GFAP, β-3tubulin, NF200, GAP43 and MAP2, in induced 3T6 cells at this time point. Conclusion Overall, these findings indicated that the P. yezoensis polysaccharide-based non-viral gene co-delivery system is a promising strategy for the generation of neural cells, which might facilitate the developments in the recovery of neural injuries.

  12. DC electric springs with DC/DC converters

    DEFF Research Database (Denmark)

    Wang, Qingsong; Cheng, Ming; Jiang, Yunlei

    2016-01-01

    The concept of DC electric spring (DCES) was recently proposed to solve the stability issue caused by integrating intermittent renewable energy sources (RESs) to DC microgrids. In this paper, a new topology of DCES is proposed based on DC/DC converters. The proposed DCES consists of a bi-directio......The concept of DC electric spring (DCES) was recently proposed to solve the stability issue caused by integrating intermittent renewable energy sources (RESs) to DC microgrids. In this paper, a new topology of DCES is proposed based on DC/DC converters. The proposed DCES consists of a bi...... and/or constant discharging for batteries is adopted and four operating modes are analyzed as charging-positive, charging-negative, discharging-positive and discharging-negative modes. An additional mechanism for fast charging or fast discharging is also designed to secure normal operation...... of batteries. With the proposed DCES, the power fluctuations due to intermittent RESs can be passed to non-critical loads (NCLs) and batteries while power on critical loads (CLs) is kept stable. This is possibly the first attempt to design a DCES with only DC/DC converters. The performances of the proposed...

  13. In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

    Science.gov (United States)

    Bez, Maxim; Sheyn, Dmitriy; Tawackoli, Wafa; Avalos, Pablo; Shapiro, Galina; Giaconi, Joseph C.; Da, Xiaoyu; Ben David, Shiran; Gavrity, Jayne; Awad, Hani A.; Bae, Hyun W.; Ley, Eric J.; Kremen, Thomas J.; Gazit, Zulma; Ferrara, Katherine W.; Pelled, Gadi; Gazit, Dan

    2017-01-01

    More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchy-mal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation. PMID:28515335

  14. Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations

    Czech Academy of Sciences Publication Activity Database

    Reschel, Tomáš; Koňák, Čestmír; Oupický, D.; Seymour, L. W.; Ulbrich, Karel

    2002-01-01

    Roč. 81, 1-2 (2002), s. 201-217 ISSN 0168-3659 R&D Projects: GA ČR GV307/96/K226; GA AV ČR IAA1050101 Institutional research plan: CEZ:AV0Z4050913 Keywords : gene delivery * self assembly * polycation/DNA complexes Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.131, year: 2002

  15. Chitosan nanoparticles as non-viral gene delivery systems: determination of loading efficiency.

    Science.gov (United States)

    Carrillo, Carolina; Suñé, Josep Maria; Pérez-Lozano, Pilar; García-Montoya, Encarna; Sarrate, Rocío; Fàbregas, Anna; Miñarro, Montserrat; Ticó, Josep Ramon

    2014-07-01

    Chitosan has been studied for use in particle delivery systems for therapeutic purposes, since one of its most important applications is as a non-viral vector in gene therapy. Due to its positive charge, it is capable of forming DNA complexes (polyplexes) obtained through several methods and with the property of protecting nucleic acids. Two methods for obtaining the nanoparticles of chitosan-nucleic acids are reported in this study: simple complexation (of depolymerized chitosan or of different chitosan salts with plasmid) and ionic gelation (by adsorption of plasmid in the nanoparticles or by encapsulation of plasmid into nanoparticles). The determination of the loading efficiency of chitosan nanoparticles with the plasmid is carried out by electrophoretic mobility of the samples on agarose gel. Furthermore, the nanoparticles have been characterized according to their morphology, size and surface charge using AFM, TEM, laser diffraction and dynamic light scattering techniques. The polyplexes obtained have been found to be spherical and nanometric in size (between 100-230nm) with a zeta potential between 37 and 48mV. Positive results have been obtained by agarose gel electrophoresis for all studied cases: a concentration of between 20 and 30μg/mL of chitosan salts is required while for the remaining chitosan samples studied, 100% loading efficiency does not occur until a concentration equal to 100μg/mL (regardless of previous depolymerisation and the method performed). Chitosan-plasmid nanocapsules have been obtained at the polymer concentrations worked with (between 0.025 and 0.2%). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  16. Tandem mass spectrometric analysis of novel peptide-modified gemini surfactants used as gene delivery vectors.

    Science.gov (United States)

    Al-Dulaymi, M; El-Aneed, A

    2017-06-01

    Diquaternary ammonium gemini surfactants have emerged as effective gene delivery vectors. A novel series of 11 peptide-modified compounds was synthesized, showing promising results in delivering genetic materials. The purpose of this work is to elucidate the tandem mass spectrometric (MS/MS) dissociation behavior of these novel molecules establishing a generalized MS/MS fingerprint. Exact mass measurements were achieved using a hybrid quadrupole orthogonal time-of-flight mass spectrometer, and a multi-stage MS/MS analysis was conducted using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were operated in the positive ionization mode and are equipped with electrospray ionization. Abundant triply charged [M+H] 3+ species were observed in the single-stage analysis of all the evaluated compounds with mass accuracies of less than 8 ppm in mass error. MS/MS analysis showed that the evaluated gemini surfactants exhibited peptide-related dissociation characteristics because of the presence of amino acids within the compounds' spacer region. In particular, diagnostic product ions were originated from the neutral loss of ammonia from the amino acids' side chain resulting in the formation of pipecolic acid at the N-terminus part of the gemini surfactants. In addition, a charge-directed amide bond cleavage was initiated by the amino acids' side chain producing a protonated α-amino-ε-caprolactam ion and its complimentary C-terminus ion that contains quaternary amines. MS/MS and MS 3 analysis revealed common fragmentation behavior among all tested compounds, resulting in the production of a universal MS/MS fragmentation pathway. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  17. 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.

  18. Ab-externo AAV-mediated gene delivery to the suprachoroidal space using a 250 micron flexible microcatheter.

    Directory of Open Access Journals (Sweden)

    Marc C Peden

    2011-02-01

    Full Text Available The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter.6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP.Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically.The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.

  19. Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide.

    Science.gov (United States)

    Ko, Y T; Hartner, W C; Kale, A; Torchilin, V P

    2009-01-01

    The treatment of myocardial ischemia using gene therapy is a rather novel but promising approach. Gene delivery to target cells may be enhanced by using double-targeted delivery systems simultaneously capable of extracellular accumulation and intracellular penetration. With this in mind, we have used low cationic liposomes-plasmid DNA complexes (lipoplexes) modified with cell-penetrating transactivating transcriptional activator (TAT) peptide (TATp) and/or with monoclonal anti-myosin monoclonal antibody 2G4 (mAb 2G4) specific toward cardiac myosin, for targeted gene delivery to ischemic myocardium. In vitro transfection of both normoxic and hypoxic cardiomyocytes was enhanced by the presence of TATp as determined by fluorescence microscopy and ELISA. The in vitro transfection was further enhanced by the additional modification with mAb 2G4 antibody in the case of hypoxic, but not normoxic cardiomyocytes. However, we did not observe a synergism between TATp and mAb 2G4 ligands under our experimental condition. In in vivo experiments, we have clearly demonstrated an increased accumulation of mAb 2G4-modified TATp lipoplexes in the ischemic rat myocardium and significantly enhanced transfection of cardiomyocytes in the ischemic zone. Thus, the genetic transformation of normoxic and hypoxic cardiomyocytes can be enhanced by using lipoplexes modified with TATp and/or mAb 2G4. Such complexes also demonstrate an increased accumulation in the ischemic myocardium and effective transfection of hypoxic cardiomyocytes in vivo.

  20. α, ω-Cholesterol-Functionalized Low Molecular Weight Polyethylene Glycol as a Novel Modifier of Cationic Liposomes for Gene Delivery

    Directory of Open Access Journals (Sweden)

    Cui-Cui Ma

    2014-11-01

    Full Text Available Here, three novel cholesterol (Ch/low molecular weight polyethylene glycol (PEG conjugates, termed α, ω-cholesterol-functionalized PEG (Ch2-PEGn, were successfully synthesized using three kinds of PEG with different average molecular weight (PEG600, PEG1000 and PEG2000. The purpose of the study was to investigate the potential application of novel cationic liposomes (Ch2-PEGn-CLs containing Ch2-PEGn in gene delivery. The introduction of Ch2-PEGn affected both the particle size and zeta potential of cationic liposomes. Ch2-PEG2000 effectively compressed liposomal particles and Ch2-PEG2000-CLs were of the smallest size. Ch2-PEG1000 and Ch2-PEG2000 significantly decreased zeta potentials of Ch2-PEGn-CLs, while Ch2-PEG600 did not alter the zeta potential due to the short PEG chain. Moreover, the in vitro gene transfection efficiencies mediated by different Ch2-PEGn-CLs also differed, in which Ch2-PEG600-CLs achieved the strongest GFP expression than Ch2-PEG1000-CLs and Ch2-PEG2000-CLs in SKOV-3 cells. The gene delivery efficacy of Ch2-PEGn-CLs was further examined by addition of a targeting moiety (folate ligand in both folate-receptor (FR overexpressing SKOV-3 cells and A549 cells with low expression of FR. For Ch2-PEG1000-CLs and Ch2-PEG2000-CLs, higher molar ratios of folate ligand resulted in enhanced transfection efficacies, but Ch2-PEG600-CLs had no similar in contrast. Additionally, MTT assay proved the reduced cytotoxicities of cationic liposomes after modification by Ch2-PEGn. These findings provide important insights into the effects of Ch2-PEGn on cationic liposomes for delivering genes, which would be beneficial for the development of Ch2-PEGn-CLs-based gene delivery system.

  1. Expression of the Grape VaSTS19 Gene in Arabidopsis Improves Resistance to Powdery Mildew and Botrytis cinerea but Increases Susceptibility to Pseudomonas syringe pv Tomato DC3000.

    Science.gov (United States)

    Wang, Yaqiong; Wang, Dejun; Wang, Fan; Huang, Li; Tian, Xiaomin; van Nocker, Steve; Gao, Hua; Wang, Xiping

    2017-09-17

    Stilbene synthase (STS) is a key enzyme that catalyzes the biosynthesis of resveratrol compounds and plays an important role in disease resistance. The molecular pathways linking STS with pathogen responses and their regulation are not known. We isolated an STS gene, VaSTS19 , from a Chinese wild grape, Vitis amurensis Rupr. cv. "Tonghua-3", and transferred this gene to Arabidopsis . We then generated VaSTS19 -expressing Arabidopsis lines and evaluated the functions of VaSTS19 in various pathogen stresses, including powdery mildew, B. cinerea and Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000). VaSTS19 enhanced resistance to powdery mildew and B. cinerea , but increased susceptibility to Pst DC3000. Aniline blue staining revealed that VaSTS19 transgenic lines accumulated more callose compared to nontransgenic control plants, and showed smaller stomatal apertures when exposed to pathogen-associated molecular patterns (flagellin fragment (flg22) or lipopolysaccharides (LPS)). Analysis of the expression of several disease-related genes suggested that VaSTS19 expression enhanced defense responses though salicylic acid (SA) and/or jasmonic acid (JA) signaling pathways. These findings provide a deeper insight into the function of STS genes in defense against pathogens, and a better understanding of the regulatory cross talk between SA and JA pathways.

  2. Expression of the Grape VaSTS19 Gene in Arabidopsis Improves Resistance to Powdery Mildew and Botrytis cinerea but Increases Susceptibility to Pseudomonas syringe pv Tomato DC3000

    Directory of Open Access Journals (Sweden)

    Yaqiong Wang

    2017-09-01

    Full Text Available Stilbene synthase (STS is a key enzyme that catalyzes the biosynthesis of resveratrol compounds and plays an important role in disease resistance. The molecular pathways linking STS with pathogen responses and their regulation are not known. We isolated an STS gene, VaSTS19, from a Chinese wild grape, Vitis amurensis Rupr. cv. “Tonghua-3”, and transferred this gene to Arabidopsis. We then generated VaSTS19-expressing Arabidopsis lines and evaluated the functions of VaSTS19 in various pathogen stresses, including powdery mildew, B. cinerea and Pseudomonas syringae pv. tomato DC3000 (PstDC3000. VaSTS19 enhanced resistance to powdery mildew and B. cinerea, but increased susceptibility to PstDC3000. Aniline blue staining revealed that VaSTS19 transgenic lines accumulated more callose compared to nontransgenic control plants, and showed smaller stomatal apertures when exposed to pathogen-associated molecular patterns (flagellin fragment (flg22 or lipopolysaccharides (LPS. Analysis of the expression of several disease-related genes suggested that VaSTS19 expression enhanced defense responses though salicylic acid (SA and/or jasmonic acid (JA signaling pathways. These findings provide a deeper insight into the function of STS genes in defense against pathogens, and a better understanding of the regulatory cross talk between SA and JA pathways.

  3. Balancing Cell Migration with Matrix Degradation Enhances Gene Delivery to Cells Cultured Three-Dimensionally Within Hydrogels

    Science.gov (United States)

    Shepard, Jaclyn A.; Huang, Alyssa; Shikanova, Ariella; Shea, Lonnie D.

    2010-01-01

    In regenerative medicine, hydrogels are employed to fill defects and support the infiltration of cells that can ultimately regenerate tissue. Gene delivery within hydrogels targeting infiltrating cells has the potential to promote tissue formation, but the delivery efficiency of nonviral vectors within hydrogels is low hindering their applicability in tissue regeneration. To improve their functionality, we have conducted a mechanistic study to investigate the contribution of cell migration and matrix degradation on gene delivery. In this report, lipoplexes were entrapped within hydrogels based on poly(ethylene glycol) (PEG) crosslinked with peptides containing matrix metalloproteinase degradable sequences. The mesh size of these hydrogels is substantially less than the size of the entrapped lipoplexes, which can function to retain vectors. Cell migration and transfection were simultaneously measured within hydrogels with varying density of cell adhesion sites (Arg-Gly-Asp peptides) and solids content. Increasing RGD density increased expression levels up to 100-fold, while greater solids content sustained expression levels for 16 days. Increasing RGD density and decreasing solids content increased cell migration, which indicates expression levels increase with increased cell migration. Initially exposing cells to vector resulted in transient expression that declined after 2 days, verifying the requirement of migration to sustain expression. Transfected cells were predominantly located within the population of migrating cells for hydrogels that supported cell migration. Although the small mesh size retained at least 70% of the lipoplexes in the absence of cells after 32 days, the presence of cells decreased retention to 10% after 16 days. These results indicate that vectors retained within hydrogels contact migrating cells, and that persistent cell migration can maintain elevated expression levels. Thus matrix degradation and cell migration are fundamental design

  4. Freeze-dried allograft-mediated gene or protein delivery of growth and differentiation factor 5 reduces reconstructed murine flexor tendon adhesions

    DEFF Research Database (Denmark)

    Svensson, Sys Hasslund; Dadali, Tulin; Ulrich-Vinther, Michael

    2014-01-01

    Advances in allograft processing have opened new horizons for clinical adaptation of flexor tendon allografts as delivery scaffolds for antifibrotic therapeutics. Recombinant adeno-associated-virus (rAAV) gene delivery of the growth and differentiation factor 5 (GDF-5) has been previously...

  5. Protein kinase A inhibition modulates the intracellular routing of gene delivery vehicles in HeLa cells, leading to productive transfection

    NARCIS (Netherlands)

    Rehman, Zia Ur; Hoekstra, Dick; Zuhorn, Inge S.

    2011-01-01

    Cellular entry of nanoparticles for drug- and gene delivery relies on various endocytic pathways, including clathrin-and caveolae-mediated endocytosis. To improve delivery, i.e., the therapeutic and/or cell biological impact, current efforts are aimed at avoiding processing of the carriers along the

  6. Recombinant AAV serotype and capsid mutant comparison for pulmonary gene transfer of alpha-1-antitrypsin using invasive and noninvasive delivery.

    Science.gov (United States)

    Liqun Wang, Rejean; McLaughlin, Thomas; Cossette, Travis; Tang, Qiushi; Foust, Kevin; Campbell-Thompson, Martha; Martino, Ashley; Cruz, Pedro; Loiler, Scott; Mueller, Christian; Flotte, Terence R

    2009-01-01

    Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing alpha-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 x 10(10) vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT ("long serpin") indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency-related emphysema.

  7. Recombinant AAV Serotype and Capsid Mutant Comparison for Pulmonary Gene Transfer of α-1-Antitrypsin Using Invasive and Noninvasive Delivery

    Science.gov (United States)

    Liqun Wang, Rejean; McLaughlin, Thomas; Cossette, Travis; Tang, Qiushi; Foust, Kevin; Campbell-Thompson, Martha; Martino, Ashley; Cruz, Pedro; Loiler, Scott; Mueller, Christian; Flotte, Terence R

    2008-01-01

    Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing α-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 × 1010 vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT (“long serpin”) indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency–related emphysema. PMID:18941444

  8. Significant inhibition of Tembusu virus envelope and NS5 gene using an adenovirus-mediated short hairpin RNA delivery system.

    Science.gov (United States)

    Wang, Hongzhi; Feng, Qiang; Wei, Lei; Zhuo, Liling; Chen, Hao; Diao, Youxiang; Tang, Yi

    2017-10-01

    Tembusu virus (TMUV) is a mosquito-borne flavivirus, which was first isolated in the tropics during the 1970s. Recently, a disease characterized by ovarian haemorrhage and neurological symptoms was observed in ducks in China, which threatens poultry production. However, there is no suitable vaccination strategy or effective antiviral drugs to combat TMUV infections. Consequently, there is an urgent need to develop a new anti-TMUV therapy. In this study, we report an efficient short hairpin RNA (shRNA) delivery strategy for the inhibition of TMUV production using an adenovirus vector system. Using specifically designed shRNAs based on the E and NS5 protein genes of TMUV, the vector-expressed viral genes, TMUV RNA replication and infectious virus production were downregulated at different levels in Vero cells, where the shRNA (NS52) was highly effective in inhibiting TMUV. Using the human adenovirus type 5 shRNA delivery system, the recombinant adenovirus (rAd-NS52) inhibited TMUV multiplication with high efficiency. Furthermore, the significant dose-dependent inhibition of viral RNA copies induced by rAd-NS52 was found in TMUV-infected cells, which could last for at least 96h post infection. Our results indicated that the adenovirus-mediated delivery of shRNAs could play an active role in future TMUV antiviral therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. 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.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

  11. Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis

    Directory of Open Access Journals (Sweden)

    Qiu Ri-Xiang

    2010-11-01

    Full Text Available Abstract Background Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection in vivo by the combination of ultrasound-targeted microbubble destruction (UTMD with polyethylenimine (PEI in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA interference therapy targeting human survivin by this novel technique. Methods Two different expression vectors (pCMV-LUC and pSIREN were incubated with PEI to prepare cationic complexes (PEI/DNA and confirmed by the gel retardation assay. Human cervical carcinoma (Hela tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected. Results Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression. Conclusions This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration

  12. Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    Directory of Open Access Journals (Sweden)

    Zhao W

    2016-11-01

    Full Text Available Wen Zhao, Yifan Zhang, Xueyun Jiang, Chunying Cui School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, China Abstract: Small interfering RNA (siRNA delivery is a prospective method in gene therapy, but it has application limitations such as negative charge, water solubility and high molecular weight. In this study, a safe and efficient nano-vector, CRS, was designed and synthesized to facilitate siRNA delivery. Physical and chemical properties of VEGF-siRNA/CRS were characterized by methods including scanning electron microscopy (SEM, transmission electron microscopy, zeta potential (ζ measurement, drug-releasing rate measurement, gel electrophoresis and confocal microscopy. The biological activities were evaluated using cell viability assay, gene-silencing efficacy assay in vitro, real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA and antitumor tests in vivo. The mean nanoparticle size of VEGF-siRNA/CRS was 121.4±0.3 nm with positive ζ potential of 7.69±4.47 mV. The release rate of VEGF-siRNA from VEGF-siRNA/CRS was 82.50% sustained for 48 h in Tris-ethylenediaminetetraacetic acid buffer (pH 8.0. Real-time polymerase chain reaction was used to analyze the efficiency of the transfection, and the result showed that VEGF mRNA expression had been knocked down by 82.36%. The expression of VEGF protein was also recorded to be downregulated to 14.83% using ELISA. The results of cytotoxicity measured by Cell Counting Kit-8 assay showed that VEGF-siRNA/CRS had significant inhibitory effect on HeLa cells. The results of antitumor assays indicated that VEGF-siRNA/CRS exhibited tumor cell growth inhibition in vivo. The results demonstrated that VEGF-siRNA could be delivered and transported by the designed carrier, while siRNA could be released constantly and led to an increasing gene-silencing effect against VEGF gene. In conclusion, VEGF-siRNA/CRS is a promising carrier for si

  13. Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers.

    Science.gov (United States)

    Mahajan, Vivek; Gaymalov, Zagit; Alakhova, Daria; Gupta, Richa; Zucker, Irving H; Kabanov, Alexander V

    2016-01-01

    Intramuscular administration of plasmid DNA (pDNA) with non-ionic Pluronic block copolymers increases gene expression in injected muscles and lymphoid organs. We studied the role of immune cells in muscle transfection upon inflammation. Local inflammation in murine hind limb ischemia model (MHLIM) drastically increased DNA, RNA and expressed protein levels in ischemic muscles injected with pDNA/Pluronic. The systemic inflammation (MHLIM or peritonitis) also increased expression of pDNA/Pluronic in the muscles. When pDNA/Pluronic was injected in ischemic muscles the reporter gene, Green Fluorescent Protein (GFP) co-localized with desmin(+) muscle fibers and CD11b(+) macrophages (MØs), suggesting transfection of MØs along with the muscle cells. P85 enhanced (∼ 4 orders) transfection of MØs with pDNA in vitro. Moreover, adoptively transferred MØs were shown to pass the transgene to inflamed muscle cells in MHLIM. Using a co-culture of myotubes (MTs) and transfected MØs expressing a reporter gene under constitutive (cmv-luciferase) or muscle specific (desmin-luciferase) promoter we demonstrated that P85 enhances horizontal gene transfer from MØ to MTs. Therefore, MØs can play an important role in muscle transfection with pDNA/Pluronic during inflammation, with both inflammation and Pluronic contributing to the increased gene expression. pDNA/Pluronic has potential for therapeutic gene delivery in muscle pathologies that involve inflammation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation

    Directory of Open Access Journals (Sweden)

    Kate E. Broderick

    2013-08-01

    Full Text Available The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21 of enhanced gene delivery with electroporation (EP was performed to observe the localization of green fluorescent protein (GFP expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design

  15. Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Kenneth C. McCullough

    2014-10-01

    Full Text Available Dendritic cells (DC play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.

  16. The effect of side-chain functionality and hydrophobicity on the gene delivery capabilities of cationic helical polypeptides.

