Sample records for gdf5 gene delivery

  1. Cloning of Integral Mature Peptide Gene of Human GDF-5

    Institute of Scientific and Technical Information of China (English)

    王万山; 顾为望; 王启伟; 朴仲贤; 朴英杰


    Summary: The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.

  2. Cushing proximal symphalangism and the NOG and GDF5 genes

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    Plett, Sara K. [Columbia University, College of Physicians and Surgeons, New York, NY (United States); Berdon, Walter E.; Oklu, Rahmi [Columbia Presbyterian Medical Center, Department of Radiology, New York, NY (United States); Cowles, Robert A. [Morgan Stanley Children' s Hospital of New York-Presbyterian, Division of Pediatric Surgery, Department of Surgery, New York, NY (United States); Campbell, John B. [Arnold Palmer Hospital for Children, Department of Radiology, Orlando, FL (United States)


    Proximal symphalangism (SYM1) is an autosomal-dominant developmental disorder of joint fusion. This disorder is best known from famous historical descriptions of two large kindred: Cushing's description in 1916 of the ''straight-fingered'' Brown family of Virginia and Drinkwater's description in 1917 of the British Talbot family of noble blood, descended from the English war hero John Talbot, the first Earl of Shrewsbury (1388-1453). Recent genetic studies link this phenotype to expression of abnormal genes at future joint sites: too little expression of NOG, a growth antagonist, or overexpression of GDF5, a growth agonist, results in cartilage overgrowth and bony fusion. This review unites in depth the first historical accounts of SYM1 with a clinical description and reviews the current understanding of the molecular mechanism underlying what is likely the oldest dominant trait ever studied. (orig.)

  3. Human chondrocytes respond discordantly to the protein encoded by the osteoarthritis susceptibility gene GDF5.

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    Madhushika Ratnayake

    Full Text Available A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis

  4. Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells. (United States)

    Wang, Haili; Kroeber, Markus; Hanke, Michael; Ries, Rainer; Schmid, Carsten; Poller, Wolfgang; Richter, Wiltrud


    To develop new therapeutic options for the treatment of disc degeneration we tested the possibility of overexpression of active growth and differentiation factor (GDF) 5 and of transforming growth factor (TGF) beta(1) by adenoviral gene transfer and characterized its effect on cell proliferation and matrix synthesis of cultured rabbit and human intervertebral disc cells. Recombinant adenovirus encoding for GDF-5 or TGF-beta(1) was developed and transgene expression characterized by RT-PCR, western blot and ELISA. Growth and matrix synthesis of transduced cells was measured by [(3)H]thymidine or [(35)S]sulfate incorporation. Disc cells expressed the receptors BMPR1A, BMPR1B, and BMPR2, which are relevant for GDF-5 action. Adenovirus efficiently transferred the GDF-5 gene or the TGF-beta(1) gene to rabbit and human intervertebral disc cells. About 50 ng GDF-5 protein/10(6 )cells per 24 h or 7 ng TGF-beta(1) protein/10(6 )cells per 24 h was produced. According to western blotting, two GDF-5 forms, with molecular weights consistent with the activated GDF-5 dimer and the proform, were secreted over the 3 weeks following gene transfer. Overexpressed GDF-5 and TGF-beta(1) were bioactive and promoted growth of rabbit disc cells in monolayer culture. Our results suggest that ex vivo gene delivery of GDF-5 and TGF-beta(1) is an attractive approach for the release of mature and pre-GDF-5 in surrounding tissue. This leads us to hope that it will prove possible to improve the treatment of degenerative disc disease by means of ex vivo gene transfer of single or multiple growth factors.

  5. Positive selection on the osteoarthritis-risk and decreased-height associated variants at the GDF5 gene in East Asians.