    Science.gov (United States)

    Zhang, Rujing; Zheng, Nan; Song, Ziyuan; Yin, Lichen; Cheng, Jianjun

    2014-03-01

    The rational design of effective and safe non-viral gene vectors is largely dependent on the understanding of the structure-property relationship. We herein report the design of a new series of cationic, α-helical polypeptides with different side charged groups (amine and guanidine) and hydrophobicity, and mechanistically unraveled the effect of polypeptide structure on the gene delivery capability. Guanidine-containing polypeptides displayed superior membrane activities to their amine-containing analogues via the pore formation mechanism, and thus possessed notably higher transfection efficiencies. Elongating the hydrophobic side chain also potentiated the membrane activities of the polypeptides, while at the meantime caused higher cytotoxicities. Upon an optimal balance between membrane activity and cytotoxicity, maximal transfection efficiency was achieved which outperformed commercial reagent Lipofectamine™ 2000 (LPF2000) by 3-6 folds. This study thus provides mechanistic insights into the rational design of non-viral gene delivery vectors, and the best-performing materials identified also serve as a promising addition to the existing systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution

    Directory of Open Access Journals (Sweden)

    Dachs Gabi U

    2004-11-01

    Full Text Available Abstract Background Electroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal. Methods Intravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared. Results Electroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses. Conclusions Intravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous

  18. Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution

    International Nuclear Information System (INIS)

    Cemazar, Maja; Wilson, Ian; Dachs, Gabi U; Tozer, Gillian M; Sersa, Gregor

    2004-01-01

    Electroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal. Intravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP) and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared. Electroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses. Intravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous distribution of gene expression in the syngeneic P22 rat tumor model

  19. Attenuation of endoplasmic reticulum stress-related myocardial apoptosis by SERCA2a gene delivery in ischemic heart disease.

    Science.gov (United States)

    Xin, Wei; Lu, Xiaochun; Li, Xiaoying; Niu, Kun; Cai, Jimei

    2011-01-01

    Previous studies suggested that endoplasmic reticulum (ER) stress-associated apoptosis plays an important role in the pathogenesis of ischemic heart disease. Gene transfer of sarco/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) attenuates myocardial apoptosis in a variety of heart failure models. This study is to investigate the effects of SERCA2a gene delivery on the myocardial apoptosis and ER stress pathway in a porcine ischemic heart disease model. Eighteen pigs were either subjected to ameroid implantation in the coronary artery or sham operation. Eight wks after gene delivery, the protein level and activity of SERCA2a were measured. Myocardial apoptosis was determined using terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling assay. Regional myocardial perfusion and function were evaluated by (99m)Tc-sestamibi ((99m)Tc-MIBI) single photon emission computed tomography and echocardiography. The ER stress signaling was assessed by Western blot. SERCA2a protein level and activity were significantly decreased in the ischemic myocardium and restored to normal after SERCA2a gene transfer. Restoration of SERCA2a expression significantly improved the cardiac function, although no improvement of regional myocardial perfusion was detected. Restoration of SERCA2a significantly attenuated myocardial apoptosis and reversed the activation of unfolded protein response (UPR) pathway and the ER stress-associated apoptosis pathways. These findings demonstrate a robust role of SERCA2a in attenuation of ischemic myocardial apoptosis, correlating with reverse activation of the ER stress-associated apoptosis pathways, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of ER stress-associated myocardial apoptosis.

  20. siRNA delivery using polyelectrolyte-gold nanoassemblies in neuronal cells for BACE1 gene silencing.

    Science.gov (United States)

    Chaudhary, Aparna; Garg, Sanjeev

    2017-11-01

    Small interfering RNA (siRNA) mediated RNA interference is a versatile therapeutic tool for many intractable genetic disorders. Various nanoassemblies specifically designed to deliver the siRNAs could be utilized for efficient siRNA delivery which is one of the major concern for the success of this therapeutic. Thus, in the present study, polyelectrolyte-gold nanoassemblies (PE-Gold NAs) were selected for siRNA delivery of an in vitro verified siRNA. Three different polyelectrolytes (polyethyleneimine, citraconic anhydride modified poly (allylamine) hydrochloride and poly l-arginine) were used to formulate the PE-Gold NAs using the layer-by-layer technique. Successful physico-chemical characterizations of these PE-Gold NAs were performed using UV-Visible, FTIR, 1 H-NMR spectroscopies, XRD, TEM, DLS and Zeta potential measurements. In vitro studies for the cytotoxicity, the uptake of these nanoassemblies and the gene silencing were carried out using these PE-Gold NAs in N2a and NB4 1A3 (murine neuronal) cell lines. The three selected PE-Gold NAs showed significant BACE1 (β-site APP cleaving enzyme 1) gene silencing (50-60%). This work demonstrates the potential of PE-Gold NAs to deliver siRNA targeting BACE1 in neuronal cells. Finally, it was concluded that different polyelectrolytes used in the PE-Gold NAs achieve different gene silencing due to the variation in their delivery efficiencies. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry

    International Nuclear Information System (INIS)

    Przybylski, Cédric; Benito, Juan M.; Bonnet, Véronique; Mellet, Carmen Ortiz; García Fernández, José M.

    2016-01-01

    Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps. - Highlights: • The first ETD-MS/MS characterization of polycationic carbohydrate based non-viral gene delivery

  2. Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Przybylski, Cédric, E-mail: cedric.przybylski@upmc.fr [Université d’Evry-Val-d’Essonne, Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement, CNRS UMR 8587, Bâtiment Maupertuis, Bld F. Mitterrand, F-91025 Evry (France); Benito, Juan M. [Instituto de Investigaciones Químicas (IIQ), CSIC−Universidad de Sevilla, Américo Vespucio 49, Isla de la Cartuja, E-41092 Sevilla (Spain); Bonnet, Véronique [Université de Picardie Jules Verne, Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources, CNRS UMR 7378, 80039 Amiens (France); Mellet, Carmen Ortiz [Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, E-41012 Sevilla (Spain); García Fernández, José M. [Instituto de Investigaciones Químicas (IIQ), CSIC−Universidad de Sevilla, Américo Vespucio 49, Isla de la Cartuja, E-41092 Sevilla (Spain)

    2016-12-15

    Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps. - Highlights: • The first ETD-MS/MS characterization of polycationic carbohydrate based non-viral gene delivery

  3. Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Ahn Jae

    2010-05-01

    Full Text Available Abstract Background Mesenchymal stem cells (MSCs are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP and brain-derived neurotropfic factor (BDNF plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells. Results Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83% and only minimal cell damage than when conventional liposome-based reagent (in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells. Conclusion Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.

  4. 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.

  5. AC133+ progenitor cells as gene delivery vehicle and cellular probe in subcutaneous tumor models: a preliminary study

    Directory of Open Access Journals (Sweden)

    Knight Robert A

    2009-03-01

    Full Text Available Abstract Background Despite enormous progress in gene therapy for breast cancer, an optimal systemic vehicle for delivering gene products to the target tissue is still lacking. The purpose of this study was to determine whether AC133+ progenitor cells (APC can be used as both gene delivery vehicles and cellular probes for magnetic resonance imaging (MRI. In this study, we used superparamagentic iron oxide (SPIO-labeled APCs to carry the human sodium iodide symporter (hNIS gene to the sites of implanted breast cancer in mouse model. In vivo real time tracking of these cells was performed by MRI and expression of hNIS was determined by Tc-99m pertechnetate (Tc-99m scan. Results Three million human breast cancer (MDA-MB-231 cells were subcutaneously implanted in the right flank of nude mice. APCs, isolated from fresh human cord blood, were genetically transformed to carry the hNIS gene using adenoviral vectors and magnetically labeled with ferumoxides-protamine sulfate (FePro complexes. Magnetically labeled genetically transformed cells were administered intravenously in tumor bearing mice when tumors reached 0.5 cm in the largest dimension. MRI and single photon emission computed tomography (SPECT images were acquired 3 and 7 days after cell injection, with a 7 Tesla animal MRI system and a custom built micro-SPECT using Tc-99m, respectively. Expression of hNIS in accumulated cells was determined by staining with anti-hNIS antibody. APCs were efficiently labeled with ferumoxide-protamine sulfate (FePro complexes and transduced with hNIS gene. Our study showed not only the accumulation of intravenously administered genetically transformed, magnetically labeled APCs in the implanted breast cancer, but also the expression of hNIS gene at the tumor site. Tc-99m activity ratio (tumor/non-tumor was significantly different between animals that received non-transduced and transduced cells (P Conclusion This study indicates that genetically transformed

  6. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    Science.gov (United States)

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated.

  7. Three-layered polyplex as a microRNA targeted delivery system for breast cancer gene therapy

    Science.gov (United States)

    Li, Yan; Dai, Yu; Zhang, Xiaojin; Chen, Jihua

    2017-07-01

    MicroRNAs (miRNAs), small non-coding RNAs, play an important role in modulating cell proliferation, migration, and differentiation. Since miRNAs can regulate multiple cancer-related genes simultaneously, regulating miRNAs could target a set of related oncogenic genes or pathways. Owing to their reduced immune response and low toxicity, miRNAs with small size and low molecular weight have become increasingly promising therapeutic drugs in cancer therapy. However, one of the major challenges of miRNAs-based cancer therapy is to achieve specific, effective, and safe delivery of therapeutic miRNAs into cancer cells. Here we provide a strategy using three-layered polyplex with folic acid as a targeting group to systemically deliver miR-210 into breast cancer cells, which results in breast cancer growth being inhibited.

  8. DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

    Science.gov (United States)

    Cole, Grace; Ali, Ahlam A; McCrudden, Cian M; McBride, John W; McCaffrey, Joanne; Robson, Tracy; Kett, Vicky L; Dunne, Nicholas J; Donnelly, Ryan F; McCarthy, Helen O

    2018-03-03

    Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 μg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer. Copyright © 2018. Published by Elsevier B.V.

  9. Development of Gold Nanoparticle towards Radioenhancement Therapy, Renal Clearance, siRNA Delivery and Light-Controlled Gene Silencing

    Science.gov (United States)

    Wang, Jianxin

    Gold nanoparticles (GNPs) have been widely studied and used in research for diagnostic, prophylactic or therapeutic purposes. However, they still face many technical challenges before they can be used to effectively address unmet biomedical needs. The theme of this dissertation is focused on addressing challenges of GNPs in clinical translation, and to improve their potential for application in radioenhancement therapy and siRNA delivery. We demonstrate the facile self-assembly of micellar gold nanocapsules using zwitterionic surfactants, with hydrodynamic diameters below 10 nm, which holds promise for good renal clearance to promote the excretion of GNPs in human body. We also prepared PEI- and PEG-coated GNPs and demonstrated their uptake into HeLa cells with exposure to soft X-rays (120 kVp), based on the consideration that the proximity of GNPs to nuclear DNA may be beneficial for enhancing low-energy ionizing radiotherapy. GNP-mediated siRNA delivery may be challenged by nonspecific siRNA desorption during circulation, which can cause off-target effects and immunogenicity. The use of gold nanorods (GNRs) for siRNA delivery also faces challenges like reduced dispersion stability during siRNA functionalization. We developed an effective way to load siRNA onto GNRs at high density, using oleylsulfobetaine (OSB) as an intermediate surfactant and dithiocarbamates (DTCs) as desorption-resistant anchors for siRNA. The GNR?siRNA complexes provided excellent control for laser-triggered gene silencing.

  10. The novel albumin-chitosan core-shell nanoparticles for gene delivery: preparation, optimization and cell uptake investigation

    Energy Technology Data Exchange (ETDEWEB)

    Karimi, Mahdi [Tarbiat Modares University, Department of Nanobiotechnology, Faculty of Biological Sciences (Iran, Islamic Republic of); Avci, Pinar [Massachusetts General Hospital, Wellman Center for Photomedicine (United States); Mobasseri, Rezvan [Tarbiat Modares University, Department of Nanobiotechnology, Faculty of Biological Sciences (Iran, Islamic Republic of); Hamblin, Michael R. [Massachusetts General Hospital, Wellman Center for Photomedicine (United States); Naderi-Manesh, Hossein, E-mail: naderman@modares.ac.ir [Tarbiat Modares University, Department of Nanobiotechnology, Faculty of Biological Sciences (Iran, Islamic Republic of)

    2013-05-15

    Natural polymers and proteins such as chitosan (CS) and albumin (Alb) have recently attracted much attention both in drug delivery and gene delivery. The underlying rationale is their unique properties such as biodegradability, biocompatibility and controlled release. This study aimed to prepare novel albumin-chitosan-DNA (Alb-CS-DNA) core-shell nanoparticles as a plasmid delivery system and find the best conditions for their preparation. Phase separation method and ionic interaction were used for preparation of Alb nanoparticles and Alb-CS-DNA core-shell nanoparticles, respectively. The effects of three important independent variables (1) CS/Alb mass ratio, (2) the ratios of moles of the amine groups of cationic polymers to those of the phosphate groups of DNA (N/P ratio), and (3) Alb concentration, on the nanoparticle size and loading efficiency of the plasmid were investigated and optimized through Box-Behnken design of response surface methodology (RSM). The optimum conditions were found to be CS/Alb mass ratio = 3, N/P ratio = 8.24 and Alb concentration = 0.1 mg/mL. The most critical factors for the size of nanoparticles and loading efficiency were Alb concentration and N/P ratio. The optimized nanoparticles had an average size of 176 {+-} 3.4 nm and loading efficiency of 80 {+-} 3.9 %. Cytotoxicity experiments demonstrated that the prepared nanoparticles were not toxic. The high cellular uptake of nanoparticles ({approx}85 %) was shown by flow cytometry and fluorescent microscopy.

  11. Development of Novel Polymeric Materials for Gene Therapy and pH-Sensitive Drug Delivery: Modeling, Synthesis, Characterization, and Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Brian Curtis [Iowa State Univ., Ames, IA (United States)

    2002-01-01

    The underlying theme of this thesis is the use of polymeric materials in bioapplications. Chapters 2-5 either develop a fundamental understanding of current materials used for bioapplications or establish protocols and procedures used in characterizing and synthesizing novel materials. In chapters 6 and 7 these principles and procedures are applied to the development of materials to be used for gene therapy and drug delivery. Chapter one is an introduction to the ideas that will be necessary to understand the subsequent chapters, as well as a literature review of these topics. Chapter two is a paper that has been published in the ''Journal of Controlled Release'' that examines the mechanism of drug release from a polymer gel, as well as experimental design suggestions for the evaluation of water soluble drug delivery systems. Chapter three is a paper that has been published in the ''Journal of Pharmaceutical Sciences'' that discusses the effect ionic salts have on properties of the polymer systems examined in chapter two. Chapter four is a paper published in the Materials Research Society Fall 2000 Symposium Series dealing with the design and synthesis of a pH-sensitive polymeric drug delivery device. Chapter five is a paper that has been published in the journal ''Biomaterials'' proposing a novel polymer/metal composite for use as a biomaterial in hip arthroplasty surgery. Chapter six is a paper that will appear in an upcoming volume of the Journal ''Biomaterials'' dealing with the synthesis of a novel water soluble cationic polymer with possible applications in non-viral gene therapy. Chapter seven is a paper that has been submitted to ''Macromolecules'' discussing several novel block copolymers based on poly(ethylene glycol) and poly(diethylamino ethyl methacrylate) that possess both pH-sensitive and temperature sensitive properties. Chapter eight contains a

  12. Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells.

    Science.gov (United States)

    Li, Bo-Chao; Chang, Hao; Ren, Ke-Feng; Ji, Jian

    2016-11-01

    Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. 76 FR 31462 - Airworthiness Directives; The Boeing Company Model DC-10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10...

    Science.gov (United States)

    2011-06-01

    ... Model DC-10-10, DC- 10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F.... Applicability (c) This AD applies to all The Boeing Company Model DC-10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10- 40, DC-10-40F, MD-10-10F, MD-10-30F, MD-11, and MD-11F airplanes...

  14. Discovery of Antibiotics-derived Polymers for Gene Delivery using Combinatorial Synthesis and Cheminformatics Modeling

    Science.gov (United States)

    Potta, Thrimoorthy; Zhen, Zhuo; Grandhi, Taraka Sai Pavan; Christensen, Matthew D.; Ramos, James; Breneman, Curt M.; Rege, Kaushal

    2014-01-01

    We describe the combinatorial synthesis and cheminformatics modeling of aminoglycoside antibiotics-derived polymers for transgene delivery and expression. Fifty-six polymers were synthesized by polymerizing aminoglycosides with diglycidyl ether cross-linkers. Parallel screening resulted in identification of several lead polymers that resulted in high transgene expression levels in cells. The role of polymer physicochemical properties in determining efficacy of transgene expression was investigated using Quantitative Structure-Activity Relationship (QSAR) cheminformatics models based on Support Vector Regression (SVR) and ‘building block’ polymer structures. The QSAR model exhibited high predictive ability, and investigation of descriptors in the model, using molecular visualization and correlation plots, indicated that physicochemical attributes related to both, aminoglycosides and diglycidyl ethers facilitated transgene expression. This work synergistically combines combinatorial synthesis and parallel screening with cheminformatics-based QSAR models for discovery and physicochemical elucidation of effective antibiotics-derived polymers for transgene delivery in medicine and biotechnology. PMID:24331709

  15. Discovery of antibiotics-derived polymers for gene delivery using combinatorial synthesis and cheminformatics modeling.

    Science.gov (United States)

    Potta, Thrimoorthy; Zhen, Zhuo; Grandhi, Taraka Sai Pavan; Christensen, Matthew D; Ramos, James; Breneman, Curt M; Rege, Kaushal

    2014-02-01

    We describe the combinatorial synthesis and cheminformatics modeling of aminoglycoside antibiotics-derived polymers for transgene delivery and expression. Fifty-six polymers were synthesized by polymerizing aminoglycosides with diglycidyl ether cross-linkers. Parallel screening resulted in identification of several lead polymers that resulted in high transgene expression levels in cells. The role of polymer physicochemical properties in determining efficacy of transgene expression was investigated using Quantitative Structure-Activity Relationship (QSAR) cheminformatics models based on Support Vector Regression (SVR) and 'building block' polymer structures. The QSAR model exhibited high predictive ability, and investigation of descriptors in the model, using molecular visualization and correlation plots, indicated that physicochemical attributes related to both, aminoglycosides and diglycidyl ethers facilitated transgene expression. This work synergistically combines combinatorial synthesis and parallel screening with cheminformatics-based QSAR models for discovery and physicochemical elucidation of effective antibiotics-derived polymers for transgene delivery in medicine and biotechnology. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA.

    Science.gov (United States)

    Deng, Xin; Zhang, Guoli; Zhang, Ling; Feng, Yan; Li, Zehong; Wu, GuangMou; Yue, Yuhuan; Li, Gensong; Cao, Yu; Zhu, Ping

    2015-01-01

    Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.

  17. Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

    International Nuclear Information System (INIS)

    Peng, Jing; Zhang, Xinming; Li, Zhaoyang; Liu, Yunde; Yang, Xianjin

    2015-01-01

    Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression

  18. Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jing, E-mail: pengjingtd@163.com [Airport College, Civil Aviation University of China, Tianjin 300300 (China); Zhang, Xinming, E-mail: xinmingmail@163.com [Tianjin Product Quality Inspection Technology Research Institute, Tianjin 300384 (China); School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Li, Zhaoyang [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Liu, Yunde [School of Medical Laboratory, Tianjin Medical University, Tianjin 300203 (China); Yang, Xianjin [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China)

    2015-11-01

    Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression.

  19. A novel plasmid and SonoVue formulation plus ultrasound sonication for effective gene delivery in nude mice.

    Science.gov (United States)

    Wang, Xin-Lu; Zhao, Xiao-Yun; Li, Shuo; Jia, Chang-Jun; Jiang, Luo; Shi, Tei-Mei; Ren, Wei-Dong

    2013-10-11

    This study aimed to identify the effectiveness of gene therapy mediated by ultrasound-targeted SonoVue using the herpes simplex virus-thymidine kinase (TSV-TK) driven by the kinase insert domain receptor (KDR) promoter (KDR-TK) for the treatment of ovarian carcinomas in nude mice. The optimized conditions for gene transfection were also explored. In this study, we developed a novel technique to deliver a plasmid vector-carried gene into tumor xenografts in sixty nude mice. We first mixed plasmid DNA with SonoVue to form microbubbles and then treated the mice with ultrasound sonication to enhance plasmid gene transfection and expression in tumor xenografts. The data showed that injection of pBluescript-KDR-TK cDNA mixed with SonoVue into nude mice plus ultrasound sonication significantly (Group E) increased the transfection efficiency and expression of KDR-TK mRNA in tumor xenografts. The growth of tumor xenografts in nude mice was significantly suppressed in Group E compared to the other four control groups (Groups A, B, C, and D, namely, treatment with phosphate-buffered saline (PBS), KDR-TK+PBS, KDR-TK+SonoVue, KDR-TK+PBS+ultrasound sonication, respectively). TUNEL staining showed that SonoVue plus ultrasound sonication significantly induced apoptosis and reduced microvessel density (MVD). This study revealed that the formulation of plasmid with SonoVue plus ultrasound could provide efficient gene delivery into tumor xenografts. Increased gene expression was observed in vivo, which effectively reduced the tumor growth and MVD of tumor xenografts and induced apoptosis in tumor cells. Future clinical trials are necessary to further analyze the relevance of this technique. © 2013.

  20. Charge pump DC-DC converter comprising solid state batteries

    NARCIS (Netherlands)

    Reefman, D.; Roozeboom, F.; Notten, P.H.L.; Klootwijk, J.H.

    2013-01-01

    An electronic device is provided which comprises a DC-DC converter. The DC-DC converter comprises at least one solid-state rechargeable battery (B1, B2) for storing energy for the DC-DC conversion and an output capacitor (C2).

  1. DC source assemblies

    Science.gov (United States)

    Campbell, Jeremy B; Newson, Steve

    2013-02-26

    Embodiments of DC source assemblies of power inverter systems of the type suitable for deployment in a vehicle having an electrically grounded chassis are provided. An embodiment of a DC source assembly comprises a housing, a DC source disposed within the housing, a first terminal, and a second terminal. The DC source also comprises a first capacitor having a first electrode electrically coupled to the housing, and a second electrode electrically coupled to the first terminal. The DC source assembly further comprises a second capacitor having a first electrode electrically coupled to the housing, and a second electrode electrically coupled to the second terminal.

  2. Reduction-sensitive lipopolyamines as a novel nonviral gene delivery system for modulated release of DNA with improved transgene expression.

    Science.gov (United States)

    Byk, G; Wetzer, B; Frederic, M; Dubertret, C; Pitard, B; Jaslin, G; Scherman, D

    2000-11-16

    We have designed and synthesized original cationic lipids for modulated release of DNA from cationic lipid/DNA complexes. Our rationale was that modulated degradation of the lipids during or after penetration into the cell could improve the trafficking of DNA to the nucleus resulting in increased transgene expression. The new reduction-sensitive lipopolyamines (RSL) harbor a disulfide bridge within different positions in the backbone of the lipids as biosensitive function. A useful synthetic method was developed to obtain, with very good yields and reproducibility, unsymmetrical disulfide-bridged molecules, starting from symmetrical disulfides and thiols. The new lipopolyamines are good candidates as carriers of therapeutic genes for in vivo gene delivery. To optimize the transfection efficiency in these novel series, we have carried out structure-activity relationship studies by placing the disulfide bridge at different positions in the backbone of the cationic lipid and by systematic variation of lipid chain length. Results indicate that the transfection level can be modulated as a function of the location of the disulfide bridge in the molecule. We suggest that an early release of DNA during or after penetration into the cell, probably promoted by reduction of a disulfide bridge placed between the polyamine and the lipid, implies a total loss of transfection efficiency. On the other hand, proper modulation of DNA release by inserting the disulfide bridge between one lipid chain and the rest of the molecule brings about increased transfection efficiency as compared to previously described nondegradable lipopolyamine analogues. Finally, preliminary physicochemical characterization of the complexes demonstrates that DNA release from complexes can be modulated as a function of the surrounding reducing conditions of the complexes and of the localization of the disulfide bridge within the lipopolyamine. Our results suggest that RSL is a promising new approach for gene

  3. Gene delivery by cationic lipids : in and out of an endosome

    NARCIS (Netherlands)

    Hoekstra, D.; Rejman, J.; Wasungu, L.; Shi, F.; Zuhorn, I.

    Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in

  4. DC-SCRIPT is a novel regulator of the tumor suppressor gene CDKN2B and induces cell cycle arrest in ERα-positive breast cancer cells

    NARCIS (Netherlands)

    M. Ansems (Marleen); J.N. Søndergaard (Jonas Nørskov); A.M. Sieuwerts (Anieta); M.W.G. Looman (Maaike W. G.); M. Smid (Marcel); A.M.A. de Graaf (Annemarie M. A.); V. de Weerd (Vanja); M. Zuidscherwoude (Malou); J.A. Foekens (John); J.W.M. Martens (John); G.J. Adema (Gosse J.)

    2015-01-01

    textabstractBreast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERα) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERα and as a strong and independent

  5. DC-SCRIPT is a novel regulator of the tumor suppressor gene CDKN2B and induces cell cycle arrest in ERalpha-positive breast cancer cells

    NARCIS (Netherlands)

    Ansems, M.; Sondergaard, J.N.; Sieuwerts, A.M.; Looman, M.W.G.; Smid, M.; Graaf, A.M.A. de; Weerd, V. de; Zuidscherwoude, M.; Foekens, J.A.; Martens, J.W.; Adema, G.J.

    2015-01-01

    Breast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERalpha) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERalpha and as a strong and independent

  6. IL-10/IFNγ co-expressing CD4(+) T cells induced by IL-10 DC display a regulatory gene profile and downmodulate T cell responses

    NARCIS (Netherlands)

    Boks, Martine A.; Kager-Groenland, Judith R.; van Ham, S. Marieke; ten Brinke, Anja

    2016-01-01

    Induced regulatory T cells (iTreg) are imperative for tolerance induction and spreading of infectious tolerance. Ex vivo generated tolerogenic dendritic cells (tDCs) have strong therapeutic potential to induce antigen-specific iTreg. We previously demonstrated that IL-10 tDC-primed T cells are very

  7. CISH is induced during DC development and regulates DC-mediated CTL activation.

    Science.gov (United States)

    Miah, Mohammad Alam; Yoon, Cheol-Hee; Kim, Joonoh; Jang, Jinah; Seong, Young-Rim; Bae, Yong-Soo

    2012-01-01

    The cytokine inducible SH2-domain protein (CISH) is a well-known STAT5 target gene, but its role in the immune system remains uncertain. In this study, we found that CISH is predominantly induced during dendritic cell (DC) development from mouse bone marrow (BM) cells and plays a crucial role in type 1 DC development and DC-mediated CTL activation. CISH knockdown reduced the expression of MHC class I, co-stimulatory molecules and pro-inflammatory cytokines in BMDCs. Meanwhile, the DC yield was markedly enhanced by CISH knockdown via cell-cycle activation and reduction of cell apoptosis. Down-regulation of cell proliferation at the later stage of DC development was found to be associated with CISH-mediated negative feedback regulation of STAT5 activation. In T-cell immunity, OT-1 T-cell proliferation was significantly reduced by CISH knockdown in DCs, whereas OT-2 T-cell proliferation was not affected by CISH knockdown. CTLs generated by DC vaccination were also markedly reduced by CISH knockdown, followed by significant impairment of DC-based tumor immunotherapy. Taken together, our data suggest that CISH expression at the later stage of DC development triggers the shutdown of DC progenitor cell proliferation and facilitates DC differentiation into a potent stimulator of CTLs. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles.

    Science.gov (United States)

    Mout, Rubul; Rotello, Vincent M

    2017-10-20

    In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users' need. Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al ., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al ., 2017). We report here only the generation of the delivery vehicle i.e. , the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

  9. Delivery of Adeno-Associated Virus Gene Therapy by Intravascular Limb Infusion Methods.

    Science.gov (United States)

    Gruntman, Alisha M; Flotte, Terence R

    2015-09-01

    Recombinant adeno-associated virus (rAAV) can be delivered to the skeletal muscle of the limb (pelvic or thoracic) by means of regional intravascular delivery. This review summarizes the evolution of this technique to deliver rAAV either via the arterial blood supply or via the peripheral venous circulation. The focus of this review is on applications in large animal models, including preclinical studies. Based on this overview of past research, we aim to inform the design of preclinical and clinical studies.

  10. Rapid screening of gene function by systemic delivery of morpholino oligonucleotides to live mouse embryos.

    Directory of Open Access Journals (Sweden)

    Kathryn S McClelland

    Full Text Available Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9 and pancreas (Sox9. We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis.

  11. Silk fibroin layer-by-layer microcapsules for localized gene delivery.

    Science.gov (United States)

    Li, Linhao; Puhl, Sebastian; Meinel, Lorenz; Germershaus, Oliver

    2014-09-01

    Herein, we describe the delivery of plasmid DNA (pDNA) using silk fibroin (SF) layer-by-layer assembled microcapsules. Deposition of fluorescently labeled SF onto polystyrene (PS) template particles resulted in increasing fluorescence intensity and decreasing surface charge in correlation to SF layer number. After removal of the PS core, hollow, monodisperse, and structurally stable SF microcapsules of variable size and shell thickness were obtained. Plasmid DNA encoding for enhanced green fluorescent protein (eGFP) was loaded onto 1 or 4 μm capsules, either by incorporation of pDNA within the innermost layer of the shell or by adsorption to the microcapsules surface, and in vitro pDNA release, cytotoxicty and eGFP expression were studied. Sustained pDNA release over 3 days was observed using both loading techniques, being accelerated in the presence of protease. DNA loaded SF microcapsules resulted in efficient cell transfection along with low cytotoxicity after 3 days incubation compared to treatment with pDNA/branched polyethylenimine complexes. Among the tested conditions highest transfection efficiencies were achieved using 1 μm capsules where pDNA was adsorbed to the capsule surface. Our results suggest that SF microcapsules are suitable for the localized delivery of pDNA, combining low cytotoxicity and high transfection efficiency. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Treatment of Hemophilia A in Utero and Postnatally using Sheep as a Model for Cell and Gene Delivery

    Science.gov (United States)

    Porada, Christopher D.; Almeida-Porada, Graça

    2012-01-01

    Hemophilia A represents the most common inheritable deficiency of the coagulation proteins. Current state-of- the-art treatment consists of frequent prophylactic infusions of plasma-derived or recombinant FVIII protein to maintain hemostasis, and has greatly increased life expectancy and quality of life for many hemophilia A patients. This treatment approach is, however, far from ideal, due to the need for lifelong intravenous infusions, the high treatment cost, and the fact that it is unavailable to a large percentage of the world’s hemophiliacs. There is thus a need for novel treatments that can promise long-term or permanent correction. In contrast to existing protein based therapeutics, gene therapy offers to provide a permanent cure following few, or even a single, treatment. In the present paper, we review ongoing work towards this end, focusing on studies we have performed in a large animal model. Some of the key topics covered in this review include the unique opportunities sheep offer as a model system, the re-establishment and clinical and molecular characterization of a line of sheep with severe hemophilia A, the advantages and feasibility of treating a disease like hemophilia A in utero, and the use of Mesenchymal Stem Cells (MSC) as cellular delivery vehicles for the FVIII gene. The review finishes with a brief discussion of our recent success correcting ovine hemophilia A with a postnatal transplant with gene-modified MSC, and the limitations of this approach that remain to be overcome. PMID:23264887

  13. Efficacy of In Vivo Electroporation-Mediated IL-10 Gene Delivery on Survival of Skin Flaps.

    Science.gov (United States)

    Seyed Jafari, S Morteza; Shafighi, Maziar; Beltraminelli, Helmut; Weber, Benedikt; Schmid, Ralph A; Geiser, Thomas; Gazdhar, Amiq; Hunger, Robert E

    2018-04-01

    Despite advances in understanding the underlying mechanisms of flap necrosis and improvement in surgical techniques, skin flap necrosis after reconstructive surgery remains a crucial issue. We investigated the efficacy of electroporation-mediated IL-10 gene transfer to random skin flap with an aim to accelerate wound healing and improve skin flap survival. Nine male Wistar rats (300-330 g) were divided in two groups (a) control group (n = 5), only surgery no gene transfer, and (b) experimental group, received electroporation-mediated IL-10 gene transfer 24 h before the surgery as prophylaxis (n = 4). Random skin flap (McFarlane) was performed in both groups. Planimetry, Laser Doppler imaging, and immunohistochemistry were used to evaluate the effect of IL-10 gene transfer between study groups at day 7. Electroporation-mediated IL-10 gene transfer decreased percentage of flap necrosis (p value = 0.0159) and increased cutaneous perfusion compared to the control group (p value = 0.0159). In addition, Spearman's rank correlation showed a significant negative correlation between percentage of flap necrosis and Laser Index (p value = 0.0083, r -0.83, respectively). Furthermore, significantly higher mean CD31 + vessel density was detected in the experimental group compared to the control group (p value = 0.0159). Additionally, semi-quantitative image analysis showed lower inflammatory cell count in experimental group compared to control group (p value = 0.0317). In vivo electroporation-mediated IL-10 gene transfer reduced necrosis, enhanced survival and vascularity in the ischemic skin flap.

  14. Targeted Gene Delivery to Macrophages by Biodegradable Star-Shaped Polymers.

    Science.gov (United States)

    Zhang, Yajie; Wang, Yafeng; Zhang, Chi; Wang, Jin; Pan, Dejing; Liu, Jianghuai; Feng, Fude

    2016-02-17

    In this report, two biodegradable star-shaped polyasparamide derivatives and four analogues modified with either mannose or folic acid moiety for preferential targeting of a difficult-to-transfect immune cell type, i.e., macrophage, have been synthesized. Each of the prepared star polymers complexes with plasmid DNA to form nanosized particles featuring a core-shell-like morphology. Mannose or folate functionalized star polymers can greatly improve the transfection performance on a macrophage cell line RAW 264.7. As a result, a combination of targeting ligand modification and topological structures of gene carriers is a promising strategy for immune cells-based gene therapy.

  15. BDNF gene delivery within and beyond templated agarose multi-channel guidance scaffolds enhances peripheral nerve regeneration

    Science.gov (United States)

    Gao, Mingyong; Lu, Paul; Lynam, Dan; Bednark, Bridget; Campana, W. Marie; Sakamoto, Jeff; Tuszynski, Mark

    2016-12-01

    Objective. We combined implantation of multi-channel templated agarose scaffolds with growth factor gene delivery to examine whether this combinatorial treatment can enhance peripheral axonal regeneration through long sciatic nerve gaps. Approach. 15 mm long scaffolds were templated into highly organized, strictly linear channels, mimicking the linear organization of natural nerves into fascicles of related function. Scaffolds were filled with syngeneic bone marrow stromal cells (MSCs) secreting the growth factor brain derived neurotrophic factor (BDNF), and lentiviral vectors expressing BDNF were injected into the sciatic nerve segment distal to the scaffold implantation site. Main results. Twelve weeks after injury, scaffolds supported highly linear regeneration of host axons across the 15 mm lesion gap. The incorporation of BDNF-secreting cells into scaffolds significantly increased axonal regeneration, and additional injection of viral vectors expressing BDNF into the distal segment of the transected nerve significantly enhanced axonal regeneration beyond the lesion. Significance. Combinatorial treatment with multichannel bioengineered scaffolds and distal growth factor delivery significantly improves peripheral nerve repair, rivaling the gold standard of autografts.

  16. Seeing engineered loops in a gene delivery vehicle by cryoEM

    OpenAIRE

    Zhou, Z. Hong

    2012-01-01

    In this issue of Structure, Lerch and colleagues present a 4.5 Å cryo electron microscopy (cryoEM) structure of a variant of an adeno-associated virus that has been genetically engineered for liver gene therapy. The identification of two structurally distinct loops flanking the highly conserved jellyroll β barrel highlights the potentials of high-resolution cryoEM.

  17. Gene delivery of the therapeutic polypeptide erythropoietin to primary brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Moos, Torben

    2016-01-01

    The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this pass......The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints......, however, recently shown that non-viral gene therapy to non-mitotic BCECs cultured in an in-vitro BBB model was possible without disrupting the barrier properties of the BCECs, and that this was as effective as transfection of dividing BCECs. The transfection efficiency is, however, low, questioning...... for its neuroprotective potential, from the BCECs. Our study opens for knowledge on, how non-viral gene therapy to BCECs can lead to protein secretion with the perspective of enabling therapeutic proteins to target neurons inside the CNS otherwise inhibited to access due to the restraints of the BBB...

  18. The use of Listeria monocytogenes as a DNA delivery vector for cancer gene therapy.

    LENUS (Irish Health Repository)

    Tangney, Mark

    2012-01-31

    Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.

  19. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

    Science.gov (United States)

    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  20. Noninvasive delivery of gene targeting probes to live brains for transcription MRI

    Science.gov (United States)

    Liu, Christina H.; You, Zerong; Ren, JiaQian; Kim, Young R.; Eikermann-Haerter, Katharina; Liu, Philip K.

    2009-01-01

    We aimed to test the feasibility of detecting gliosis in living brains when the blood-brain barrier (BBB) is disrupted. We designed a novel magnetic resonance (MR) probe that contains superparamagnetic iron oxide nanoparticles (SPION, a T2 susceptibility contrast agent) linked to a short DNA sequence complementary to the cerebral mRNA of glial fibrillary acidic protein (GFAP) found in glia and astrocytes. As a control, we also used a sequence complementary to the mRNA of β-actin. Our objectives are to demonstrate that this new probe, SPION-gfap, could be delivered to the brain when administered by eyedrop solution to the conjunctival sac. We induced BBB leakage by puncture wound, global cerebral ischemia, and cortical spreading depression in C57BL6 mice; 1 day after probe delivery we acquired T2* MR images and R2* (R2*=1/T2*) maps using a transcription MRI technique in live mice. We found that the SPION-gfap probe reported foci with elevated signal in subtraction R2* maps and that these foci matched areas identified as having extensive glial network (gliosis) in postmortem immunohistochemistry. Similarly, animals administered the control probe exhibited foci of R2* elevation that matched β-actin-expressing endothelia in the vascular wall. We conclude that our modular MR probe, delivered in an eyedrop solution, effectively reports gliosis associated with acute neurological disorders in living animals. As BBB leakage is often observed in acute neurological disorders, this study also served to validate noninvasive delivery of MR probes to the brains of live animals after acute neurological disorders. PMID:18029447

  1. Monocyte Trafficking, Engraftment, and Delivery of Nanoparticles and an Exogenous Gene into the Acutely Inflamed Brain Tissue - Evaluations on Monocyte-Based Delivery System for the Central Nervous System.

    Directory of Open Access Journals (Sweden)

    Hsin-I Tong

    Full Text Available The ability of monocytes and monocyte-derived macrophages (MDM to travel towards chemotactic gradient, traverse tissue barriers, and accumulate precisely at diseased sites makes them attractive candidates as drug carriers and therapeutic gene delivery vehicles targeting the brain, where treatments are often hampered by the blockade of the blood brain barrier (BBB. This study was designed to fully establish an optimized cell-based delivery system using monocytes and MDM, by evaluating their homing efficiency, engraftment potential, as well as carriage and delivery ability to transport nano-scaled particles and exogenous genes into the brain, following the non-invasive intravenous (IV cell adoptive transfer in an acute neuroinflammation mouse model induced by intracranial injection of Escherichia coli lipopolysaccharides. We demonstrated that freshly isolated monocytes had superior inflamed-brain homing ability over MDM cultured in the presence of macrophage colony stimulating factor. In addition, brain trafficking of IV infused monocytes was positively correlated with the number of adoptive transferred cells, and could be further enhanced by transient disruption of the BBB with IV administration of Mannitol, Bradykinin or Serotonin right before cell infusion. A small portion of transmigrated cells was detected to differentiate into IBA-1 positive cells with microglia morphology in the brain. Finally, with the use of superparamagnetic iron oxide nanoparticles SHP30, the ability of nanoscale agent-carriage monocytes to enter the inflamed brain region was validated. In addition, lentiviral vector DHIV-101 was used to introduce green fluorescent protein (GFP gene into monocytes, and the exogenous GFP gene was detected in the brain at 48 hours following IV infusion of the transduced monocytes. All together, our study has set up the optimized conditions for the more-in-depth tests and development of monocyte-mediated delivery, and our data supported

  2. Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery.

    Science.gov (United States)

    Rajagopalan, Rukkumani; Xavier, Jennifer; Rangaraj, Nandini; Rao, Nalam Madhusudhana; Gopal, Vijaya

    2007-04-01

    The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety. Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently. Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone. Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes

  3. Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors

    Directory of Open Access Journals (Sweden)

    Cristian Capasso

    2013-12-01

    Full Text Available Viral vectors have been extensively studied due to their great transduction efficiency compared to non-viral vectors. These vectors have been used extensively in gene therapy, enabling the comprehension of, not only the advantages of these vectors, but also the limitations, such as the activation of the immune system after vector administration. Moreover, the need to control the target of the vector has led to the development of chemical and non-chemical modifications of the vector surface, allowing researchers to modify the tropism and biodistribution profile of the vector, leading to the production of viral vectors able to target different tissues and organs. This review describes recent non-genetic modifications of the surfaces of viral vectors to decrease immune system activation and to control tissue targeting. The developments described herein provide opportunities for applications of gene therapy to treat acquired disorders and genetic diseases and to become useful tools in regenerative medicine.

  4. Gene delivery of therapeutic polypeptides to brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Moos, Torben

    Background: The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-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 into the brain. Aim: The aim of the present study was to investigate the possibility of transfection to primary rat brain capillary endothelial cells (RBEC) for recombinant protein synthesis...... of the proteins. Morphological examination of the protein expression was determined using immunofluorescence detecting FLAG. Additionally, the transfection efficiency were determined by Flow cytometry. Perspective: Our study opens for knowledge on how non-viral gene therapy to BCECs can lead to protein secretion...

  5. 75 FR 63040 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-10-10, DC-10-10F, DC-10-30, DC...

    Science.gov (United States)

    2010-10-14

    ... Corporation Model DC- 10-10, DC-10-10F, DC-10-30, DC-10-30F (KDC-10), DC-10-40, and DC-10-40F Airplanes AGENCY...-10F, DC-10-30, DC-10-30F (KDC-10), DC-10-40, and DC-10- 40F airplanes, certificated in any category...

  6. Comparison of Gene Delivery Methods for Transformation of Robinia pseudoacacia L.by Ti-plasmid

    OpenAIRE

    Ren, Chen; Koishiro, Gyokusen; Akira, Saito; Department of Forestry, Faculty of Agiculture, Kyushu University; Department of Forestry, Faculty of Agiculture, Kyushu University; Department of Forestry, Faculty of Agiculture, Kyushu University

    2000-01-01

    The bjnary vector plasmid plG121-Hm carrying the chimeric neomycin phosphotransferase (npt II),β-glucuronidase (gusA) andhygromycin phosphotransferase (hpt) genes was delivered into Robinia pseudoacacia L. callus using the Agrobacterium tuntefaciens-mediated transformation method and particle gun transformation method. It was determined that adding 10 mg/L of acetosyringone to Agrobacterium infection medium and callus-Agrobacterium co-culture medium could increase Agrobacterium-mediated trans...

  7. Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors

    OpenAIRE

    Capasso, Cristian; Hirvinen, Mari; Cerullo, Vincenzo

    2013-01-01

    Viral vectors have been extensively studied due to their great transduction efficiency compared to non-viral vectors. These vectors have been used extensively in gene therapy, enabling the comprehension of, not only the advantages of these vectors, but also the limitations, such as the activation of the immune system after vector administration. Moreover, the need to control the target of the vector has led to the development of chemical and non-chemical modifications of the vector surface, a...

  8. Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors

    Directory of Open Access Journals (Sweden)

    Nicolas eMerienne

    2013-07-01

    Full Text Available Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during CNS development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain.

  9. Egr2 induced during DC development acts as an intrinsic negative regulator of DC immunogenicity.

    Science.gov (United States)

    Miah, Mohammad Alam; Byeon, Se Eun; Ahmed, Md Selim; Yoon, Cheol-Hee; Ha, Sang-Jun; Bae, Yong-Soo

    2013-09-01

    Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig.

    Science.gov (United States)

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-06-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

  11. Therapeutic Efficacy of Vectored PGT121 Gene Delivery in HIV-1-Infected Humanized Mice.

    Science.gov (United States)

    Badamchi-Zadeh, Alexander; Tartaglia, Lawrence J; Abbink, Peter; Bricault, Christine A; Liu, Po-Ting; Boyd, Michael; Kirilova, Marinela; Mercado, Noe B; Nanayakkara, Ovini S; Vrbanac, Vladimir D; Tager, Andrew M; Larocca, Rafael A; Seaman, Michael S; Barouch, Dan H

    2018-04-01

    Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Copyright © 2018 Badamchi-Zadeh et al.