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    Dong-Dong Wu

    Full Text Available GDF5 is a member of the bone morphogenetic protein (BMP gene family, and plays an important role in the development of the skeletal system. Variants of the gene are associated with osteoarthritis and height in some human populations. Here, we resequenced the gene in individuals from four geographically separated human populations, and found that the evolution of the promoter region deviated from neutral expectations, with the sequence evolution driven by positive selection in the East Asian population, especially the haplotypes carrying the derived alleles of 5' UTR SNPs rs143384 and rs143383. The derived alleles of rs143384 and rs143383, which are associated with a risk of osteoarthritis and decreased height, have high frequencies in non-Africans and show strong extended haplotype homozygosity and high population differentiation in East Asian. It is concluded that positive selection has driven the rapid evolution of the two osteoarthritis osteoarthritis-risk and decreased height associated variants of the human GDF5 gene, and supports the suggestion that the reduction in body size during the terminal Pleistocene and Holocene period might have been an adaptive process influenced by genetic factors.

  6. A GDF5 point mutation strikes twice--causing BDA1 and SYNS2.

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    Elisa Degenkolbe

    Full Text Available Growth and Differentiation Factor 5 (GDF5 is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2. Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1 caused by a single point mutation in GDF5 (p.W414R. Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5(W414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C or SYNS2 (p.E491K revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A, is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.

  7. Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury

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    Saiga, Kenta [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Furumatsu, Takayuki, E-mail: [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Yoshida, Aki; Masuda, Shin; Takihira, Shota [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Abe, Nobuhiro [Department of Intelligent Orthopaedic System Development, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Ozaki, Toshifumi [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan)


    Research highlights: {yields} bFGF/GDF-5 treatment increases cellular proliferation and migration of MCL fibroblasts. {yields} bFGF/GDF-5 hydrogels stimulate the healing of MCL injury in vivo. {yields} bFGF/GDF-5 hydrogels stimulate Col1a1 expression and type I collagen synthesis. {yields} Combined use of bFGF/GDF-5 enhances MCL healing. -- Abstract: Basic fibroblast growth factor (bFGF) and growth and differentiation factor (GDF)-5 stimulate the healing of medial collateral ligament (MCL) injury. However, the effect of isolated and combined use of bFGF/GDF-5 remains still unclear. We investigated cellular proliferation and migration responding to bFGF/GDF-5 using rabbit MCL fibroblasts. Rabbit MCL injury was treated by bFGF and/or GDF-5 with peptide hydrogels. Gene expression and deposition of collagens in healing tissues were evaluated. bFGF/GDF-5 treatment additively enhanced cell proliferation and migration. bFGF/GDF-5 hydrogels stimulated Col1a1 expression without increasing Col3a1 expression. Combined use of bFGF/GDF-5 stimulated type I collagen deposition and the reorganization of fiber alignment, and induced better morphology of fibroblasts in healing MCLs. Our study indicates that combined use of bFGF/GDF-5 might enhance MCL healing by increasing proliferation and migration of MCL fibroblasts, and by regulating collagen synthesis and connective fiber alignment.

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

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


    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...... associated with antifibrotic effects in a mouse model of flexor tendoplasty. In this study, we compared the effects of loading freeze-dried allografts with different doses of GDF-5 protein or rAAV-Gdf5 on flexor tendon healing and adhesions. We first optimized the protein and viral loading parameters using...... reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and in vivo bioluminescent imaging. We then reconstructed flexor digitorum longus (FDL) tendons of the mouse hindlimb with allografts loaded with low and high doses of recombinant GDF-5 protein and r...

  9. Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

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    Christian Bucher


    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.

  10. Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems

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    James, R [Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908 (United States); Kumbar, S G; Laurencin, C T [Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT 06030 (United States); Balian, G; Chhabra, A B, E-mail: [Department of Orthopaedic Surgery, University of Virginia, Charlottesville, VA 22908 (United States)


    Tendon tissue engineering with a biomaterial scaffold that mimics the tendon extracellular matrix (ECM) and is biomechanically suitable, and when combined with readily available autologous cells, may provide successful regeneration of defects in tendon. Current repair strategies using suitable autografts and freeze-dried allografts lead to a slow repair process that is sub-optimal and fails to restore function, particularly in difficult clinical situations such as zone II flexor tendon injuries of the hand. We have investigated the effect of GDF-5 on cell proliferation and gene expression by primary rat adipose-derived stem cells (ADSCs) that were cultured on a poly(dl-lactide-co-glycolide) PLAGA fiber scaffold and compared to a PLAGA 2D film scaffold. The electrospun scaffold mimics the collagen fiber bundles present in native tendon tissue, and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis, the neotendon marker, was upregulated seven- to eightfold at 1 week with GDF-5 treatment when cultured on a 3D electrospun scaffold, and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. Expression of the genes that encode the major tendon ECM protein, collagen type I, was increased by fourfold starting at 1 week on treatment with 100 ng mL{sup -1} GDF-5, and at all time points the expression was significantly higher compared to 2D films irrespective of GDF-5 treatment. Thus stimulation with GDF-5 can modulate primary ADSCs on a PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration.