  12. Theranostic nanoparticles based on bioreducible polyethylenimine-coated iron oxide for reduction-responsive gene delivery and magnetic resonance imaging

    Directory of Open Access Journals (Sweden)

    Li D

    2014-07-01

    Full Text Available Dan Li,1,* Xin Tang,2,* Benjamin Pulli,1 Chao Lin,2 Peng Zhao,2 Jian Cheng,2 Zhongwei Lv,1 Xueyu Yuan,1 Qiong Luo,1 Haidong Cai,1 Meng Ye1 1Department of Nuclear Medicine, Shanghai 10th People’s Hospital, 2Shanghai East Hospital, The Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Tongji University, People’s Republic of China *These authors contributed equally to this work Abstract: Theranostic nanoparticles based on superparamagnetic iron oxide (SPIO have a great promise for tumor diagnosis and gene therapy. However, the availability of theranostic nanoparticles with efficient gene transfection and minimal toxicity remains a big challenge. In this study, we construct an intelligent SPIO-based nanoparticle comprising a SPIO inner core and a disulfide-containing polyethylenimine (SSPEI outer layer, which is referred to as a SSPEI-SPIO nanoparticle, for redox-triggered gene release in response to an intracellular reducing environment. We reveal that SSPEI-SPIO nanoparticles are capable of binding genes to form nano-complexes and mediating a facilitated gene release in the presence of dithiothreitol (5–20 mM, thereby leading to high transfection efficiency against different cancer cells. The SSPEI-SPIO nanoparticles are also able to deliver small interfering RNA (siRNA for the silencing of human telomerase reverse transcriptase genes in HepG2 cells, causing their apoptosis and growth inhibition. Further, the nanoparticles are applicable as T2-negative contrast agents for magnetic resonance (MR imaging of a tumor xenografted in a nude mouse. Importantly, SSPEI-SPIO nanoparticles have relatively low cytotoxicity in vitro at a high concentration of 100 µg/mL. The results of this study demonstrate the utility of a disulfide-containing cationic polymer-decorated SPIO nanoparticle as highly potent and low-toxic theranostic nano-system for specific nucleic acid delivery inside cancer cells. Keywords

  13. Nano and Microtechnologies for the Delivery of Oligonucleotides with Gene Silencing Properties

    Directory of Open Access Journals (Sweden)

    Giuseppe De Rosa

    2009-07-01

    Full Text Available Oligonucleotides (ONs are synthetic fragments of nucleic acid designed to modulate the expression of target proteins. DNA-based ONs (antisense, antigene, aptamer or decoy and more recently a new class of RNA-based ONs, the small interfering RNAs (siRNAs, have gained great attention for the treatment of different disease states, such as viral infections, inflammation, diabetes, and cancer. However, the development of therapeutic strategies based on ONs is hampered by their low bioavailability, poor intracellular uptake and rapid degradation in biological fluids. The use of a non-viral carrier can be a powerful tool to overcome these drawbacks. Lipid or polymer-based nanotechnologies can improve biological stability and cellular uptake of ONs, with possibility of tissue and/or cellular targeting. The use of polymeric devices can also produce a prolonged release of the ON, thus reducing the need of frequent administrations. This review summarizes advantages and issues related to the main non-viral vectors used for ON delivery.

  14. Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice

    Science.gov (United States)

    Goudy, Kevin; Song, Sihong; Wasserfall, Clive; Zhang, Y. Clare; Kapturczak, Matthias; Muir, Andrew; Powers, Matthew; Scott-Jorgensen, Marda; Campbell-Thompson, Martha; Crawford, James M.; Ellis, Tamir M.; Flotte, Terence R.; Atkinson, Mark A.

    2001-01-01

    The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders. PMID:11717448

  15. Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Joana S Boura

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSC constitutively express low levels of human leukocyte antigen-G (HLA-G, which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immunomodulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1 and a viral system (Lv-HLA-G1 using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK cell–mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties.

  16. Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry.

    Science.gov (United States)

    Sakashita, Mizuha; Mochizuki, Shinichi; Sakurai, Kazuo

    2014-10-01

    Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed 'lipoplex', we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Nanoparticle mediated PPARγ gene delivery on dental implants improves osseointegration via mitochondrial biogenesis in diabetes mellitus rat model.

    Science.gov (United States)

    Lee, Young-Hee; Kim, Jeong-Seok; Kim, Ji-Eun; Lee, Min-Ho; Jeon, Jae-Gyu; Park, Il-Song; Yi, Ho-Keun

    2017-07-01

    Diabetes mellitus (DM) has a detrimental effect on osseointegration, stability and longevity of implants due to osteoporosis. In this study, PPARγ-loaded dental implants were investigated for the improvement of osseointegration and peri-implantitis. Chitosan gold nanoparticles conjugated with PPARγ cDNA were introduced on titanium mini-implant surfaces for PPARγ release to rat mandibular. DM-induced rat mandible showed structural changes such as decreased bone mass and increased inflammatory molecules, and diminution of PPARγ expression and bone formation molecules compared to normal rats. PPARγ induced bone formation via reduction of inflammatory molecules even under glucose oxidative stress. Furthermore, PPARγ strongly activated mitochondrial biogenesis and cell viability via p-AMK and Wnt/β-catenin signaling. Consequently, PPARγ gene delivery on regional dental implants contributed osseointegration, new bone formation and mineralization in DM-induced rats. This study demonstrates that PPARγ can be used as a therapeutic gene with dental implantation in diabetic patients since regional PPARγ expression enhances osseointegration and implant longevity. Copyright © 2017. Published by Elsevier Inc.

  18. Reversing Bacterial Resistance to Antibiotics by Phage-Mediated Delivery of Dominant Sensitive Genes

    Science.gov (United States)

    Edgar, Rotem; Friedman, Nir; Molshanski-Mor, Shahar

    2012-01-01

    Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene's sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage's ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant. PMID:22113912

  19. Gene delivery into primary brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Lichota, Jacek

    model was established by co-culturing primary BCECs together with primary astrocytes, both of which were isolated from rats. This was done in order to study the possibility of using gene transfection in an environment closer to the in-vivo BBB situation. The in-vitro BBB barrier model showed trans......-endothelial electrical resistance above 200 ohm*cm2, indicating that the BCECs formed a tight polar monolayer with functional tight junctions. This was confirmed by immunostaining for the thigh junction protein ZO-1. Rat BCECs were transfected with a red fluorescence protein Hc-RED for 24 hours. Positive transfection...

  20. Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery.

    Science.gov (United States)

    Dai, Chenkai; Lehar, Mohamed; Sun, Daniel Q; Rvt, Lani Swarthout; Carey, John P; MacLachlan, Tim; Brough, Doug; Staecker, Hinrich; Della Santina, Alexandra M; Hullar, Timothy E; Della Santina, Charles C

    2017-08-01

    Sensorineural losses of hearing and vestibular sensation due to hair cell dysfunction are among the most common disabilities. Recent preclinical research demonstrates that treatment of the inner ear with a variety of compounds, including gene therapy agents, may elicit regeneration and/or repair of hair cells in animals exposed to ototoxic medications or other insults to the inner ear. Delivery of gene therapy may also offer a means for treatment of hereditary hearing loss. However, injection of a fluid volume sufficient to deliver an adequate dose of a pharmacologic agent could, in theory, cause inner ear trauma that compromises functional outcome. The primary goal of the present study was to assess that risk in rhesus monkeys, which closely approximates humans with regard to middle and inner ear anatomy. Secondary goals were to identify the best delivery route into the primate ear from among two common surgical approaches (i.e., via an oval window stapedotomy and via the round window) and to determine the relative volumes of rhesus, rodent, and human labyrinths for extrapolation of results to other species. We measured hearing and vestibular functions before and 2, 4, and 8 weeks after unilateral injection of phosphate-buffered saline vehicle (PBSV) into the perilymphatic space of normal rhesus monkeys at volumes sufficient to deliver an atoh1 gene therapy vector. To isolate effects of injection, PBSV without vector was used. Assays included behavioral observation, auditory brainstem responses, distortion product otoacoustic emissions, and scleral coil measurement of vestibulo-ocular reflexes during whole-body rotation in darkness. Three groups (N = 3 each) were studied. Group A received a 10 μL transmastoid/trans-stapes injection via a laser stapedotomy. Group B received a 10 μL transmastoid/trans-round window injection. Group C received a 30 μL transmastoid/trans-round window injection. We also measured inner ear fluid space volume via 3D

  1. Efficient Gene Delivery Mediated by a Helical Polypeptide: Controlling the Membrane Activity via Multivalency and Light-Assisted Photochemical Internalization (PCI).

    Science.gov (United States)

    Xu, Xin; Li, Yongjuan; Liang, Qiujun; Song, Ziyuan; Li, Fangfang; He, Hua; Wang, Jinhui; Zhu, Lipeng; Lin, Zhifeng; Yin, Lichen

    2018-01-10

    The development of robust and nontoxic membrane-penetrating materials is highly demanded for nonviral gene delivery. Herein, a photosensitizer (PS)-embedded, star-shaped helical polypeptide was developed, which combines the advantages of multivalency-enhanced intracellular DNA uptake and light-strengthened endosomal escape to enable highly efficient gene delivery with low toxicity. 5,10,15,20-Tetrakis-(4-aminophenyl) porphyrin as a selected PS initiated ring-opening polymerization of N-carboxyanhydride and yielded a star-shaped helical polypeptide after side-chain functionalization with guanidine groups. The star polypeptide afforded a notably higher transfection efficiency and lower cytotoxicity than those of its linear analogue. Light irradiation caused almost complete (∼90%) endosomal release of the DNA cargo via the photochemical internalization (PCI) mechanism and further led to a 6-8-fold increment of the transfection efficiency in HeLa, B16F10, and RAW 264.7 cells, outperforming commercial reagent 25k PEI by up to 3 orders of magnitude. Because the PS and DNA cargoes were compartmentalized distantly in the core and polypeptide layers, respectively, the generated reactive oxygen species caused minimal damage to DNA molecules to preserve their transfection potency. Such multivalency- and PCI-potentiated gene delivery efficiency was also demonstrated in vivo in melanoma-bearing mice. This study thus provides a promising strategy to overcome the multiple membrane barriers against nonviral gene delivery.

  2. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    Science.gov (United States)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (Pspermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (Pspermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

  3. Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia

    Directory of Open Access Journals (Sweden)

    Alizée Latour

    2015-03-01

    Full Text Available Hyperhomocysteinemia results from hepatic metabolism dysfunction and is characterized by a high plasma homocysteine level, which is also an independent risk factor for cardiovascular disease. Elevated levels of homocysteine in plasma lead to hepatic lesions and abnormal lipid metabolism. Therefore, lowering homocysteine levels might offer therapeutic benefits. Recently, we were able to lower plasma homocysteine levels in mice with moderate hyperhomocysteinemia using an adenoviral construct designed to restrict the expression of DYRK1A, a serine/threonine kinase involved in methionine metabolism (and therefore homocysteine production, to hepatocytes. Here, we aimed to extend our previous findings by analyzing the effect of hepatocyte-specific Dyrk1a gene transfer on intermediate hyperhomocysteinemia and its associated hepatic toxicity and liver dysfunction. Commensurate with decreased plasma homocysteine and alanine aminotransferase levels, targeted hepatic expression of DYRK1A in mice with intermediate hyperhomocysteinemia resulted in elevated plasma paraoxonase-1 and lecithin:cholesterol acyltransferase activities and apolipoprotein A–I levels. It also rescued hepatic apolipoprotein E, J, and D levels. Further, Akt/GSK3/cyclin D1 signaling pathways in the liver of treated mice were altered, which may help prevent homocysteine-induced cell cycle dysfunction. DYRK1A gene therapy could be useful in the treatment of hyperhomocysteinemia in populations, such as end-stage renal disease patients, who are unresponsive to B-complex vitamin therapy.

  4. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    Energy Technology Data Exchange (ETDEWEB)

    Katebi, Samira; Esmaeili, Abolghasem, E-mail: aesmaeili@sci.ui.ac.ir; Ghaedi, Kamran

    2016-03-15

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

  5. 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.

  6. Programmable dc motor controller

    Science.gov (United States)

    Hopwood, J. E.

    1982-11-01

    A portable programmable dc motor controller, with features not available on commercial instruments was developed for controlling fixtures during welding processes. The controller can be used to drive any dc motor having tachometer feedback and motor requirements not exceeding 30 volts, 3 amperes. Among the controller's features are delayed start time, upslope time, speed, and downslope time.

  7. Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies

    Directory of Open Access Journals (Sweden)

    Beatriz Santos-Carballal

    2018-04-01

    Full Text Available Non-viral gene delivery vectors have lagged far behind viral ones in the current pipeline of clinical trials of gene therapy nanomedicines. Even when non-viral nanovectors pose less safety risks than do viruses, their efficacy is much lower. Since the early studies to deliver pDNA, chitosan has been regarded as a highly attractive biopolymer to deliver nucleic acids intracellularly and induce a transgenic response resulting in either upregulation of protein expression (for pDNA, mRNA or its downregulation (for siRNA or microRNA. This is explained as the consequence of a multi-step process involving condensation of nucleic acids, protection against degradation, stabilization in physiological conditions, cellular internalization, release from the endolysosome (“proton sponge” effect, unpacking and enabling the trafficking of pDNA to the nucleus or the siRNA to the RNA interference silencing complex (RISC. Given the multiple steps and complexity involved in the gene transfection process, there is a dearth of understanding of the role of chitosan’s structural features (Mw and degree of acetylation, DA% on each step that dictates the net transfection efficiency and its kinetics. The use of fully characterized chitosan samples along with the utilization of complementary biophysical and biological techniques is key to bridging this gap of knowledge and identifying the optimal chitosans for delivering a specific gene. Other aspects such as cell type and administration route are also at play. At the same time, the role of chitosan structural features on the morphology, size and surface composition of synthetic virus-like particles has barely been addressed. The ongoing revolution brought about by the recent discovery of CRISPR-Cas9 technology will undoubtedly be a game changer in this field in the short term. In the field of rare diseases, gene therapy is perhaps where the greatest potential lies and we anticipate that chitosans will be key players

  8. Multilevel DC link inverter

    Science.gov (United States)

    Su, Gui-Jia

    2003-06-10

    A multilevel DC link inverter and method for improving torque response and current regulation in permanent magnet motors and switched reluctance motors having a low inductance includes a plurality of voltage controlled cells connected in series for applying a resulting dc voltage comprised of one or more incremental dc voltages. The cells are provided with switches for increasing the resulting applied dc voltage as speed and back EMF increase, while limiting the voltage that is applied to the commutation switches to perform PWM or dc voltage stepping functions, so as to limit current ripple in the stator windings below an acceptable level, typically 5%. Several embodiments are disclosed including inverters using IGBT's, inverters using thyristors. All of the inverters are operable in both motoring and regenerating modes.

  9. Cloning of replication-incompetent herpes simplex viruses as bacterial artificial chromosomes to facilitate development of vectors for gene delivery into differentiated neurons.

    Science.gov (United States)

    Schmeisser, Falko; Weir, Jerry P

    2006-01-01

    We have previously described the adaptation of a tetracycline-regulated system of gene expression for herpes simplex virus (HSV) vectors and demonstrated that such a system was capable of inducible foreign gene expression in irreversibly differentiated neurons. These studies suggested that such gene delivery vectors would be especially useful for studying the neuron in vitro. Here, we describe the cloning of a replication-incompetent HSV vector as a bacterial artificial chromosome (BAC) to facilitate vector construction. Using prokaryotic genetic techniques for allele replacement, we demonstrate the ease of manipulation of the BAC-containing vector, including the construction of vector mutations for which there is no simple phenotypic selection. Such constructions include the insertion of a tetracycline-regulated gene cassette into the UL41 gene for regulated gene expression and the mutation of the UL48 gene to reduce vector toxicity. In addition, HSV vectors cloned as BACs can be sequentially modified to make multiple changes to the vector platform. Finally, using the BAC system, we constructed an HSV vector that expressed an inducible human superoxide dismutase-1 (SOD1) gene for delivery into differentiated human NT-neurons (cells of the human embryonal carcinoma cell line NT2, which differentiate irreversibly into postmitotic neuron-like cells after treatment with retinoic acid). The results indicated that there is appreciable expression of SOD1 from this HSV vector in the presence of doxycycline and that vector-expressed SOD1 interacts with endogenous SOD1. Thus, the BAC system provides a practicable platform for construction and manipulation of HSV vectors that are suitable for gene delivery into postmitotic neurons in vitro.

  10. Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics.

    Science.gov (United States)

    Candolfi, Marianela; Xiong, Weidong; Yagiz, Kader; Liu, Chunyan; Muhammad, A K M G; Puntel, Mariana; Foulad, David; Zadmehr, Ali; Ahlzadeh, Gabrielle E; Kroeger, Kurt M; Tesarfreund, Matthew; Lee, Sharon; Debinski, Waldemar; Sareen, Dhruv; Svendsen, Clive N; Rodriguez, Ron; Lowenstein, Pedro R; Castro, Maria G

    2010-11-16

    Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.

  11. Controlled shielding and deshielding of gene delivery polyplexes using hydroxyethyl starch (HES) and alpha-amylase.

    Science.gov (United States)

    Noga, Matthäus; Edinger, Daniel; Rödl, Wolfgang; Wagner, Ernst; Winter, Gerhard; Besheer, Ahmed

    2012-04-10

    The non-viral delivery of nucleic acids faces many extracellular and intracellular hurdles on the way from injection site to the site of action. Among these, aggregation in the blood stream and rapid elimination by the mononuclear phagocytic system (MPS) represent strong obstacles towards successful development of these promising therapeutic modalities. Even the state-of-the-art solutions using PEGylation show low transfection efficiency due to limited uptake and hindered endosomal escape. Engineering the carriers with sheddable coats reduces aggregation and phagocytosis due to the effective shielding, while the controlled deshielding at the desired site of action enhances the uptake and intracellular release. This work reports for the first time the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. HES, with different molar masses, was grafted to polyethylenimine (PEI) and characterized using (1)H NMR, colorimetric copper-assay, and SEC. HES-PEI conjugates were used to generate polyplexes with the luciferase-expressing plasmid DNA pCMVluc, and were characterized by DLS and zeta potential measurements. Deshielding was tested in vitro by zeta potential measurements and, erythrocyte aggregation assay upon addition of α-amylase (AA) to the HES-decorated particles. The addition of AA led to gradual increase in the zeta potential of the nanoparticles over 0.5 to 1h and to a higher aggregation tendency for erythrocytes due to the degradation of the HES-coat and exposure of the polyplexes' positive charge. In vitro transfection experiments were conducted in 2 cell-lines±AA in the culture medium. The amylase-treated HES-decorated complexes showed up to 2 orders of magnitude higher transfection levels compared to the untreated HES-shielded particles, while AA had no effect on the transfection of PEG-coated or uncoated polyplexes. Finally, flow cytometry showed that the addition of AA increased the amount of delivered DNA per cell for

  12. 75 FR 38943 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-10-10, DC-10-10F, DC-10-30, DC...

    Science.gov (United States)

    2010-07-07

    ... Corporation Model DC- 10-10, DC-10-10F, DC-10-30, DC-10-30F (KDC-10), DC-10-40, and DC-10-40F Airplanes AGENCY... propose to adopt a new airworthiness directive (AD) for certain Model DC-10-10, DC-10-10F, DC-10-30, DC-10-30F (KDC-10), DC-10- 40, and DC-10-40F airplanes. This proposed AD would require installing a support...

  13. Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

    Directory of Open Access Journals (Sweden)

    Sun X

    2012-06-01

    Full Text Available Xun Sun, Simu Chen, Jianfeng Han, Zhirong ZhangKey Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of ChinaBackground: To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG and a series of its mannosylated derivatives.Methods: PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs using flow cytometry.Results: PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation.Conclusion: These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.Keywords: dendritic cells, DCs, mannose, polyethyleneimine, PEI, gene delivery

  14. rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.

    Science.gov (United States)

    Rocca, C J; Ur, S N; Harrison, F; Cherqui, S

    2014-06-01

    Effective gene therapy strategies for the treatment of kidney disorders remain elusive. We report an optimized kidney-targeted gene delivery strategy using recombinant adeno-associated virus (rAAV) administered via retrograde renal vein injection in mice. Renal vein injection of rAAV consistently resulted in superior kidney transduction compared with tail vein injection using as little as half the tail vein dose. We compared rAAV5, 6, 8 and 9, containing either green fluorescent protein (GFP) or luciferase reporter genes driven by the Cytomegalovirus promoter. We demonstrated that although rAAV6 and 8 injected via renal vein transduced the kidney, transgene expression was mainly restricted to the medulla. Transgene expression was systematically low after rAAV5 injection, attributed to T-cell immune response, which could be overcome by transient immunosuppression. However, rAAV9 was the only serotype that permitted high-transduction efficiency of both the cortex and medulla. Moreover, both the glomeruli and tubules were targeted, with a higher efficiency within the glomeruli. To improve the specificity of kidney-targeted gene delivery with rAAV9, we used the parathyroid hormone receptor 'kidney-specific' promoter. We obtained a more efficient transgene expression within the kidney, and a significant reduction in other tissues. Our work represents the first comprehensive and clinically relevant study for kidney gene delivery.

  15. A Biomimic Reconstituted High-Density-Lipoprotein-Based Drug and p53 Gene Co-delivery System for Effective Antiangiogenesis Therapy of Bladder Cancer

    Science.gov (United States)

    Ouyang, Qiaohong; Duan, Zhongxiang; Jiao, Guangli; Lei, Jixiao

    2015-07-01

    A biomimic reconstituted high-density-lipoprotein-based drug and p53 gene co-delivery system (rHDL/CD-PEI/p53 complexes) was fabricated as a targeted co-delivery nanovector of drug and gene for potential bladder cancer therapy. Here, CD-PEI was utilized to effectively condense the p53 plasmid, to incorporate the plasmid into rHDL, and to act as an antitumor drug to suppress tumor angiogenesis. The rHDL/CD-PEI/p53 complexes exhibited desirable and homogenous particle size, neutral surface charge, and low cytotoxicity in vitro. The results of confocal laser scanning microscopy and flow cytometry confirmed that SR-BI-targeted function induced specific cytoplasmic delivery and high gene transfection efficiency in MBT-2 murine bladder cells. In addition, rHDL/CD-PEI/p53 complexes co-delivering CD and p53 gene achieved synergistic angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein via different pathways in vitro. In vivo investigation on C3H/He mice bearing MBT-2 tumor xenografts revealed that rHDL/CD-PEI/p53 complexes possessed strong antitumor activity. These findings suggested that rHDL/CD-PEI/p53 complexes could be an ideal tumor-targeting system for simultaneous transfer of drug and gene, which might be a new promising strategy for effective bladder cancer therapy.

  16. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

    Science.gov (United States)

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-02-26

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Development of amino acid substituted gemini surfactant-based mucoadhesive gene delivery systems for potential use as noninvasive vaginal genetic vaccination.

    Science.gov (United States)

    Singh, Jagbir; Michel, Deborah; Getson, Heather M; Chitanda, Jackson M; Verrall, Ronald E; Badea, Ildiko

    2015-02-01

    Recently, we synthesized amino acid- and peptide-substituted gemini surfactants, 'biolipids' that exhibited high transfection efficiency in vitro. In this study, we developed these plasmid DNA and gemini surfactant lipid particles for noninvasive administration in vaginal cavity. Novel formulations of these gene delivery systems were prepared with poloxamer 407 to induce in situ gelling of the formulation and diethylene glycol monoethyl ether to improve their penetration across mucosal tissue. Poloxamer at 16% w/v concentration in diethylene glycol monoethyl ether aqueous solution produced dispersions that gelled near body temperature and had a high yield value, preventing leakage of the formulation from the vaginal cavity. Intravaginal administration in rabbits showed that the glycyl-lysine-substituted gemini surfactant led to a higher gene expression compared with the parent unsubstituted gemini surfactant. This provides a proof-of-concept that amino acid substituted gemini surfactants can be used as noninvasive mucosal (vaginal) gene delivery systems to treat diseases associated with mucosal epithelia.

  18. Starch-Chitosan Polyplexes: A Versatile Carrier System for Anti-Infectives and Gene Delivery

    Directory of Open Access Journals (Sweden)

    Hanzey Yasar

    2018-03-01

    Full Text Available Despite the enormous potential of nanomedicine, the search for materials from renewable resources that balance bio-medical requirements and engineering aspects is still challenging. This study proposes an easy method to make nanoparticles composed of oxidized starch and chitosan, both isolated from natural biopolymers. The careful adjustment of C/N ratio, polymer concentration and molecular weight allowed for tuning of particle characteristics. The system’s carrier capability was assessed both for anti-infectives and for nucleic acid. Higher starch content polyplexes were found to be suitable for high encapsulation efficiency of cationic anti-infectives and preserving their bactericidal function. A cationic carrier was obtained by coating the anionic polyplex with chitosan. Coating allowed for a minimal amount of cationic polymer to be employed and facilitated plasmid DNA loading both within the particle core and on the surface. Transfection studies showed encouraging result, approximately 5% of A549 cells with reporter gene expression. In summary, starch-chitosan complexes are suitable carriers with promising perspectives for pharmaceutical use.

  19. Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti-transferrin receptor antibodies and an immunoglobulin promoter.

    Science.gov (United States)

    Leoh, Lai Sum; Morizono, Kouki; Kershaw, Kathleen M; Chen, Irvin S Y; Penichet, Manuel L; Daniels-Wells, Tracy R

    2014-01-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, as a result of widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of a dual targeted lentiviral-mediated gene therapy strategy to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter. Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored using the MTS or WST-1 viability assay. We found that EGFP was specifically expressed in a panel of human malignant B-cell lines, but not in human malignant T-cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC and the FCU1 gene. The present study demonstrates that gene expression can be restricted to malignant B cells and suggests that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches. Copyright © 2014 John Wiley & Sons, Ltd.

  20. Microneedle assisted micro-particle delivery from gene guns: experiments using skin-mimicking agarose gel.

    Science.gov (United States)

    Zhang, Dongwei; Das, Diganta B; Rielly, Chris D

    2014-02-01

    A set of laboratory experiments has been carried out to determine if micro-needles (MNs) can enhance penetration depths of high-speed micro-particles delivered by a type of gene gun. The micro-particles were fired into a model target material, agarose gel, which was prepared to mimic the viscoelastic properties of porcine skin. The agarose gel was chosen as a model target as it can be prepared as a homogeneous and transparent medium with controllable and reproducible properties allowing accurate determination of penetration depths. Insertions of various MNs into gels have been analysed to show that the length of the holes increases with an increase in the agarose concentration. The penetration depths of micro-particle were analysed in relation to a number of variables, namely the operating pressure, the particle size, the size of a mesh used for particle separation and the MN dimensions. The results suggest that the penetration depths increase with an increase of the mesh pore size, because of the passage of large agglomerates. As these particles seem to damage the target surface, then smaller mesh sizes are recommended; here, a mesh with a pore size of 178 μm was used for the majority of the experiments. The operating pressure provides a positive effect on the penetration depth, that is it increases as pressure is increased. Further, as expected, an application of MNs maximises the micro-particle penetration depth. The maximum penetration depth is found to increase as the lengths of the MNs increase, for example it is found to be 1272 ± 42, 1009 ± 49 and 656 ± 85 μm at 4.5 bar pressure for spherical micro-particles of 18 ± 7 μm diameter when we used MNs of 1500, 1200 and 750 μm length, respectively. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  1. Oil components modulate physical characteristics and function of the natural oil emulsions as drug or gene delivery system.

    Science.gov (United States)

    Chung, H; Kim, T W; Kwon, M; Kwon, I C; Jeong, S Y

    2001-04-28

    Oil-in-water (o/w) type lipid emulsions were formulated by using 18 different natural oils and egg phosphatidylcholine (egg PC) to investigate how emulsion particle size and stability change with different oils. Cottonseed, linseed and evening primrose oils formed emulsions with very large and unstable particles. Squalene, light mineral oil and jojoba bean oil formed stable emulsions with small particles. The remaining natural oils formed moderately stable emulsions. Emulsions with smaller initial particle size were more stable than those with larger particles. The correlation between emulsion size made with different oils and two physical properties of the oils was also investigated. The o/w interfacial tension and particle size of the emulsion were inversely proportional. The effect of viscosity was less pronounced. To study how the oil component in the emulsion modulates the in vitro release characteristics of lipophilic drugs, three different emulsions loaded with two different drugs were prepared. Squalene, soybean oil and linseed oil emulsions represented the most, medium and the least stable systems, respectively. For the lipophilic drugs, release was the slowest from the most stable squalene emulsion, followed by soybean oil and then by linseed oil emulsions. Cationic emulsions were also prepared with the above three different oils as gene carriers. In vitro transfection activity was the highest for the most stable squalene emulsion followed by soybean oil and then by linseed oil emulsions. Even though the in vitro transfection activity of emulsions were lower than the liposome in the absence of serum, the activity of squalene emulsion, for instance, was ca. 30 times higher than that of liposome in the presence of 80% (v/v) serum. In conclusion, the choice of oil component in o/w emulsion is important in formulating emulsion-based drug or gene delivery systems.

  2. Nanosized hydroxyapatite and other calcium phosphates: chemistry of formation and application as drug and gene delivery agents.

    Science.gov (United States)

    Uskoković, Vuk; Uskoković, Dragan P

    2011-01-01

    The first part of this review looks at the fundamental properties of hydroxyapatite (HAP), the basic mineral constituent of mammalian hard tissues, including the physicochemical features that govern its formation by precipitation. A special emphasis is placed on the analysis of qualities of different methods of synthesis and of the phase transformations intrinsic to the formation of HAP following precipitation from aqueous solutions. This serves as an introduction to the second part and the main subject of this review, which relates to the discourse regarding the prospects of fabrication of ultrafine, nanosized particles based on calcium phosphate carriers with various therapeutic and/or diagnostic agents coated on and/or encapsulated within the particles. It is said that the particles could be either surface-functionalized with amphiphiles, peptides, proteins, or nucleic acids or injected with therapeutic agents, magnetic ions, or fluorescent molecules. Depending on the additive, they could be subsequently used for a variety of applications, including the controlled delivery and release of therapeutic agents (extracellularly or intracellularly), magnetic resonance imaging and hyperthermia therapy, cell separation, blood detoxification, peptide or oligonucleotide chromatography and ultrasensitive detection of biomolecules, and in vivo and in vitro gene transfection. Calcium phosphate nanoparticles as carriers of therapeutic agents that would enable a controlled drug release to treat a given bone infection and at the same be resorbed in the body so as to regenerate hard tissue lost to disease are emphasized hereby as one of the potentially attractive smart materials for the modern medicine. © 2010 Wiley Periodicals, Inc.

  3. Biopolymers/poly(ε-caprolactone)/polyethylenimine functionalized nano-hydroxyapatite hybrid cryogel: Synthesis, characterization and application in gene delivery.

    Science.gov (United States)

    Simionescu, Bogdan C; Drobota, Mioara; Timpu, Daniel; Vasiliu, Tudor; Constantinescu, Cristina Ana; Rebleanu, Daniela; Calin, Manuela; David, Geta

    2017-12-01

    Nano-hydroxyapatite (nHAp), surface functionalized with linear polyethylenimine (LPEI), was used for the preparation of biocomposites in combination with biopolymers and poly(ε-caprolactone) (PCL), by cryogelation technique, to yield biomimetic scaffolds with controlled interconnected macroporosity, mechanical stability, and predictable degradation behavior. The structural characteristics, swelling and degradation behavior of hydroxyapatite and hydroxyapatite/β-tricalcium phosphate (β-TCP) filled matrices were investigated as compared to the corresponding naked polymer 3D system. It was found that the homogeneity and cohesivity of the composite are significantly dependent on the size and amount of the included inorganic particles, which are thus determining the structural parameters. Surface modification with LPEI and nanodimensions favored the nHAp integration in the organic matrix, with preferential location along protein fibers, while β-TCP microparticles induced an increased disorder in the hybrid system. The biocomposite including nHAp only was further investigated targeting biomedical uses, and proved to be non-cytotoxic and capable of acting as gene-activated matrix (GAM). It allowed sustained delivery over time (until 22days) of embedded PEI 25 -pDNA polyplexes at high levels of transgene expression, while insuring a decrease in cytotoxicity as compared to polyplexes alone. Experimental data recommend such biocomposite as an attractive material for regenerative medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Single Tyrosine Mutation in AAV8 Vector Capsid Enhances Gene Lung Delivery and Does Not Alter Lung Morphofunction in Mice

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2014-08-01

    Full Text Available Background/Aims: Vectors derived from adeno-associated viruses (AAVs are important gene delivery tools for treating pulmonary diseases. Phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. We evaluated the pulmonary transduction efficiency of AAV8 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Male C57BL/6 mice (20-25 g, n=24 were randomly assigned into three groups: control group animals received intratracheal (i.t. instillation of saline (50 μl, wild-type AAV8 group, and capsid mutant Y733F AAV8 group, which received (i.t. AAV8 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics and morphometry, vector transduction (immunohistochemistry and mRNA expression of eGFP, and inflammatory cytokines and growth factor expression were analyzed. Results: Tyrosine-mutant AAV8 vectors displayed significantly increased transduction efficiency in the lung compared with their wild-type counterparts. No significant differences were observed in lung mechanics and morphometry between experimental groups. There was no evidence of inflammatory response in any group. Conclusion: AAV8 vectors may be useful for new therapeutic strategies for the treatment of pulmonary diseases.

  5. Surface-mediated functional gene delivery: an effective strategy for enhancing competitiveness of endothelial cells over smooth muscle cells.

    Science.gov (United States)

    Chang, Hao; Ren, Ke-feng; Wang, Jin-Lei; Zhang, He; Wang, Bai-liang; Zheng, Shan-mei; Zhou, Yuan-yuan; Ji, Jian

    2013-04-01

    The non-biorecognition of general biomaterials and inherent biospecificity of biological systems pose key challenges to the optimal functions of medical devices. In this study, we constructed the surface-mediated functional gene delivery through layer-by-layer self-assembly of protamine sulfate (PrS) and plasmid DNA encoding hepatocyte growth factor (HGF), aiming at specific enhancing endothelial cells (EC) compeititiveness over smooth muscle cells (SMC). Characterizations of the (PrS/HGF-pDNA) multilayered films present the linear buildup with homogeneous and flat topographical feature. The amount of DNA can be easily controlled. By using these multilayered films, both human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (HUASMC) can be directly transfected when they contact with the multilayered films. On transfection, increasing secretion of HGF has been detected in both HUVEC and HUASMC culture, which leads to selective promotion of HUVEC proliferation. In the co-culture experiment, we also exhibit the promoted and hindered growth of HUVEC and HUASMC, respectively, which could be attributed to the inverse influence of HUVEC on HUASMC. These results collectively demonstrate that our system can be served as a powerful tool for enhancing competitiveness of EC over SMC, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Protamine/DNA/Niosome Ternary Nonviral Vectors for Gene Delivery to the Retina: The Role of Protamine.

    Science.gov (United States)

    Puras, G; Martínez-Navarrete, G; Mashal, M; Zárate, J; Agirre, M; Ojeda, E; Grijalvo, S; Eritja, R; Diaz-Tahoces, A; Avilés-Trigueros, M; Fernández, E; Pedraz, J L

    2015-10-05

    The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1:1:5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released, and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability, and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated with the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.

  7. Design, synthesis, and evaluation of gadolinium cationic lipids as tools for biodistribution studies of gene delivery complexes.

    Science.gov (United States)

    Leclercq, Francoise; Cohen-Ohana, Mirit; Mignet, Nathalie; Sbarbati, Andrea; Herscovici, Jean; Scherman, Daniel; Byk, Gerardo

    2003-01-01

    Gadolinium-chelating cationic lipids have been synthesized to obtain lipoplexes with MRI contrast properties. These compounds were designed to follow the biodistribution of synthetic DNA for gene delivery by nuclear magnetic resonance imaging. The lipid MCO-I-68 was synthesized, and chelate complexes with gadolinium were formed and characterized in terms of physicochemical and DNA binding properties. The transfection activity of MCO-I-68-Gd/DNA complexes was assayed in vitro on NIH 3T3. Different formulations of the product were tested. When up to 5% of the gadolinium lipid complexes were co-formulated with the cationic lipid RPR120535 used as a reference, the transfection levels were maintained as compared to RPR120535 alone. To date, only a liposomal formulation of a gadolinium-cationic lipid chelate without DNA had been observed using magnetic resonance imaging. In vivo intratumoral administration of MCO-I-68-Gd/DNA lipoplexes to tumor model led to an important increase of the NMR signal. It was demonstrated that the new complexes also acted as transfection carriers when they were formulated from liposomes.

  8. Multiplex RT-PCR amplification of HIV genes to create a completely autologous DC-based immunotherapy for the treatment of HIV infection.

    Directory of Open Access Journals (Sweden)

    Irina Tcherepanova

    Full Text Available BACKGROUND: Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. METHODOLOGY/PRINCIPAL FINDINGS: The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. CONCLUSION/SIGNIFICANCE: This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce

  9. Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection

    Science.gov (United States)

    Tcherepanova, Irina; Harris, Jason; Starr, Aijing; Cleveland, Jaclyn; Ketteringham, Helen; Calderhead, David; Horvatinovich, Joe; Healey, Don; Nicolette, Charles A.

    2008-01-01

    Background Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. Methodology/Principal Findings The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. Conclusion/Significance This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral

  10. Forback DC-to-DC converter

    Science.gov (United States)

    Lukemire, Alan T.

    1995-05-01

    A pulse-width modulated DC-to-DC power converter including a first inductor, i.e. a transformer or an equivalent fixed inductor equal to the inductance of the secondary winding of the transformer, coupled across a source of DC input voltage via a transistor switch which is rendered alternately conductive (ON) and nonconductive (OFF) in accordance with a signal from a feedback control circuit is described. A first capacitor capacitively couples one side of the first inductor to a second inductor which is connected to a second capacitor which is coupled to the other side of the first inductor. A circuit load shunts the second capacitor. A semiconductor diode is additionally coupled from a common circuit connection between the first capacitor and the second inductor to the other side of the first inductor. A current sense transformer generating a current feedback signal for the switch control circuit is directly coupled in series with the other side of the first inductor so that the first capacitor, the second inductor and the current sense transformer are connected in series through the first inductor. The inductance values of the first and second inductors, moreover, are made identical. Such a converter topology results in a simultaneous voltsecond balance in the first inductance and ampere-second balance in the current sense transformer.

  11. Generation of a Vero-Based Packaging Cell Line to Produce SV40 Gene Delivery Vectors for Use in Clinical Gene Therapy Studies

    Directory of Open Access Journals (Sweden)

    Miguel G. Toscano

    2017-09-01

    Full Text Available Replication-defective (RD recombinant simian virus 40 (SV40-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles compared to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

  12. A star-shaped porphyrin-arginine functionalized poly(L-lysine) copolymer for photo-enhanced drug and gene co-delivery.

    Science.gov (United States)

    Ma, Dong; Lin, Qian-Ming; Zhang, Li-Ming; Liang, Yuan-Yuan; Xue, Wei

    2014-05-01

    The co-delivery of drug and gene has become the primary strategy in cancer and other disease therapy. To co-deliver hydrophobic drug and functional gene efficiently into tumor cells, a star-shaped copolymer (PP-PLLD-Arg) with a photochemical internalization effect consisting of a porphyrin (PP) core and arginine-functionalized poly(L-lysine) dendron (PLLD-Arg) arms has been designed, and used to co-deliver docetaxel (DOC) and MMP-9 shRNA plasmid for nasopharyngeal cancer therapy. It was found that PP-PLLD-Arg/MMP-9 nanocomplex showed the photo-enhanced gene transfection efficiency in vitro, and could mediate a significant reduce of MMP-9 protein expression in HNE-1 cells. For co-delivery analysis, the obtained PP-PLLD-Arg/DOC/MMP-9 complexes could induce a more significant apoptosis than DOC or MMP-9 used only, and decreased invasive capacity of HNE-1 cells. Moreover, the star-shaped copolymer exhibited better blood compatibility and lower cytotoxicity compared to PEI-25k in the hemolysis and MTT assays, and also showed a good biocompatibility in vivo. Therefore, PP-PLLD-Arg with suited irradiation is a promising non-toxic and photo-inducible effective drug and gene delivery strategy, which should be encouraged in tumor therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. A DC Transformer

    Data.gov (United States)

    National Aeronautics and Space Administration — The goal of the project was to demonstrate a true direct current (DC) transformer, a new electro-mechanical component with potentially high power applications; in...

  14. Synchronous DC Power Supply.

    Science.gov (United States)

    The patent describes a synchronous direct current (dc) power supply which has the power supply input drive synchronized with the pulse repetition...frequency (PRF) of the amplifying or load circuit requiring the dc power for operation. This limits the occurrence of ripple components in the power ... supply output to the spectral positions of the PRF lines, eliminating ripple interference with signal processing in the load. An astable multivibrator is

  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. Three new DC-to-DC Single-Switch Converters

    Directory of Open Access Journals (Sweden)

    Barry W. Williams

    2017-06-01

    Full Text Available This paper presents a new family of three previously unidentified dc-to-dc converters, buck, boost, and buck-boost voltage-transfer-function topologies, which offer advantageous transformer coupling features and low capacitor dc voltage stressing. The three single-switch, single-diode, converters offer the same features as basic dc-to-dc converters, such as the buck function with continuous output current and the boost function with continuous input current. Converter time-domain simulations and experimental results (including transformer coupling support and extol the dc-to-dc converter concepts and analysis presented.

  17. Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

    Directory of Open Access Journals (Sweden)

    Ma G

    2013-03-01

    Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

  18. Novel non-viral vectors for gene delivery: synthesis of a second-generation library of mono-functionalized poly-(guanidinium)amines and their introduction into cationic lipids.

    Science.gov (United States)

    Byk, G; Soto, J; Mattler, C; Frederic, M; Scherman, D

    1998-01-01

    The development of new gene delivery technologies is a prerequisite towards gene therapy clinical trials. Because gene delivery mediated by viral vectors remains of limited scope due to immunological and propagation risks, the development of new non-viral gene delivery systems is of crucial importance. We have synthesized a secondary library of mono-functionalized poly-(guanidinium)amines generated from a library of mono-functionalized polyamines applying the concept of "libraries from libraries." The method allows a quick and easy access to mono-functionalized geometrically varied poly-(guanidinium)amines. The new building blocks were introduced into cationic lipids to obtain novel poly-(guanidinium)amine lipids, which are potential DNA vectors for gene delivery. Copyright 1998 John Wiley & Sons, Inc.

  19. Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

    OpenAIRE

    Peluffo, Hugo; Acarin, Laia; Arís, Anna; González, Pau; Villaverde, Antoni; Castellano, Bernardo; González, Berta

    2006-01-01

    Abstract Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn S...

  20. 75 FR 68246 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-10-10, DC-10-10F, DC-10-15, DC...

    Science.gov (United States)

    2010-11-05

    ... Corporation Model DC- 10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F... to supersede an existing airworthiness directive (AD) that applies to all Model DC-10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F, MD-10- 10F, MD-10-30F, MD-11...

  1. 75 FR 20790 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-10-10, DC-10-10F, DC-10-15, DC...

    Science.gov (United States)

    2010-04-21

    ... Corporation Model DC- 10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F... (AD) for certain Model DC-10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC- 10A and KDC-10), DC-10-40, DC-10-40F, MD-10-10F, MD-10-30F, MD-11, and MD-11F airplanes. This proposed AD would require...

  2. 75 FR 23571 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-10-10, DC-10-10F, DC-10-15, DC...

    Science.gov (United States)

    2010-05-04

    ... Corporation Model DC- 10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F.... ACTION: Final rule. SUMMARY: We are adopting a new airworthiness directive (AD) for certain Model DC-10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F, MD-10-10F, MD...

  3. 75 FR 60602 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-10-10, DC-10-10F, DC-10-15, DC...

    Science.gov (United States)

    2010-10-01

    ... Corporation Model DC- 10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F.... ACTION: Final rule. SUMMARY: We are adopting a new airworthiness directive (AD) for certain Model DC-10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F, MD-10-10F, MD...

  4. Synthesis and Characterization of Cationic Glycidyl-Based Poly(aminoester-Folic Acid Targeting Conjugates and Study on Gene Delivery

    Directory of Open Access Journals (Sweden)

    Yu Che Hsiao

    2012-07-01

    Full Text Available A new poly(aminoester (EPAE-FA containing folic acid and amino groups in the backbone and side chain was synthesized. EPAE-FA self-assembled readily with the plasmid DNA (pCMV-βgal in HEPES buffer and was characterized by dynamic light scattering, zeta potential, fluorescence images, and XTT cell viability assays. To evaluate the transfection effect of graft ratio of FA on the EPAE system, EPAE-FA polymers with two different graft ratios (EPAE-FA12k and EPAE-FA14k were also prepared. This study found that all EPAE-FA polymers were able to bind plasmid DNA and yielded positively charged complexes with nano-sized particles ( < 200 nm. To assess the transfection efficiency mediated by EPAE and EPAE-FA polymers, we performed in vitro transfection activity assays using FR-negative (COS-7 and FR-positive (HeLa cells. The EPAE-FA12k/DNA and EPAE-FA14k/DNA complexes were able to transfect HeLa cell in vitro with higher transfection efficiency than PEI25k/DNA at the similar weight ratio. These results demonstrated that the introduction of FA into EPAE system had a significant effect on transferring ability for FR-positive cells (HeLa. Examination of the cytotoxicity of PEI25k and EPAE-FA system revealed that EPAE-FA system had lower cytotoxicity. In this paper, EPAE-FA seemed to be a novel cationic poly(aminoester for gene delivery and an interesting candidate for further study.