  11. The identification of trans-acting factors that regulate the expression of GDF5 via the osteoarthritis susceptibility SNP rs143383.

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    Catherine M Syddall


    Full Text Available rs143383 is a C to T transition SNP located in the 5'untranslated region (5'UTR of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with increased risk of osteoarthritis (OA in Europeans and in Asians. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE. The aim of this study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1 competition and supershift electrophoretic mobility shift assays (EMSAs and 2 an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP, and the functional effects of candidate proteins investigated by RNA interference (RNAi and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15, and DEAF-1 were identified as interacting with the GDF5 5'UTR. Knockdown and over expression of the factors demonstrated that Sp1, Sp3, and DEAF-1 are repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. In conclusion, we have identified four trans-acting factors that are binding to GDF5, three of which are modulating GDF5 expression via the OA susceptibility locus rs143383.

  12. Expression of GDF-5 during Limb Skeletal Development of Mice and the effect of GDF-5 on bone marrow mesenchymal stem cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yukun Zhang; Shuhua Yang; Li Sun; Cao Yang; Zhewei Ye; Dehao Fu


    Objective: To investigate the expression of growth differentiation factor 5 (GDF-5) during limb skeletal development of mice and the effect of GDF-5 on bone marrow mesenchymal stem cells in vitro. Methods: The expression of GDF-5 mRNA and protein in mouse fetal limb buds were detected in embryonic day 11.5-15.5 (El1.5-15.5) by RT-PCR and Western blotting respectively. Type Ⅱ collagen protein was examined with immunocytochemistry and the sulfate glycosaminoglycan was measured by Alcian blue. Results: During early stage of developmental skeletogenesis, the expression of GDF-5mRNA was constant and began with embryos E11.5, highlighted at embryos E12.5 and E13.5, subsequently dropped at embryos E14.5 and E15.5.There was very significant difference (P < 0.01) in average light density ratio of GDF-5/β-actin between E12.5-13.5 and the other three days. The expression of GDF-5 protein had a similar change with mRNA during limb skeletogenesis. Immunocytochemistry showed that GDF-5 could promote expression of Type Ⅱ collagen protein and histological staining of proteoglycan with Alcian blue revealed the deposition of typical cartilage extracellular matrix components. Conclusion: GDF-5 can enhance chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro, which plays an important role in limb skeletal development and joint formation.

  13. Differentiation of SOX-9 and GDF-5 co-transfected bone marrow mesenchymal stem cells into nucleus pulposus cells%SOX-9和GDF-5共同转染骨髓间充质干细胞向类髓核细胞的分化

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    杜志才; 银和平; 李树文; 武海军; 白明; 曹振华; 孟格东