  5. Magnetic resonance-imaging of the effect of targeted antiangiogenic gene delivery in a melanoma tumour model

    Energy Technology Data Exchange (ETDEWEB)

    Hundt, Walter [Stanford School of Medicine, Department of Radiology, Lucas MRS Research Center, Stanford, CA (United States); Philipps University Marburg, Department of Radiology, Marburg (Germany); Steinbach, Silke [Philipps University Marburg, Department of Otolaryngology Head and Neck Surgery, Marburg (Germany); Mayer, Dirk; Guccione, Samira [Stanford School of Medicine, Department of Radiology, Lucas MRS Research Center, Stanford, CA (United States); Burbelko, Mykhaylo; Kiessling, Andreas; Figiel, Jens [Philipps University Marburg, Department of Radiology, Marburg (Germany)

    2015-04-01

    We investigated the effect of targeted gene therapy to melanoma tumours (M21) by MR-imaging. M21 and M21-L tumours were grown to a size of 850 mm{sup 3}. M21 and M21-L tumours were intravenously treated with an αvβ3-integrin-ligand-coupled nanoparticle (RGDNP)/RAF(-) complex five times every 72 hours. MRI was performed at set time intervals 24h and 72h after the i.v. injection of the complex. The MRI protocol was T1-wt-SE±CM, T2-wt-FSE, DCE-MRI, Diffusion-wt-STEAM-sequence, T2-time obtained on a 1.5-T-GE-MRI device. The size of the treated M21 tumours kept nearly constant during the treatment phase (847.8±31.4 mm{sup 3} versus 904.8±44.4 mm{sup 3}). The SNR value (T2-weighted images) of the tumours was 36.7±0.6 and dropped down to 30.6±1.9 (p=0.004). At the beginning the SNR value (T1-weighted images) of the tumours after contrast medium application was 42.3±1.9 and dropped down to 28.5±3.0 (p<0.001). In the treatment group the diffusion coefficient increased significantly under therapy (0.54±0.01x10{sup -3} mm{sup 2}/s versus 0.67±0.04x10{sup -3} mm{sup 2}/s). The DCE-MRI showed a reduction of the slope and of the Akep of 67.8±4.3 % respectively 64.8±3.3 % compared to baseline. Targeted gene delivery therapy induces significant changes in MR-imaging. MRI showed a significant reduction of contrast medium uptake parameters and increase of the diffusion coefficient of the tumours. (orig.)

  6. From bacterial avirulence genes to effector functions via the hrp delivery system: an overview of 25 years of progress in our understanding of plant innate immunity.

    Science.gov (United States)

    Mansfield, John W

    2009-11-01

    Cloning the first avirulence (avr) gene has led not only to a deeper understanding of gene-for-gene interactions in plant disease, but also to fundamental insights into the suppression of basal defences against microbial attack. This article (focusing on Pseudomonas syringae) charts the development of ideas and research progress over the 25 years following the breakthrough achieved by Staskawicz and coworkers. Advances in gene cloning technology underpinned the identification of both avr and hrp genes, the latter being required for the activation of the defensive hypersensitive reaction (HR) and pathogenicity. The delivery of Avr proteins through the type III secretion machinery encoded by hrp gene clusters was demonstrated, and the activity of the proteins inside plant cells as elicitors of the HR was confirmed. Key roles for avr genes in pathogenic fitness have now been established. The rebranding of Avr proteins as effectors, proteins that suppress the HR and cell wall-based defences, has led to the ongoing search for their targets, and is generating new insights into the co-ordination of plant resistance against diverse microbes. Bioinformatics-led analysis of effector gene distribution in genomes has provided a remarkable view of the interchange of effectors and also their functional domains, as the arms race of attack and defence drives the evolution of microbial pathogenicity. The application of our accrued knowledge for the development of disease control strategies is considered.

  7. Design and Testing of Boost Type DC/DC Converter for DC Motor Control Applications

    OpenAIRE

    Samman, Faizal Arya; Akil, Yusri Syam; Noor, Nirwan A.

    2017-01-01

    in The Proceeding of The 2nd International Symposium on Smart Material and Mechatronics 2015 This paper presents the design and testing of a boost type DC/DC converter circuit, which can be used for DC motor control applications. The Boost converter is designed using DC chopper and DC chopper cascade configurations. The experimental setup was made by connecting the boost converter circuit with four types of DC motor, i.e. self-excited DC motor shunt, series, compound and separately exci...

  8. Overview of Multi-DC-Bus Solutions for DC Microgrids

    DEFF Research Database (Denmark)

    Ricchiuto, D.; Mastromauro, R.A.; Liserre, Marco

    2013-01-01

    DC Microgrids have recently received a lot of attention in the last years due to high penetration of renewable energy sources as well as distributed energy storage systems. In the future DC microgrids could be preferable respect to AC microgrids in terms of redundancy since multi-DC-Bus solutions...... could provide a continuative power supply to the loads. An overview of Multi-DC-Bus solutions is presented in this paper. The performances are compared on the basis of possible DC microgrid configurations, redundancy, different DC voltage levels....

  9. Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells.

    Science.gov (United States)

    Venturelli, D; Martinez, R; Melotti, P; Casella, I; Peschle, C; Cucco, C; Spampinato, G; Darzynkiewicz, Z; Calabretta, B

    1995-01-01

    Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:7638209

  10. Proceedings of the International Summit on Human Gene Editing: a global discussion-Washington, D.C., December 1-3, 2015.

    Science.gov (United States)

    LaBarbera, Andrew R

    2016-09-01

    The US Academies of Sciences and Medicine, the Royal Society, and the Chinese Academy of Sciences convened a summit of experts in biology, medicine, law, ethics, sociology, and journalism, in December 2015 to review the state of the art in gene editing technology and discuss the medical and social ramifications of the technologies. The summit concluded with the following consensus recommendations: (1) intensive basic and preclinical research in animal and human models should proceed with appropriate legal and ethical oversight; (2) clinical applications in somatic cells must be rigorously evaluated within existing and evolving regulatory frameworks for gene therapy; (3) it would be irresponsible to proceed with any clinical use of germline editing until relevant safety and efficacy issues have been resolved and there is broad societal consensus about such a use; and (4) the international community should strive to establish generally acceptable uses of human germline editing.

  11. Proceedings of the International Summit on Human Gene Editing: a global discussion—Washington, D.C., December 1–3, 2015

    OpenAIRE

    LaBarbera, Andrew R.

    2016-01-01

    The US Academies of Sciences and Medicine, the Royal Society, and the Chinese Academy of Sciences convened a summit of experts in biology, medicine, law, ethics, sociology, and journalism, in December 2015 to review the state of the art in gene editing technology and discuss the medical and social ramifications of the technologies. The summit concluded with the following consensus recommendations: (1) intensive basic and preclinical research in animal and human models should proceed with appr...

  12. The Effects of Targeted Deliveries of Lovastatin and Tocotrienol on Ossification-Related Gene Expressions in Fracture Healing in an Osteoporosis Rat Model

    Directory of Open Access Journals (Sweden)

    Nurul ‘Izzah Ibrahim

    2015-10-01

    Full Text Available Osteoporotic drugs are used to prevent fragility fractures, but their role in fracture healing still remains unknown. Thus, alternative agents with suitable mode of delivery are needed to promote fracture healing. This study was performed to investigate the effects of direct deliveries of lovastatin and tocotrienol to fracture sites on ossification-related gene expression in fracture healing in a postmenopausal osteoporosis model. Forty-eight Sprague Dawley female rats were divided into six groups. Group I comprised the sham-operated rats, while Groups II–VI were ovariectomized rats. After 8 weeks, the right tibiae of all rats were fractured and stabilized. Group I and Group II were given two single injections of lovastatin and tocotrienol carriers. Group III was given an estrogen preparation at 64.5 µg/kg daily via oral gavages. Group IV was injected with lovastatin particles (750 µg/kg, while Group V was injected with tocotrienol particles (60 mg/kg. Group VI received two single injections of 750 µg/kg lovastatin particles and 60 mg/kg tocotrienol particles. After 4 weeks, the gene expressions were measured. Group VI showed significantly higher gene expressions of osteocalcin, BMP-2, VEGF-α, and RUNX-2 compared to Group II. In conclusion, combined treatment of lovastatin and tocotrienol upregulated the expression of genes related to fracture healing.

  13. Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains

    Directory of Open Access Journals (Sweden)

    Mir A Hossain

    2016-01-01

    Full Text Available Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult β-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex. We generated two synthetic zinc-finger DNA-binding domains (ZF-DBDs targeting this sequence. The -567Gγ ZF-DBDs associated with high affinity and specificity with the target site in the γ-globin gene promoter. We delivered the -567Gγ ZF-DBDs directly to primary erythroid cells. Exposure of these cells to the recombinant -567Gγ ZF-DBDs led to increased expression of the γ-globin gene. Direct protein delivery of ZF-DBDs that compete with transcription regulatory proteins will have broad implications for modulating gene expression in analytical or therapeutic settings.

  14. DC arc weld starter

    Science.gov (United States)

    Campiotti, Richard H.; Hopwood, James E.

    1990-01-01

    A system for starting an arc for welding uses three DC power supplies, a high voltage supply for initiating the arc, an intermediate voltage supply for sustaining the arc, and a low voltage welding supply directly connected across the gap after the high voltage supply is disconnected.

  15. Systemic siRNA Delivery via Peptide-Tagged Polymeric Nanoparticles, Targeting PLK1 Gene in a Mouse Xenograft Model of Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Meenakshi Malhotra

    2013-01-01

    Full Text Available Polymeric nanoparticles were developed from a series of chemical reactions using chitosan, polyethylene glycol, and a cell-targeting peptide (CP15. The nanoparticles were complexed with PLK1-siRNA. The optimal siRNA loading was achieved at an N : P ratio of 129.2 yielding a nanoparticle size of >200 nm. These nanoparticles were delivered intraperitoneally and tested for efficient delivery, cytotoxicity, and biodistribution in a mouse xenograft model of colorectal cancer. Both unmodified and modified chitosan nanoparticles showed enhanced accumulation at the tumor site. However, the modified chitosan nanoparticles showed considerably, less distribution in other organs. The relative gene expression as evaluated showed efficient delivery of PLK1-siRNA (0.5 mg/kg with 50.7±19.5% knockdown (P=0.031 of PLK1 gene. The in vivo data reveals no systemic toxicity in the animals, when tested for systemic inflammation and liver toxicity. These results indicate a potential of using peptide-tagged nanoparticles for systemic delivery of siRNA at the targeted tumor site.

  16. Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo.

    Science.gov (United States)

    Gratton, Jean-Philippe; Yu, Jun; Griffith, Jason W; Babbitt, Roger W; Scotland, Ramona S; Hickey, Reed; Giordano, Frank J; Sessa, William C

    2003-03-01

    Small polybasic peptides derived from the transduction domains of certain proteins, such as the third alpha-helix of the Antennapedia (Antp) homeodomain, can cross the cell membrane through a receptor-independent mechanism. These cell-permeable molecules have been used as 'Trojan horses' to introduce biologically active cargo molecules such as DNA, peptides or proteins into cells. Using these cell-permeable peptides, we have developed an efficient and simple method to increase virally mediated gene delivery and protein expression in vitro and in vivo. Here, we show that cell-permeable peptides increase viral cell entry, improve gene expression at reduced titers of virus and improve efficacy of therapeutically relevant genes in vivo.

  17. dc Arc Fault Effect on Hybrid ac/dc Microgrid

    Science.gov (United States)

    Fatima, Zahra

    The advent of distributed energy resources (DER) and reliability and stability problems of the conventional grid system has given rise to the wide spread deployment of microgrids. Microgrids provide many advantages by incorporating renewable energy sources and increasing the reliability of the grid by isolating from the main grid in case of an outage. AC microgrids have been installed all over the world, but dc microgrids have been gaining interest due to the advantages they provide over ac microgrids. However the entire power network backbone is still ac and dc microgrids require expensive converters to connect to the ac power network. As a result hybrid ac/dc microgrids are gaining more attention as it combines the advantages of both ac and dc microgrids such as direct integration of ac and dc systems with minimum number of conversions which increases the efficiency by reducing energy losses. Although dc electric systems offer many advantages such as no synchronization and no reactive power, successful implementation of dc systems requires appropriate protection strategies. One unique protection challenge brought by the dc systems is dc arc faults. A dc arc fault is generated when there is a gap in the conductor due to insulation degradation and current is used to bridge the gap, resulting in an arc with very high temperature. Such a fault if it goes undetected and is not extinguished can cause damage to the entire system and cause fires. The purpose of the research is to study the effect of the dc arc fault at different locations in the hybrid ac/dc microgrid and provide insight on the reliability of the grid components when it is impacted by arc faults at various locations in the grid. The impact of dc arc fault at different locations on the performance of the PV array, wind generation, and constant power loads (CPL) interfaced with dc/dc converters is studied. MATLAB/Simulink is used to model the hybrid ac/dc microgrid and arc fault.

  18. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

    Directory of Open Access Journals (Sweden)

    Ruben R Bender

    2016-06-01

    Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

  19. Cationic lipid-coated PEI/DNA polyplexes with improved efficiency and reduced cytotoxicity for gene delivery into mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Song HM

    2012-08-01

    Full Text Available Hongmei Song, Gang Wang, Bin He, Li Li, Caixia Li, Yusi Lai, Xianghui Xu, Zhongwei GuNational Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan, People's Republic of ChinaBackground: Effective gene transfection without serum deprivation is a prerequisite for successful stem cell-based gene therapy. Polyethylenimine (PEI is an efficient nonviral gene vector, but its application has been hindered by serum sensitivity and severe cytotoxicity.Methods: To solve this problem, a new family of lipopolyplexes was developed by coating PEI/DNA polyplexes with three serum-resistant cationic lipids, namely, lysinylated, histidylated, and arginylated cholesterol. The physical properties, transfection efficiency, cellular uptake, subcellular distribution, and cytotoxicity of the lipopolyplexes was investigated.Results: The outer coat composed of lysinylated or histidylated cholesterol remarkably improved the transfection efficiency of the polyplex with a low PEI/DNA ratio of 2 in the presence of serum. The resulting lysinylated and histidylated cholesterol lipopolyplexes were even more efficient than the best performing polyplex with a high PEI/DNA ratio of 10. Results from cellular uptake and subcellular distribution studies suggest that their higher transfection efficiency may result from accelerated DNA nuclear localization. The superiority of the lipopolyplexes over the best performing polyplex was also confirmed by delivering the therapeutic gene, hVEGF165. Equally importantly, the lipid coating removed the necessity of introducing excess free PEI chains into the transfection solution for higher efficiency, generating lipopolyplexes with no signs of cytotoxicity.Conclusion: Noncovalent modification of polyplexes with lysinylated and histidylated cholesterol lipids can simultaneously improve efficiency and reduce the toxicity of gene delivery under serum conditions, showing great promise for genetic modification of bone

  20. Early Oscillation Detection for DC/DC Converter Fault Diagnosis

    Science.gov (United States)

    Wang, Bright L.

    2011-01-01

    The electrical power system of a spacecraft plays a very critical role for space mission success. Such a modern power system may contain numerous hybrid DC/DC converters both inside the power system electronics (PSE) units and onboard most of the flight electronics modules. One of the faulty conditions for DC/DC converter that poses serious threats to mission safety is the random occurrence of oscillation related to inherent instability characteristics of the DC/DC converters and design deficiency of the power systems. To ensure the highest reliability of the power system, oscillations in any form shall be promptly detected during part level testing, system integration tests, flight health monitoring, and on-board fault diagnosis. The popular gain/phase margin analysis method is capable of predicting stability levels of DC/DC converters, but it is limited only to verification of designs and to part-level testing on some of the models. This method has to inject noise signals into the control loop circuitry as required, thus, interrupts the DC/DC converter's normal operation and increases risks of degrading and damaging the flight unit. A novel technique to detect oscillations at early stage for flight hybrid DC/DC converters was developed.

  1. 75 FR 6160 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-10-10, DC-10-10F, DC-10-15, DC...

    Science.gov (United States)

    2010-02-08

    ... Douglas Corporation Model DC- 10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC-10A and KDC-10), DC-10-40, DC-10-40F, MD-10-10F, MD-10-30F, MD-11, and MD-11F Airplanes AGENCY: Federal Aviation... airworthiness directive (AD) for certain Model DC-10-10, DC-10-10F, DC-10-15, DC-10-30, DC-10-30F (KC- 10A and...

  2. A new optimized formulation of cationic solid lipid nanoparticles intended for gene delivery: development, characterization and DNA binding efficiency of TCERG1 expression plasmid.

    Science.gov (United States)

    Fàbregas, Anna; Sánchez-Hernández, Noemí; Ticó, Josep Ramon; García-Montoya, Encarna; Pérez-Lozano, Pilar; Suñé-Negre, Josep M; Hernández-Munain, Cristina; Suñé, Carlos; Miñarro, Montserrat

    2014-10-01

    Solid lipid nanoparticles (SLNs) are being considered as a new approach for therapeutics for many known diseases. In addition to drug delivery, their use as non-viral vectors for gene delivery can be achieved by the inclusion of cationic lipids, which provide a positive surface potential that favours binding to the DNA backbone. This work is based on the idea that the optimization of the components is required as the first step in simplifying the qualitative and quantitative composition of SLNs as much as possible without affecting the essential properties that define SLNs as optimal non-viral vectors for gene delivery. We selected the best lipids and surfactants in terms of particle size and zeta potential and characterized the properties of the resulting nanoparticles using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The SLNs had a particle size of approximately 120 nm and a positive surface charge of 42 mV. In addition, we analysed the main physicochemical characteristics of the bulk components of the nanoparticles using X-ray diffraction (XRD), differential scanning calorimetry (DSC) and mass spectrometry (MS). The suitability of the optimized SLNs for DNA binding was evaluated after the lyophilisation process using a carboxyl-terminal region of the TCERG1 gene, a human factor that has been implicated in several diseases. We show that the SLNs presented high efficiency in the binding of DNA, and importantly, they presented no toxicity when assayed in an in vivo system. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase

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    Kelly M Podetz-Pedersen

    2016-01-01

    Full Text Available The Sleeping Beauty (SB transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg, we observed a broad range of pCMV-SB100x doses (0.1–2.5 µg conferring optimal levels of long-term expression (>1011 photons/second/cm2. At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>1010 photons/second/cm2 was achieved at a transposon dose of 5–125 μg. We also found that in the linear range of transposon doses (100 ng, co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver.

  4. TOPOLOGICAL REVIEW AND ANALYSIS OF DC-DC BOOST CONVERTERS

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    V. INDRA GANDHI

    2017-06-01

    Full Text Available DC voltage boost up is essential in numerous applications; especially considering Photovoltaic (PV based renewable power generation system. The conventional DC-DC boost converter is the most admired configuration for this scheme, even if the converter efficiency is restricted at duty cycle near to maximum value. In order to find solution to the problem and improve its conversion capability, many converter configurations have been implemented so far. With this circumstance, this research work proposes to give overview of a few most imperative research works related to DC-DC boost converters. Some configurations are covered and classified basically based on the application. The major benefits and disadvantages related to the available techniques are also briefly conveyed. At last, a proper evaluation is recognized among the important types of DC-DC boost converters in terms of efficiency, number of components, and stability.

  5. A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Chicoine Louis G

    2007-09-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1 A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2 an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3 the use of viral doses to accommodate current limitations imposed by vector production methods; 4 and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation. Methods The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a that recognizes the N-terminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL. The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD. Results Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month, 91.3 ± 3.1 (2 months, and 89.6 ± 1.6% (3 months. rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8 (1 month, 78.9 ± 7.4 (2 months, and 81.2 ± 6.2% (3 months transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 ± 0.3 (1 month, 2.1 ± 0.8 (2 months, and 2.1 ± 0.7% (3 months] by vascular delivery. Micro

  6. Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

    Science.gov (United States)

    Bode, Nadine J; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen; Pearson, Melanie M

    2015-06-01

    The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. The fate of ribosomal RNA genes in spontaneous polyploid dogrose hybrids (Rosa L. sect. Caninae (DC.) Ser.) exhibiting non-symmetrical meiosis.

    Science.gov (United States)

    Herklotz, Veit; Kovařík, Aleš; Lunerová, Jana; Lippitsch, Susan; Groth, Marco; Ritz, Christiane M

    2018-01-31

    Dogroses represent an exceptional system for studying the effects of genome doubling and hybridization: their asymmetrical meiosis enables recombination in bi-parentally inherited chromosomes but prevents it in maternally inherited ones. We employed fluorescent in situ hybridization, genome skimming, amplicon sequencing of genomic and cDNA as well as conventional cloning of nuclear ribosomal DNA in two phylogenetically distinct pentaploid (2n = 5x = 35) species, Rosa canina and R. inodora, and their naturally occurring reciprocal hybrids, R. dumalis (5x) and R. agrestis (5x, 6x). Both progenitor species differed in composition, meiotic behaviour and expression of rDNA loci: R. canina (five 18S and 5-8 5S loci) was dominated by the Canina ribotypes but R. inodora (four 18S loci and 7-8 5S loci) by the Rubiginosa ribotype. The co-localised 5S/18S loci occurred on either bivalent-forming (R. canina) or univalent-forming (R. inodora) chromosomes. Ribosomal DNA loci were additively inherited, however, the Canina ribotypes were dominantly expressed, even in genotypes with relatively low copy number of these genes. Moreover, we observed rDNA homogenization towards the paternally transmitted Canina ribotype in 6x R. agrestis. The here observed variation in arrangement and composition of rDNA types between R. canina and R. inodora suggests involvement of different genomes in bivalent formation. We hypothesize that the asymmetrical meiosis arose at least twice by independent ancient hybridization events. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Li, X.L. [Department of Dermatology, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); He, X.J. [Department of Orthopedics, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Wu, B.J.; Xu, M. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Chang, H.M. [Department of Otolaryngology - Head and Neck Surgery, Affiliated Hospital of Xi' an Medical University, Xi' an (China); Zhang, X.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Xing, Z. [Department of Clinical Dentistry, Faculty of Dentistry, Center for Clinical Dental Research, University of Bergen, Bergen (Norway); Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China)

    2014-03-18

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  9. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    Directory of Open Access Journals (Sweden)

    Z.H. Wang

    2014-04-01

    Full Text Available SRY-related high-mobility-group box 9 (Sox9 gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs. After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  10. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    International Nuclear Information System (INIS)

    Wang, Z.H.; Li, X.L.; He, X.J.; Wu, B.J.; Xu, M.; Chang, H.M.; Zhang, X.H.; Xing, Z.; Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y.

    2014-01-01

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering

  11. An injectable elastin-based gene delivery platform for dose-dependent modulation of angiogenesis and inflammation for critical limb ischemia.

    Science.gov (United States)

    Dash, Biraja C; Thomas, Dilip; Monaghan, Michael; Carroll, Oliver; Chen, Xizhe; Woodhouse, Kimberly; O'Brien, Timothy; Pandit, Abhay

    2015-10-01

    Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 μg) and IL-10 (10 μg)/eNOS (20 μg) and reduced inflammation with IL-10 (10 μg) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. An MRI-visible non-viral vector bearing GD2 single chain antibody for targeted gene delivery to human bone marrow mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Pengfei Pang

    Full Text Available The neural ganglioside GD2 has recently been reported to be a novel surface marker that is only expressed on human bone marrow mesenchymal stem cells within normal marrow. In this study, an MRI-visible, targeted, non-viral vector for effective gene delivery to human bone marrow mesenchymal stem cells was first synthesized by attaching a targeting ligand, the GD2 single chain antibody (scAbGD2, to the distal ends of PEG-g-PEI-SPION. The targeted vector was then used to condense plasmid DNA to form nanoparticles showing stable small size, low cytotoxicity, and good biocompatibility. Based on a reporter gene assay, the transfection efficiency of targeting complex reached the highest value at 59.6% ± 4.5% in human bone marrow mesenchymal stem cells, which was higher than those obtained using nontargeting complex and lipofectamine/pDNA (17.7% ± 2.9% and 34.9% ± 3.6%, respectively (P<0.01. Consequently, compared with the nontargeting group, more in vivo gene expression was observed in the fibrotic rat livers of the targeting group. Furthermore, the targeting capacity of scAbGD2-PEG-g-PEI-SPION was successfully verified in vitro by confocal laser scanning microscopy, Prussian blue staining, and magnetic resonance imaging. Our results indicate that scAbGD2-PEG-g-PEI-SPION is a promising MRI-visible non-viral vector for targeted gene delivery to human bone marrow mesenchymal stem cells.

  13. Ex vivo bone morphogenetic protein 2 gene delivery using periodontal ligament stem cells for enhanced re-osseointegration in the regenerative treatment of peri-implantitis.

    Science.gov (United States)

    Park, Shin-Young; Kim, Kyoung-Hwa; Gwak, Eun-Hye; Rhee, Sang-Hoon; Lee, Jeong-Cheol; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

    2015-01-01

    Peri-implantitis is a chronic inflammatory process with advanced bone loss and impaired healing potential. For peri-implantitis treatment, tissue engineering can be applied to enhance bone regeneration of peri-implant defects. This study aimed to evaluate ex vivo bone morphogenetic protein 2 (BMP2) gene delivery using canine periodontal ligament stem cells (PDLSCs) for regeneration of peri-implantitis defects. Canine PDLSCs were transduced with adenoviral vectors containing BMP2 (BMP2/PDLSCs). After peri-implantitis was induced by ligature placement in six beagle dogs, regenerative procedures were performed; hydroxyapatite (HA) particles and collagen gel with autologous canine PDLSCs (PDLSC group) or BMP2/PDLSCs (BMP/PDLSC group) or without cells (control group) were grafted into the defects and covered by an absorbable membrane. Three months later, the animals were sacrificed. In vitro, BMP2/PDLSCs showed similar levels of stem cell properties to PDLSCs, such as colony-forming efficiency and expression of MSC markers STRO-1 and CD 146. BMP2/PDLSCs produced BMP-2 until day 21 at a concentration of 4-8 ng/mL. In vivo, the BMP2/PDLSC group showed significantly more new bone formation and re-osseointegration in peri-implantitis defects compared to the other groups. In conclusion, ex vivo BMP2 gene delivery using PDLSCs enhanced new bone formation and re-osseointegration in peri-implantitis defects. © 2014 Wiley Periodicals, Inc.

  14. 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.

  15. 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.

  16. Complexation of AB+, AB+C, ACB+, and A(B+-stat-C) block copolymer micelles with poly(styrene sulfonate) as models for tunable gene delivery vectors

    Science.gov (United States)

    Laaser, Jennifer; Jiang, Yaming; Lohmann, Elise; Reineke, Theresa; Lodge, Timothy

    We investigate the complexation of poly(styrene sulfonate) with micelles with mixed cationic/hydrophilic coronas as models for tunable gene delivery vectors. The micelles are self-assembled from AB+, AB+C, ACB+, and A(B+-stat-C) block polymer architectures, where the hydrophobic A blocks (poly(styrene)) form the micelle cores, and the cationic B blocks (poly(dimethylamino ethyl methacrylate)) and hydrophilic, nonionic C blocks (poly(poly(ethylene glycol) methyl ether methacrylate)) form the coronas. We find that hydrophilic units do not change the colloidal stability of the complexes, and complexes based on all four micelle architectures form broad, multimodal size distributions. While complexes based on the AB+, AB+C, and ACB+polymer architectures are kinetically trapped at low ionic strength, however, those based on the A(B+-stat-C) architecture rapidly rearrange into single-micelle complexes when the linear polyanion is in excess. This suggests that the randomly-placed hydrophilic units break up the ion pairing between the cationic and anionic chains and promote formation of over-charged complexes. Design of the micelle architecture may thus provide a powerful way control the structure and stability of micelle-polyelectrolyte complexes for gene delivery applications.

  17. Klotho gene delivery ameliorates renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats by suppressing the Rho-associated coiled-coil kinase signaling pathway.

    Science.gov (United States)

    Deng, Minghong; Luo, Yumei; Li, Yunkui; Yang, Qiuchen; Deng, Xiaoqin; Wu, Ping; Ma, Houxun

    2015-07-01

    The present study aimed to investigate whether klotho gene delivery attenuated renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats. A recombinant adeno-associated virus (rAAV) carrying mouse klotho full-length cDNA (rAAV.mKL), was constructed for in vivo investigation of klotho expression. Diabetes was induced in rats by a single tail vein injection of 60 mg/kg streptozotocin. Subsequently, the diabetic rats received an intravenous injection of rAAV.mKL, rAAV.green fluorescent protein (GFP) or phosphate-buffered saline (PBS). The Sprague-Dawley rat group received PBS and served as the control group. After 12 weeks, all the rats were sacrificed and ELISA, immunohistochemical and histological analyses, fluorescence microscopy, semi-quantitative reverse transcription-polymerase chain reaction and western blottin were performed. A single dose of rAAV.mKL was found to prevent the progression of renal hypertrophy and fibrosis for at least 12 weeks (duration of study). Klotho expression was suppressed in the diabetic rats, but was increased by rAAV.mKL delivery. rAAV.mKL significantly suppressed diabetes-induced renal hypertrophy and histopathological changes, reduced renal collagen fiber generation and decreased kidney hypertrophy index. In addition, rAAV.mKL decreased the protein expression levels of fibronectin and vimentin, while it downregulated the mRNA expression and activity of Rho-associated coiled-coil kinase (ROCK)I in the kidneys of the diabetic rats. These results indicated that klotho gene delivery ameliorated renal hypertrophy and fibrosis in diabetic rats, possibly by suppressing the ROCK signaling pathway. This may offer a novel approach for the long-term control and renoprotection of diabetes.

  18. Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition

    Science.gov (United States)

    Pozzi, D.; Marchini, C.; Cardarelli, F.; Salomone, F.; Coppola, S.; Montani, M.; Zabaleta, M. Elexpuru; Digman, M.A.; Gratton, E.; Colapicchioni, V.; Caracciolo, G.

    2014-01-01

    Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid–protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP–DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP–DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol–DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. PMID:24296066

  19. 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

    Since the first optical microscope invented by Anton van Leeuwenhoek in 1674, the great development of laser technique and its applications in biophotonics have helped us reveal the mechanisms underlying numerous biological activities gradually. The introduction of fs lasers to the studies of biology has emerged as a fast developing area calling for the efforts and skills both from optics and electric engineering and biology and medicine. Due to the fast update of laser source techniques, there has been an increasing number of commercialized fs lasers available for this growing market of biophotonics. To better utilize the potential offered by fs lasers, we studied the technique of optical gene delivery and tried to narrow the gap between laboratorial research and industrial/clinical applications, in that the strict experimental conditions of specific optical laboratorial studies are generally not appropriate for the practical biological applications. To carry out our experiments, we built a two-stage amplifier fs laser system to generate the desired pulse train. The laser pulse train was coupled into an invert fluorescence microscope for the imaging and manipulation of each cell. To overcome limitations brought by the tight focus of laser beam due to high NA objective, we introduced gold nanorods (GNRs), a metallic nanomaterial, with tunable optical property. With these additional membrane for membrane permeabilization, which could significantly improve the manipulation speed than that based on the tightly focused laser. We used GFP plasmid to demonstrate the applications of this technique in gene delivery, and successfully transfected and GFP-expressed cells were observed one day after the optical transfection. Additionally, as an important trend of biophotonics, the integration of optics with microfluidic chips has become the new frontier of both biology and engineering. Here we firstly demonstrated a technique of gene delivery by an on-chip device generating

  20. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells

    OpenAIRE

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-01-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transf...

  1. Auxiliary resonant DC tank converter

    Science.gov (United States)

    Peng, Fang Z.

    2000-01-01

    An auxiliary resonant dc tank (ARDCT) converter is provided for achieving soft-switching in a power converter. An ARDCT circuit is coupled directly across a dc bus to the inverter to generate a resonant dc bus voltage, including upper and lower resonant capacitors connected in series as a resonant leg, first and second dc tank capacitors connected in series as a tank leg, and an auxiliary resonant circuit comprising a series combination of a resonant inductor and a pair of auxiliary switching devices. The ARDCT circuit further includes first clamping means for holding the resonant dc bus voltage to the dc tank voltage of the tank leg, and second clamping means for clamping the resonant dc bus voltage to zero during a resonant period. The ARDCT circuit resonantly brings the dc bus voltage to zero in order to provide a zero-voltage switching opportunity for the inverter, then quickly rebounds the dc bus voltage back to the dc tank voltage after the inverter changes state. The auxiliary switching devices are turned on and off under zero-current conditions. The ARDCT circuit only absorbs ripples of the inverter dc bus current, thus having less current stress. In addition, since the ARDCT circuit is coupled in parallel with the dc power supply and the inverter for merely assisting soft-switching of the inverter without participating in real dc power transmission and power conversion, malfunction and failure of the tank circuit will not affect the functional operation of the inverter; thus a highly reliable converter system is expected.

  2. Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

    Directory of Open Access Journals (Sweden)

    Christian Bucher

    2013-01-01

    Full Text Available Intervertebral disc (IVD cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5 by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

  3. PENGGUNAAN FUZZY LOGIC UNTUK KONTROL PARALLEL CONVERTER DC-DC

    Directory of Open Access Journals (Sweden)

    Bambang Prio Hartono

    2012-09-01

    Full Text Available Abstract: Using system fuzzy logic as control  technology have been used on low load dc-dc converter with combined parallel compiled  dc-dc converter can  obtain big load.   With existence of differrence of component parameter and each parallel compiled converter can obtained different current  and voltage output.  Function of controller  for to do adjustment, so that current which is applied  to  load by each converter  can be obtained  difference error as small as possible or same. The object of research is developing design of large signal dc-dc converter which is  combined with using  FLC so that  obtain  better performance.  To get better performance have been made plant model and simulation with CDE method.  The more systematic  system and design is needed to overcome bigger load  on dc-dc converter, so that parallel  compiled current master slave control system on dc-dc converter with using fuzzy logic  controller is used. Result of  research showed that error or difference of  current  which is applied to load can handled by fuzzy logic  controller.  Technic of current and voltage controller co to do adjustment current and voltage distribution  equally to load.  Distribution of iL1,iL2 and  output voltage Vo on dc-dc  converter with load 2,25 until  7,875 and voltage  100  until 120 volt,  load current beetwen  12 until 48, % relatif  error  Vo  0,4% until  0,9%.

  4. A ternary-complex of a suicide gene, a RAGE-binding peptide, and polyethylenimine as a gene delivery system with anti-tumor and anti-angiogenic dual effects in glioblastoma.

    Science.gov (United States)

    Choi, Eunji; Oh, Jungju; Lee, Dahee; Lee, Jaewon; Tan, Xiaonan; Kim, Minkyung; Kim, Gyeungyun; Piao, Chunxian; Lee, Minhyung

    2018-04-13

    The receptor for advanced glycation end-products (RAGE) is involved in tumor angiogenesis. Inhibition of RAGE might be an effective anti-angiogenic therapy for cancer. In this study, a cationic RAGE-binding peptide (RBP) was produced as an antagonist of RAGE, and a ternary-complex consisting of RBP, polyethylenimine (2 kDa, PEI2k), and a suicide gene (pHSVtk) was developed as a gene delivery system with dual functions: the anti-tumor effect of pHSVtk and anti-angiogenic effect of RBP. As an antagonist of RAGE, RBP decreased the secretion of vascular-endothelial growth factor (VEGF) in activated macrophages and reduced the tube-formation of endothelial cells in vitro. In in vitro transfection assays, the RBP/PEI2k/plasmid DNA (pDNA) ternary-complex had higher transfection efficiency than the PEI2k/pDNA binary-complex. In an intracranial glioblastoma animal model, the RBP/PEI2k/pHSVtk ternary-complex reduced α-smooth muscle actin expression, suggesting that the complex has an anti-angiogenic effect. In addition, the ternary-complex had higher pHSVtk delivery efficiency than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes in an animal model. As a result, the ternary-complex induced apoptosis and reduced tumor volume more effectively than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes. In conclusion, due to its dual anti-tumor and anti-angiogenesis effects, the RBP/PEI2k/pHSVtk ternary-complex might be an efficient gene delivery system for the treatment of glioblastoma. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Electroporation-mediated in vivo gene delivery of the Na+/K+-ATPase pump reduced lung injury in a mouse model of lung contusion.

    Science.gov (United States)

    Machado-Aranda, David A; Suresh, M V; Yu, Bi; Raghavendran, Krishnan

    2012-01-01

    Lung contusion (LC) is an independent risk factor for acute respiratory distress syndrome. The final common pathway in ARDS involves accumulation of fluid in the alveoli. In this study, we demonstrate the application of a potential gene therapy approach by delivering the Na+/K+-ATPase pump subunits in a murine model of LC. We hypothesized that restoring the activity of the pump will result in removal of excess alveolar fluid and additionally reduce inflammation. Under anesthesia, C57/BL6 mice were struck along the right posterior axillary line 1 cm above the costal margin with a cortical contusion impactor. Immediately afterward, 100 μg of plasmid DNA coding for the α,β of the Na+/K+-ATPase pump were instilled into the lungs (LC-electroporation-pump group). Contusion only (LC-only) and a sham saline instillation group after contusion were used as controls (LC-electroporation-sham). By using a BTX 830 electroporator, eight electrical pulses of 200 V/cm field strength were applied transthoracically. Mice were killed at 24 hours, 48 hours, and 72 hours after delivery. Bronchial alveolar lavage was recollected to measure albumin and cytokines by enzyme-linked immunosorbent assay. Pulmonary compliance was measured, and lungs were subject to histopathologic analysis. After the electroporation and delivery of genes coding for the α,β subunits of the Na+/K+-ATPase pump, there was a significant mitigation of acute lung injury as evidenced by reduction in bronchial alveolar lavage levels of albumin, improved pressure volume curves, and reduced inflammation seen on histology. Electroporation-mediated gene transfer of the subunits of the Na+/K+-ATPase pump enhanced recovery from acute inflammatory lung injury after LC.

  6. Delivery presentations

    Science.gov (United States)

    Pregnancy - delivery presentation; Labor - delivery presentation; Occiput posterior; Occiput anterior; Brow presentation ... The mother can walk, rock, and try different delivery positions during labor to help encourage the baby ...

  7. Self-assembled nanoparticles based on the c(RGDfk peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy

    Directory of Open Access Journals (Sweden)

    Liu L

    2014-07-01

    Full Text Available Li Liu,1 Xiaoxia Liu,1 Qian Xu,1 Ping Wu,2 Xialin Zuo,3 Jingjing Zhang,1 Houliang Deng,1 Zhuomin Wu,1 Aimin Ji1 1Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 2Department of Pharmacy, Chengdu Integrated TCM & Western Medicine Hospital, Chengdu, People’s Republic of China; 3Institute of Neurosciences and the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, People’s Republic of China Abstract: The clinical application of small interfering RNA (siRNA has been restricted by their poor intracellular uptake, low serum stability, and inability to target specific cells. During the last several decades, a great deal of effort has been devoted to exploring materials for siRNA delivery. In this study, biodegradable, tumor-targeted, self-assembled peptide nanoparticles consisting of cyclo(Arg–Gly–Asp–d–Phe–Lys-8–amino–3,6–dioxaoctanoic acid–β–maleimidopropionic acid (hereafter referred to as RPM were found to be an effective siRNA carrier both in vitro and in vivo. The nanoparticles were characterized based on transmission electron microscopy, circular dichroism spectra, and dynamic light scattering. In vitro analyses showed that the RPM/VEGFR2-siRNA exhibited negligible cytotoxicity and induced effective gene silencing. Delivery of the RPM/VEGFR2 (zebrafish-siRNA into zebrafish embryos resulted in inhibition of neovascularization. Administration of RPM/VEGFR2 (mouse-siRNA to tumor-bearing nude mice led to a significant inhibition of tumor growth, a marked reduction of vessels, and a downregulation of VEGFR2 (messenger RNA and protein in tumor tissue. Furthermore, the levels of IFN-α, IFN-γ, IL-12, and IL-6 in mouse serum, assayed via enzyme-linked immunosorbent assay, did not indicate any immunogenicity of the RPM/VEGFR2

  8. Dual Delivery of rhPDGF-BB and Bone Marrow Mesenchymal Stromal Cells Expressing the BMP2 Gene Enhance Bone Formation in a Critical-Sized Defect Model

    Science.gov (United States)

    Park, Shin-Young; Kim, Kyoung-Hwa; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo

    2013-01-01

    Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis. PMID:23901900

  9. Complete Blood Count Reference Values of Cord Blood in Taiwan and the Influence of Gender and Delivery Route on Them

    Directory of Open Access Journals (Sweden)

    Yu-Hsun Chang

    2011-06-01

    Conclusion: We successfully obtained the normal CBC and WBC DC reference values of the cord blood in Taiwan. Gender and delivery routes were important confounding factors that influenced the cord blood CBC and WBC DC values.

  10. A gene delivery system containing nuclear localization signal: Increased nucleus import and transfection efficiency with the assistance of RanGAP1.

    Science.gov (United States)

    Chen, Kang; Guo, Lingling; Zhang, Jiulong; Chen, Qing; Wang, Kuanglei; Li, Chenxi; Li, Weinan; Qiao, Mingxi; Zhao, Xiuli; Hu, Haiyang; Chen, Dawei

    2017-01-15

    In the present report, a degradable gene delivery system (PAMS/DNA/10NLS) containing nucleus location signal peptide (NLS) was prepared. The agarose gel electrophoresis, particle size and zeta potential of PAMS/DNA/10NLS were similar to those of PAMS/DNA, which proved that NLS did not affect the interaction between PAMS and DNA. PAMS/DNA/10NLS exhibited marked extracellular and intracellular degradation under acidic conditions. The degradation was believed to allow NLS to come into contact with importins easily, which was able to mediate the nucleus import. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. Moreover, the transfection of PAMS/DNA/10NLS was less dependent on the breakdown of the nucleus envelope than PAMS/DNA. Considering that GTPase-activating protein 1 (RanGAP1) was able to activate the endogenous GTPase, which was necessary for NLS-mediated nucleus import, RanGAP1 overexpressed cells (RanGAP1 cells) were produced. This result showed that RanGAP1 cells had higher GTPase activities than normal cells. Both the nucleus import and transfection efficiency of PAMS/DNA/10NLS were markedly higher in RanGAP1 cells than that in normal cells. The in vivo transfection results also showed that the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. These findings showed that PAMS/DNA/10NLS is a promising gene delivery system with the assistance of RanGAP1. The present report describes the increased transfection efficiency of a degradable gene delivery system (PAMS/DNA/10NLS) containing nuclear location signal (NLS) with the assistance of GTPase-activating protein 1 (RanGAP1). The physicochemical properties of PAMS/DNA/10NLS were similar to those of PAMS/DNA. PAMS/DNA/10NLS exhibited great extracellular and intracellular degradations, which might allow NLS to contact with importins easily. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection

  11. Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: a new concept for improvement of non-viral therapeutic gene delivery.

    Science.gov (United States)

    Tolmachov, Oleg E

    2012-05-01

    The cell-specific and long-term expression of therapeutic transgenes often requires a full array of native gene control elements including distal enhancers, regulatory introns and chromatin organisation sequences. The delivery of such extended gene expression modules to human cells can be accomplished with non-viral high-molecular-weight DNA vectors, in particular with several classes of linear DNA vectors. All high-molecular-weight DNA vectors are susceptible to damage by shear stress, and while for some of the vectors the harmful impact of shear stress can be minimised through the transformation of the vectors to compact topological configurations by supercoiling and/or knotting, linear DNA vectors with terminal loops or covalently attached terminal proteins cannot be self-compacted in this way. In this case, the only available self-compacting option is self-entangling, which can be defined as the folding of single DNA molecules into a configuration with mutual restriction of molecular motion by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the entangling by bringing DNA segments into proximity and by interfering with the DNA slipping away from the entanglement. I propose that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, inter-segment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. It is expected that the non-B-DNA based entanglement of the linear DNA vectors would consist of the initial transient and co-operative non-B-DNA mediated binding events followed by tight self-ensnarement of the vector DNA. Once in the nucleoplasm of the target human cells, the DNA can be disentangled by type II

  12. Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption.

    Science.gov (United States)

    Johansen, Anne Katrine; Molenaar, Bas; Versteeg, Danielle; Leitoguinho, Ana Rita; Demkes, Charlotte; Spanjaard, Bastiaan; de Ruiter, Hesther; Akbari Moqadam, Farhad; Kooijman, Lieneke; Zentilin, Lorena; Giacca, Mauro; van Rooij, Eva

    2017-10-27

    CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized. To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice. We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6 , Sav1 , and Tbx20 , using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1 , which increased the editing efficiency. Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo. © 2017 American Heart Association, Inc.

  13. Power flow analysis for DC voltage droop controlled DC microgrids

    DEFF Research Database (Denmark)

    Li, Chendan; Chaudhary, Sanjay; Dragicevic, Tomislav

    2014-01-01

    This paper proposes a new algorithm for power flow analysis in droop controlled DC microgrids. By considering the droop control in the power flow analysis for the DC microgrid, when compared with traditional methods, more accurate analysis results can be obtained. The algorithm verification...

  14. SCM Handbooks for dc-to-dc Converters

    Science.gov (United States)

    Lee, F.; Mohmoud, M.; Yu, Y.

    1984-01-01

    Two documents aid in design of control modules for dc-to-dc converters. Features of SCM include: Adaptive stability, power component stress limiting, implementation of various control laws, unified design approach. Analysis and quidelines contained in handbooks enable engineer to design SCM circuit and confidently predict resulting overall performance.

  15. Sheppard-Taylor Isolated High Boost DC-DC Converter

    DEFF Research Database (Denmark)

    Chub, Andrii; Siwakoti, Yam Prasad; Vinnikov, Dmitri

    2017-01-01

    This paper presents a new galvanically isolated step-up dc-dc converter intended for low-power but high step-up applications. The proposed converter is capable of regulating output voltage within a wide range of the input voltage or load variations. In contrast to competitors, the converter can b...