    背景:移植间充质干细胞预防和治疗椎间盘退变是一种可行的方法,将SOX-9和GDF-5共同转染骨髓间充质干细胞,使其向髓核细胞转化,以期获得更大的髓核诱导和促增殖效应.目的:探讨SOX-9和GDF-5基因共同诱导兔骨髓间充质干细胞向类髓核细胞分化的效果.方法:提取、分离、纯化4周龄新西兰大白兔骨髓间充质干细胞,取第3代骨髓间充质干细胞分为5组体外诱导其向类髓核细胞分化,分别为未转染组、空载体转染组、SOX-9转染组、GDF-5转染组、共转染组.转染后第14 天采用RT-PCR检测SOX-9,GDF-5和Ⅱ型胶原的mRNA表达,免疫组化染色法检测髓核细胞标记物KRT19表达.结果与结论:共转染组SOX-9 mRNA表达高于转染SOX-9组,差异有显著性意义(P < 0.05);共转染组GDF-5 mRNA表达高于转染GDF-5组,差异有显著性意义(P < 0.05).共转染组Ⅱ型胶原表达高于转染SOX-9组、转染GDF-5组,差异有显著性意义(P < 0.05).SOX-9转染组及GDF-5转染组KRT19呈阳性表达,共转染组呈强阳性表达,可见被转染的骨髓间充质干细胞向类髓核细胞分化,且双基因转染诱导骨髓间充质干细胞向类髓核细胞分化的能力和分泌细胞外基质的能力明显高于单基因转染.%BACKGROUND:Transplantation of mesenchymal stem cels to prevent and treat degeneration of the intervertebral disc is a feasible method. Mesenchymal stem cels co-transfected by SRY-related high mobility group-box gene 9 (SOX-9) and growth differentiation factor-5 (GDF-5) can differentiate into nucleus pulposus cels, in order to obtain greater effect of induction and proliferation of nucleus pulposus cels. OBJECTIVE:To investigate the effect of SOX-9 and GDF-5 co-transfection on the differentiation of rabbit bone marrow mesenchymal stem cels into nucleus pulposus cels. METHODS: We separated and cultured bone marrow mesenchymal stem cels from the bone marrow of rabbit aged 4 months. Passage 3 cels

  14. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation (United States)

    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi


    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling. PMID:27030100

  15. Mutations in GDF5 reveal a key residue mediating BMP inhibition by NOGGIN.

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    Petra Seemann


    Full Text Available Signaling output of bone morphogenetic proteins (BMPs is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T in patients with multiple synostosis syndrome (SYM1 in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

  16. Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

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    Farooq, Muhammad; Nakai, Hiroyuki; Fujimoto, Atsushi;


    expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired...... in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins...

  17. Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics

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    D Wulsten


    Full Text Available This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue.Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

  18. Decationized polyplexes for gene delivery

    NARCIS (Netherlands)

    Novo, L.; Mastrobattista, E.; Nostrum, van C.F.; Lammers, T.G.G.M.; Hennink, W.E.


    Gene therapy has received much attention in the field of drug delivery. Synthetic, nonviral gene delivery systems have gained increasing attention as vectors for gene therapy mainly due to a favorable immunogenicity profile and ease of manufacturing as compared to viral vectors. The great majority o

  19. Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2

    DEFF Research Database (Denmark)

    Seemann, Petra; Schwappacher, Raphaela; Kjær, Klaus Wilbrandt


    Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b...... activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN...... was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like...

  20. A genetic association study between growth differentiation factor 5 (GDF 5 polymorphism and knee osteoarthritis in Thai population

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    Sura Thanyachai


    Full Text Available Abstract Objective Osteoarthritis (OA is a multi-factorial disease and genetic factor is one of the important etiologic risk factors. Various genetic polymorphisms have been elucidated that they might be associated with OA. Recently, several studies have shown an association between Growth Differentiation Factor 5(GDF5 polymorphism and knee OA. However, the role of genetic predisposing factor in each ethnic group cannot be replicated to all, with conflicting data in the literatures. Therefore, the aim of this study was to investigate the association between GDF5 polymorphism and knee OA in Thai population. Materials and Methods One hundred and ninety three patients aged 54-88 years who attended Ramathibodi Hospital were enrolled. Ninety cases with knee OA according to American College of Rheumatology criteria and one hundred and three cases in control group gave informed consent. Blood sample (5 ml were collected for identification of GDF5 (rs143383 single nucleotide polymorphism by PCR/RFLP according to a standard protocol. This study protocol was approved by the Ethics Committee on human experimentation of Ramathibodi Hospital Faculty of Medicine, Mahidol University. Odds ratios (OR and 95% confidence intervals were calculated for the risk of knee OA by genotype (TT, TC and CC and allele (T/C analyses. Results The baseline characteristics between two groups including job, smoking and activity were not different, except age and BMI. The entire cases and controls were in Hardy-Weinberg equilibrium (p > 0.05. The OA knee group (n = 90 had genotypic figure which has shown by TT 42.2% (n = 38, TC 45.6% (n = 41 and CC 12% (n = 11, whereas the control group (n = 103 revealed TT 32% (n = 33, TC 45.6% (n = 47, and CC 22.3% (n = 23, respectively. Genotypic TT increased risk of knee OA as compared to CC [OR = 2.41 (P = 0.04, 95%CI = 1.02-5.67]. In the allele analysis, the T allele was found to be significantly associated with knee OA [OR = 1.53 (P = 0

  1. Combination of engineered neural cell adhesion molecules and GDF-5 for improved neurite extension in nerve guide concepts. (United States)

    Niere, Marc; Braun, Bettina; Gass, Rea; Sturany, Sabine; Volkmer, Hansjürgen


    Current therapeutical approaches for the treatment of severe lesions in the peripheral nervous system rely on the use of autologous tissue or the body's own Schwann cells. However, these approaches are limited and alternative strategies for peripheral nerve regeneration are required. Here we evaluate combinations of a variety of neuronal regeneration factors including engineered cell adhesion molecules and growth factors in embryonic model neurons to test the possible improvement of artificial nerve guides by cooperative mechanisms. Cell adhesion molecules L1 and neurofascin synergistically promote neurite elongation. The outgrowth promoting properties of both proteins can be combined and further increased within one chimeric protein. Addition of growth and differentiation factor 5 (GDF-5) further enhances neurite outgrowth in a substrate-independent manner. This effect is not due to a protective mode of action of GDF-5 against pro-apoptotic stimuli. Consequently, the study supports the idea that different modes of action of pro-regenerative factors may contribute synergistically to neurite outgrowth and emphasizes the applicability of combinations of proteins specifically involved in development of the nervous system for therapeutical approaches.

  2. Electroporation-mediated gene delivery. (United States)

    Young, Jennifer L; Dean, David A


    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models.

  3. Nonviral Vectors for Gene Delivery (United States)

    Baoum, Abdulgader Ahmed


    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (˜200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was

  4. Gene doping: gene delivery for olympic victory. (United States)

    Gould, David


    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place.

  5. PDMAEMA based gene delivery materials

    Directory of Open Access Journals (Sweden)

    Seema Agarwal


    Full Text Available Gene transfection is the transfer of genetic material like DNA into cells. Cationic polymers which form nanocomplexes with DNA, so-called non-viral gene vectors, are a highly promising platform for efficient gene transfection. Despite intensive research efforts and some of the on-going clinical trials on gene transfection, none of the existing cationic polymer systems are generally acceptable for human gene therapy. Since the process of gene transfection is complex and puts different challenges and demands on the delivery system, there is a strong requirement for the design and development of a multifunctional system in a simple way. This review will discuss recent efforts in design, synthesis, and performance of poly(2-dimethylaminoethyl methacrylate (PDMAEMA nanocomplexes with DNA.

  6. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis (United States)

    Garciadiego-Cázares, David; Aguirre-Sánchez, Hilda I.; Abarca-Buis, René F.; Kouri, Juan B.; Velasquillo, Cristina; Ibarra, Clemente


    The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy

  7. Delivery systems for gene therapy

    Directory of Open Access Journals (Sweden)

    Shrikant Mali


    Full Text Available The structure of DNA was unraveled by Watson and Crick in 1953, and two decades later Arber, Nathans and Smith discovered DNA restriction enzymes, which led to the rapid growth in the field of recombinant DNA technology. From expressing cloned genes in bacteria to expressing foreign DNA in transgenic animals, DNA is now slated to be used as a therapeutic agent to replace defective genes in patients suffering from genetic disorders or to kill tumor cells in cancer patients. Gene therapy provides modern medicine with new perspectives that were unthinkable two decades ago. Progress in molecular biology and especially, molecular medicine is now changing the basics of clinical medicine. A variety of viral and non-viral possibilities are available for basic and clinical research. This review summarizes the delivery routes and methods for gene transfer used in gene therapy.

  8. Cationic Bolaamphiphiles for Gene Delivery (United States)

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


    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.