  16. Very High Frequency Half Bridge DC/DC Converter

    DEFF Research Database (Denmark)

    Madsen, Mickey Pierre; Knott, Arnold; Andersen, Michael A. E.

    2014-01-01

    This paper presents the first, off chip, class DE (resonant half bridge) converter working in the Very High Frequency (VHF) range. The benefits of using half bridge circuits both in the inverter and rectifier part of a VHF resonant dc/dc converter are analyzed and design equations for all...

  17. Effective gene delivery into adipose-derived stem cells: transfection of cells in suspension with the use of a nuclear localization signal peptide-conjugated polyethylenimine.

    Science.gov (United States)

    Park, Eulsoon; Cho, Hong-Baek; Takimoto, Koichi

    2015-05-01

    Adipose-derived stem cells have the ability to turn into several clinically important cell types. However, it is difficult to transfect these cells with the use of conventional cationic lipid-based reagents. Polyethylenimine (PEI) is considered to be an inexpensive and effective tool for delivery of nucleic acids into mammalian cells. We used a linear PEI conjugated with the nuclear localization signal (NLS) peptide of Simian vacuolating virus 40 large T antigen (PEI-NLS) for transfection of plasmid DNA into adipose-derived cells. We also tested if transfection of cells in suspension might improve the degree and duration of exogenous gene expression. Transfection of cells in suspension with the use of a PEI conjugated with an NLS peptide resulted in high levels of reporter gene expression for an extended period of time in clonal 3T3-L1 preadipocytes and native human adipose-derived stem cells. The reporter gene expression increased for 3 days after the addition of the PEI-NLS peptide-DNA mixture in cell suspension and remained significant for at least 7 days. Cell density did not influence the level of reporter gene expression. Thus, the suspension method with the use of an NLS peptide-conjugated PEI leads to a robust and sustained expression of exogenous genes in adipose-derived cells. The devised transfection method may be useful for reprogramming of adipose-derived stem cells and cell-based therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Delivery of chitosan/dsRNA nanoparticles for silencing of wing development vestigial (vg) gene in Aedes aegypti mosquitoes.

    Science.gov (United States)

    Ramesh Kumar, D; Saravana Kumar, P; Gandhi, M Rajiv; Al-Dhabi, Naif Abdullah; Paulraj, M Gabriel; Ignacimuthu, S

    2016-05-01

    RNA interference (RNAi) has been used as a gene silencing strategy by the introduction of long double stranded RNA (dsRNA) for the control of pest insects. The aim of the present study was to examine whether the expression of vg gene which is responsible for wing development, can be repressed by chitosan/dsRNA based nanoparticles in Aedes aegypti. The vestigial gene (vg) was amplified from adult mosquito and cloned in pLitmus28i vector. Genetically engineered recombinant plasmid was transformed into RNase III deficient strain for synthesis of bacterially expressed dsRNA. Nanoparticles were prepared via electrostatic interaction between cationic polymer chitosan and anionic nucleic acids (dsRNA). The formation of chitosan/dsRNAnanoparticles and their size were confirmed by Atomic force microscopy (AFM). Chitosan/dsRNA mediated knockdown of Enhanced Green Fluorescence Protein (EGFP) was demonstrated in Sf21 cells. Further, we tested whether such an approach could be used to target vg gene in Ae. aegypti. The results showed that chitosan/dsRNA caused significant mortality, delayed growth development and caused adult wing-malformation. A qRT-PCR analysis confirmed that the chitosan/dsRNA mediated transcriptional level was downregulated. Our findings suggest that vg gene intervention strategies through RNAi can emerge as viable option for pest control. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Control of improved full-bridge three-level DC/DC converter for wind turbines in a DC grid

    DEFF Research Database (Denmark)

    Deng, Fujin; Chen, Zhe

    2013-01-01

    This paper presents an improved full-bridge three-level (IFBTL) dc/dc converter for a wind turbine in a dc grid by inserting a passive filter into the dc/dc converter to improve the performance of the converter. The passive filter can effectively reduce the voltage stress of the medium frequency...

  20. A DC Transformer

    Science.gov (United States)

    Youngquist, Robert C.; Ihlefeld, Curtis M.; Starr, Stanley O.

    2013-01-01

    A component level dc transformer is described in which no alternating currents or voltages are present. It operates by combining features of a homopolar motor and a homopolar generator, both de devices, such that the output voltage of a de power supply can be stepped up (or down) with a corresponding step down (or up) in current. The basic theory for this device is developed, performance predictions are made, and the results from a small prototype are presented. Based on demonstrated technology in the literature, this de transformer should be scalable to low megawatt levels, but it is more suited to high current than high voltage applications. Significant development would be required before it could achieve the kilovolt levels needed for de power transmission.

  1. An improved dc SQUID

    International Nuclear Information System (INIS)

    Clarke, J.; Goubau, W.M.; Ketchen, M.B.

    1975-01-01

    Cylindrical thin film dc SQUIDS that make use of resistively shunted Nb-NbOx-Pb tunnel junctions have been constructed and tested. These junctions are very robust: the SQUIDs can be thermally cycled repeatedly and stored at room temperature indefinitely. The weak temperature dependence of the critical current allows SQUID operation at any temperature below about 6K. The resolution is typically 4x10 -5 phi 0 /√Hz above 2x10 -2 Hz; at lower frequencies the noise power spectrum is 1/f. The long-term drift is less than 2x10 -5 phi 0 /hr. The 1/f noise and drift are lower than any values reported for other types of SQUIDs. (Auth.)

  2. Chitosan nanoparticle-based delivery of fused NKG2D–IL-21 gene suppresses colon cancer growth in mice

    Directory of Open Access Journals (Sweden)

    Tan L

    2017-04-01

    Full Text Available Lunmei Tan,1 Sen Han,2 Shizhen Ding,2 Weiming Xiao,3,4 Yanbing Ding,3 Li Qian,2,4 Chenming Wang,1,5 Weijuan Gong1–5 1Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, 2Department of Immunology, School of Medicine, 3Department of Gastroenterology, The Second Clinical Medical College, 4Department of Integrated Chinese and Western Medicine, School of Medicine, 5Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, People’s Republic of China Abstract: Nanoparticles can be loaded with exogenous DNA for the potential expression of cytokines with immune-stimulatory function. NKG2D identifies major histocompatibility complex class I chain-related protein in human and retinoic acid early induced transcript-1 in mouse, which acts as tumor-associated antigens. Biologic agents based on interleukin 21 (IL-21 have displayed antitumor activities through lymphocyte activation. The NKG2D–IL-21 fusion protein theoretically identifies tumor cells through NKG2D moiety and activates T cells through IL-21 moiety. In this study, double-gene fragments that encode the extracellular domains of NKG2D and IL-21 genes were connected and then inserted into the pcDNA3.1(– plasmid. PcDNA3.1–dsNKG2D–IL-21 plasmid nanoparticles based on chitosan were generated. Tumor cells pretransfected with dsNKG2D–IL-21 gene nanoparticles can activate natural killer (NK and CD8+ T cells in vitro. Serum IL-21 levels were enhanced in mice intramuscularly injected with the gene nanoparticles. DsNKG2D–IL-21 gene nanoparticles accumulated in tumor tissues after being intravenously injected for ~4–24 h. Treatment of dsNKG2D–IL-21 gene nanoparticles also retarded tumor growth and elongated the life span of tumor-bearing mice by activating NK and T cells in vivo. Thus, the dsNKG2D–IL-21 gene nanoparticles exerted efficient antitumor activities and would be potentially used for tumor therapy. Keywords: NKG2

  3. Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Goya Rodolfo G

    2011-03-01

    Full Text Available Abstract Background Insulin-like growth factor-I (IGF-I exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex. Methods An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to Aequorea victoria enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1β and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65 was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test. Results IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65 to the cell nucleus. Conclusion These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory

  4. Three-port DC-DC converter with new integrated transformer for DC Distribution Systems

    DEFF Research Database (Denmark)

    Ouyang, Ziwei; Andersen, Michael A. E.

    2014-01-01

    A new integrated transformer for three-port dc-dc converter is proposed to overcome the power coupling effect existed in some known multiple inputs dc-dc converters. Orthogonal primary windings arrangement and in series connection of diagonal secondary Windings enables a fully power decoupling...... between the multiple inputs while the output power is still coupled with all inputs. The energy is accordingly allowed to deliver into the output load simultaneously or at any time-multiplexing scheme. 1-kW experimental prototypes have been built to demonstrate a well-managed power flow for photovoltaic...... (PV) and battery standalone system....

  5. The performance of the DC motor by the PID controlling PWM DC-DC boost converter

    OpenAIRE

    Can, Erol; Sayan, Hasan Hüseyin

    2017-01-01

    This paper presents the PID controlling direct current (DC) to the direct current boost converter feds DC motor which has a 3.68 kW and 240 V of DC voltage input on its characteristics. What is first formed is the boost converter mathematical model at the design stage. Secondly, a mathematical model of the DC motor is created so that the boost converter with the machine can be established and modeled at the Matlab Simulink. The PID controller is considered for arranging a pulse width modulati...

  6. Design, synthesis, and optimization of nanostructured calcium phosphates (NanoCaPs) and natural polymer based 3-D non-viral gene delivery systems

    Science.gov (United States)

    Ko, Hsu-Feng

    Sustained delivery of therapeutic genes from a three-dimensional (3-D) scaffold and subsequent gene expression capable of triggering the regeneration of damaged tissues is a tissue engineering strategy that has been gaining increased attention. Nanostructured calcium phosphates (NanoCaPs) are biocompatible and non-toxic biomaterials. Furthermore, their efficient transfection in vitro have rendered them attractive gene delivery carriers compared to other viral- or lipid-based agents that tend to be immunogenic or cytotoxic, leading to undesirable responses when utilized above a critical threshold. However, NanoCaPs are typically characterized by variable transfection and short shelf life due to particle aggregation. A viable solution to this problem is the incorporation of NanoCaPs into 3-D scaffolds. The main objectives of this research are therefore two-fold: (1) Examination of the potential of achieving optimized transfection of NanoCaPs via anionic substitution and (2) high throughput synthesis and screening of non-viral gene delivery systems (GDS) comprised of naturally-derived polymers as scaffolds containing NanoCaPs gene carriers. Results indicated that in addition to the excellent transfection levels exhibited by NanoCaPs in vitro, an additional 20-30% increase was observed for NanoCaPs with 10-25 mol% anion substitution. In contrast, high anion substitution (>60%) yielded a drastic decline in transfection. Structural characterizations verified successful anion substitution with a noticeable increase in lattice parameters indicative of an expanded unit cell due to ionic substitution. All of the anion substituted calcium phosphates exhibited the primary phase of hydroxyapatite. For the first time, GDS composed of various concentrations of alginate (AA), fibronectin (FN), and NanoCaPs-DNA complexes were demonstrated. The presence of AA and FN was effective in immobilizing NanoCaPs and reducing the aggregation. High throughput synthesis and screening

  7. Captopril-polyethyleneimine conjugate modified gold nanoparticles for co-delivery of drug and gene in anti-angiogenesis breast cancer therapy.

    Science.gov (United States)

    Li, Manhong; Li, Yong; Huang, Xiaohui; Lu, Xizhi

    2015-01-01

    Captopril-polyethyleneimine (CP) containing low molecular weight polyethyleneimine and anti-angiogenesis drug captopril conjugated via an amide bond was fabricated to modify gold nanoparticles and complex with siRNA to construct siRNA/CP/GNP complexes for the co-delivery of drug and siRNA in anti-angiogenesis breast cancer therapy. The self-assembled siRNA/CP/GNP complexes exhibited desirable and homogenous particle size, reasonable positive charges and condensation ability, and effective gene-silencing property in vitro. In addition, siRNA/CP/GNP complexes co-delivering captopril and siRNA achieved combined angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor mRNA and protein via different pathways in vitro, as compared to mono-delivery systems. In vivo investigation on nude mice bearing MDA-MB435 tumor xenografts revealed that siRNA/CP/GNP complexes possessed satisfying tumor homing ability and strong antitumor activity. These findings suggested that siRNA/CP/GNP complexes could be an ideal system for simultaneous transfer of drug and siRNA, which might be a new promising strategy for effective breast cancer therapy.

  8. Construction of an attenuated Salmonella delivery system harboring genes encoding various virulence factors of avian pathogenic Escherichia coli and its potential as a candidate vaccine for chicken colibacillosis.

    Science.gov (United States)

    Chaudhari, Atul A; Matsuda, Kiku; Lee, John Hwa

    2013-03-01

    An attenuated Salmonella (deltalon, deltacpxR, and deltaasdA16) delivery system containing the genes encoding P-fimbriae (papa and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of avian pathogenic Escherichia coli (APEC) was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. The birds were divided into three groups designated group A (nonvaccinated control), group B (given a single immunization), and group C (administered prime and boost immunizations). Prime and booster vaccinations with the constructions were administered to 1-day-old and 14-day-old birds, respectively. Immune responses were measured postimmunization, and the birds were challenged via an intra-air sac route with a virulent APEC strain at the second, third, and fourth weeks of age. Group B birds were partially protected against the challenge and showed increased levels of plasma immunoglobulin (Ig)G, mucosal IgA antibodies, and lymphocyte proliferation. Group C birds showed greater protection against the challenge, with significantly stronger immune responses compared with the birds in the other groups. Overall, our data suggest that the Salmonella delivery system with recombinant constructs is capable of inducing robust immune responses and induces effective protection against colibacillosis caused by APEC.

  9. Self-assembled biodegradable amphiphilic PEG-PCL-lPEI triblock copolymers at the borderline between micelles and nanoparticles designed for drug and gene delivery.

    Science.gov (United States)

    Endres, Thomas K; Beck-Broichsitter, Moritz; Samsonova, Olga; Renette, Thomas; Kissel, Thomas H

    2011-10-01

    Amphiphilic PEG-PCL-PEI triblock copolymers self-assemble into nano-scaled, positively charged, multifunctional carriers, suitable for drug and gene delivery. A set of block copolymers with varying hydrophilic/hydrophobic ratio (systematically altered at the borderline of micelle and particle forming polymers) was synthesized, characterized and assembled into carriers. A detailed structural characterization in the liquid state of these assemblies was carried out: carrier size was determined using dynamic light scattering, cryogenic scanning electron microscopy and atomic force microscopy. Nuclear magnetic resonance analyses elucidated carrier's core-shell structure. ζ-potential and thickness of the hydrophilic outer polymer shell were determined by laser Doppler anemometry. Subsequently the impact of carrier's structure on its features (stability and toxicity) was investigated. Polymers hydrophilic in nature formed small (polymers aggregated to larger particle-like assemblies (>100 nm). Monitoring carrier size as a function of initial polymer concentration clarified different assembly mechanisms. Shell thickness, colloidal stability and toxicity were found to depend on the length of the hydrophilic polymer block. Due to controllable size, charge, stability and toxicity, this class of novel carriers is a promising candidate for prospective co-delivery of drugs and nucleic acids. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. 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.

  11. Evaluation of the biological differences of canine and human factor VIII in gene delivery: Implications in human hemophilia treatment

    Science.gov (United States)

    The canine is the most important large animal model for testing novel hemophilia A(HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, the different biological properties between cFVIII and human FVII...

  12. A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons

    NARCIS (Netherlands)

    Fagoe, N D; Eggers, R; Verhaagen, J; Mason, M R J

    Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the

  13. DC Distribution Systems and Microgrids

    DEFF Research Database (Denmark)

    Dragicevic, Tomislav; Anvari-Moghaddam, Amjad; Quintero, Juan Carlos Vasquez

    2017-01-01

    A qualitative overview of different hardware topologies and control systems for DC MGs has been presented in this chapter. Some challenges and design considerations of DC protections systems have also been discussed. Finally, applications of DC MGs in emerging smart grid applications have been su...... in different industries and gradually lead to new ways of rethinking of the future power distribution philosophies, especially with the emergence of SSTs. Research in DC systems, especially in the power electronics-based technologies will be highly attractive in the future.......A qualitative overview of different hardware topologies and control systems for DC MGs has been presented in this chapter. Some challenges and design considerations of DC protections systems have also been discussed. Finally, applications of DC MGs in emerging smart grid applications have been...... summarized. Due to its attractive characteristics in terms of compliance with modern generation, storage and electronic load technologies, high reliability and current carrying capacity, as well as simple control, DC systems are already an indispensable part of power systems. Moreover, the existing...

  14. Synthesis of mesoporous SiO{sub 2}-ZnO nanocapsules: encapsulation of small biomolecules for drugs and 'SiOZO-plex' for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Vijay Bhooshan [School of Engineering Sciences and Technology, University of Hyderabad (India); Annamanedi, Madhavi [School of Life Sciences, University of Hyderabad, Department of Animal Sciences (India); Prashad, Muvva Durga [University of Hyderabad, Centre for Nanoscience and Nanotechnology (India); Arunasree, Kalle M. [School of Life Sciences, University of Hyderabad, Department of Animal Sciences (India); Mastai, Yitzhak; Gedanken, Aharon, E-mail: gedanken@mail.biu.ac.il [Bar-Ilan University, Department of Chemistry, Institute for Nanotechnology and Advanced Materials (Israel); Paik, Pradip, E-mail: ppse@uohyd.ernet.in [School of Engineering Sciences and Technology, University of Hyderabad (India)

    2013-09-15

    This work presents a new synthesis of mesoporous SiO{sub 2}-ZnO composite nanocapsules with sizes of 90-150 nm and represents their applications in encapsulation of small biomolecules (fluorescent molecules, drugs, and DNA) for uses in medical biotechnology (e.g., drug and gene delivery) for the first time. The nanocapsule size and morphology have been confirmed through the HRSEM and HRTEM. The mesoporous structure of the novel materials has been confirmed through both BET and HRTEM, and the pore diameter observed to be ca. 2-8 nm with an average diameter of 5.1 nm. The BET surface area of mesoporous SiO{sub 2}-ZnO was found to be {approx}230 m{sup 2} g{sup -1}. Three different types of pores were detected through HRTEM: type-I, normal pores in silica matrix, pore with ZnO nanoparticles at the boundary (type-II) and type-III, the pores with tiny ZnO nanoparticles ({approx}5-7 nm) inside them. To demonstrate the biocompatibility and cell viability of the nanocapsules, normal and cancerous lymphocyte cells have been chosen and investigated in a systematic way. Fluorescent dye (Rhodamine 6G), anticancer drug e.g., Doxorubicin (DOX) were loaded in all types of pores, and EtBr-labeled DNA molecules were loaded efficiently into the mesopores of second and third types of the composite nanocapsules to manifest the characteristic of mesoporous, and to find out its loading efficacy. The release kinetics of Rhodamine 6G and DOX were studied. The results highlight the potential of novel functional mesoporous SiO{sub 2}-ZnO nanoparticles for using as the carrier of drugs and formation of 'SiOZO-plex', a complex of mesoporous SiO{sub 2}-ZnO with DNA for gene delivery applications.Graphical Abstract.

  15. Synthesis of mesoporous SiO2–ZnO nanocapsules: encapsulation of small biomolecules for drugs and “SiOZO-plex” for gene delivery

    International Nuclear Information System (INIS)

    Kumar, Vijay Bhooshan; Annamanedi, Madhavi; Prashad, Muvva Durga; Arunasree, Kalle M.; Mastai, Yitzhak; Gedanken, Aharon; Paik, Pradip

    2013-01-01

    This work presents a new synthesis of mesoporous SiO 2 –ZnO composite nanocapsules with sizes of 90–150 nm and represents their applications in encapsulation of small biomolecules (fluorescent molecules, drugs, and DNA) for uses in medical biotechnology (e.g., drug and gene delivery) for the first time. The nanocapsule size and morphology have been confirmed through the HRSEM and HRTEM. The mesoporous structure of the novel materials has been confirmed through both BET and HRTEM, and the pore diameter observed to be ca. 2–8 nm with an average diameter of 5.1 nm. The BET surface area of mesoporous SiO 2 –ZnO was found to be ∼230 m 2 g −1 . Three different types of pores were detected through HRTEM: type-I, normal pores in silica matrix, pore with ZnO nanoparticles at the boundary (type-II) and type-III, the pores with tiny ZnO nanoparticles (∼5–7 nm) inside them. To demonstrate the biocompatibility and cell viability of the nanocapsules, normal and cancerous lymphocyte cells have been chosen and investigated in a systematic way. Fluorescent dye (Rhodamine 6G), anticancer drug e.g., Doxorubicin (DOX) were loaded in all types of pores, and EtBr-labeled DNA molecules were loaded efficiently into the mesopores of second and third types of the composite nanocapsules to manifest the characteristic of mesoporous, and to find out its loading efficacy. The release kinetics of Rhodamine 6G and DOX were studied. The results highlight the potential of novel functional mesoporous SiO 2 –ZnO nanoparticles for using as the carrier of drugs and formation of “SiOZO-plex”, a complex of mesoporous SiO 2 –ZnO with DNA for gene delivery applications.Graphical Abstract

  16. Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery

    Directory of Open Access Journals (Sweden)

    Sarah E. Sinnett

    2017-06-01

    Full Text Available Intravenous administration of adeno-associated virus serotype 9 (AAV9/hMECP2 has been shown to extend the lifespan of Mecp2−/y mice, but this delivery route induces liver toxicity in wild-type (WT mice. To reduce peripheral transgene expression, we explored the safety and efficacy of AAV9/hMECP2 injected into the cisterna magna (ICM. AAV9/hMECP2 (1 × 1012 viral genomes [vg]; ICM extended Mecp2−/y survival but aggravated hindlimb clasping and abnormal gait phenotypes. In WT mice, 1 × 1012 vg of AAV9/hMECP2 induced clasping and abnormal gait. A lower dose mitigated these adverse phenotypes but failed to extend survival of Mecp2−/y mice. Thus, ICM delivery of this vector is impractical as a treatment for Rett syndrome (RTT. To improve the safety of MeCP2 gene therapy, the gene expression cassette was modified to include more endogenous regulatory elements believed to modulate MeCP2 expression in vivo. In Mecp2−/y mice, ICM injection of the modified vector extended lifespan and was well tolerated by the liver but did not rescue RTT behavioral phenotypes. In WT mice, these same doses of the modified vector had no adverse effects on survival or neurological phenotypes. In summary, we identified limitations of the original vector and demonstrated that an improved vector design extends Mecp2−/y survival, without apparent toxicity.

  17. 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

    International Nuclear Information System (INIS)

    Ghiamkazemi, S.; Amanzadeh, A.; Dinarvand, R.; Rafiee-Tehrani, M.; Amini, M.; Ghiamkazemi, S.; Dinarvand, R.; Rafiee-Tehrani, M.; Ghiamkazemi, S.; Rafiee-Tehrani, M.; Amanzadeh, A.; Rafiee-Tehrani, M.

    2010-01-01

    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 nanoparticles could be considered as a useful non-viral vector for targeted gene delivery.

  18. Modular Power System Configured with Standard Product Hybrid DC-DC Converter