  9. Engineered nanoscaled polyplex gene delivery systems. (United States)

    Fernandez, Christian A; Rice, Kevin G


    Improving the transfection efficiencies of nonviral gene delivery requires properly engineered nanoscaled delivery carriers that can overcome the multiple barriers associated with the delivery of oligonucleotides from the site of administration to the nucleus or cytoplasm of the target cell. This article reviews the current advantages and limitation of polyplex nonviral delivery systems, including the apparent barriers that limit gene expression efficiency compared to physical methods such as hydrodynamic dosing and electroporation. An emphasis is placed on engineered nanoscaled polyplexes (NSPs) of modular design that both self-assemble and systematically disassemble at the desired stage of delivery. It is suggested that NSPs of increasingly sophisticated designs are necessary to improve the efficiency of the rate limiting steps in gene delivery.

  10. Liposomes as a gene delivery system

    Directory of Open Access Journals (Sweden)

    C. Ropert


    Full Text Available Gene therapy is an active field that has progressed rapidly into clinical trials in a relatively short time. The key to success for any gene therapy strategy is to design a vector able to serve as a safe and efficient gene delivery vehicle. This has encouraged the development of nonviral DNA-mediated gene transfer techniques such as liposomes. Many liposome-based DNA delivery systems have been described, including molecular components for targeting given cell surface receptors or for escaping from the lysosomal compartment. Another recent technology using cationic lipids has been evaluated and has generated substantial interest in this approach to gene transfer.

  11. Delivery Systems in Gene Therapy

    Institute of Scientific and Technical Information of China (English)

    Liu Hu; Anas El-Aneed; Cui Guohui


    1 Gene therapy Gene therapy includes the treatment of both genetically based and infectious diseases by introducing genetic materials which have therapeutic effects[1~3]. In its simplest terms, a wild type gene (which is non-functional in the cell leading to disease development) is introduced into the somatic cell lacking this gene to restore the normal gene function in this cell. Many gene therapy strategies, however, utilize genes to destroy specific cells.

  12. Analysis of association between IL-1β, CASP-9, and GDF5 variants and low-back pain in Chinese male soldier: clinical article. (United States)

    Mu, Jihong; Ge, Weiming; Zuo, Xincheng; Chen, Yuxuan; Huang, Changlin


    Recent studies have suggested that genetic risk factors play an important role in the occurrence of low-back pain (LBP) and lumbar disc disease. The authors' study aimed to assess the association between 3 single-nucleotide polymorphisms (SNPs) and susceptibility to LBP related to military training. In this case-control study, data from 892 Chinese male soldiers were retrospectively reviewed. The case group was made up of 305 soldiers with LBP related to military training and a control group of 587 soldiers without constant LBP for more than 2 weeks. Genomic DNA was extracted from peripheral blood leukocytes of all subjects and polymerase chain reaction products were genotyped. No association was found between the SNP of interleukin-1β (IL-1β; +3954C/T) and LBP at both the genotypic (p = 0.104) and the allelic (p = 0.098) level. However, the G allele of caspase-9 (CASP-9) and the T allele of growth differentiation factor 5 (GDF5) were more common in patients with LBP than in patients without LBP (p Chinese military cohort indicated that CASP-9 (-1263A/G) and GDF5 (+104T/C) polymorphisms are associated with a susceptibility to LBP related to military training.

  13. Gene doping: gene delivery for olympic victory



    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted...

  14. Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents. (United States)

    Fishbein, Ilia; Chorny, Michael; Adamo, Richard F; Forbes, Scott P; Corrales, Ricardo A; Alferiev, Ivan S; Levy, Robert J

    A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation.

  15. "Bronchial Artery Delivery of Viral Vectors for Gene delivery in Cystic Fibrosis; Superior to Airway Delivery?"

    Directory of Open Access Journals (Sweden)

    Coutelle Charles C


    Full Text Available Abstract Background Attempts at gene therapy for the pulmonary manifestations of Cystic Fibrosis have relied mainly on airway delivery. However the efficiency of gene transfer and expression in the airway epithelia has not reached therapeutic levels. Access to epithelial cells is not homogenous for a number of reasons and the submucosal glands cannot be reached via the airways. Presentation We propose to inject gene delivery vectors directly into bronchial arteries combined with pre-delivery of vascular endothelial growth factor to increase vascular endothelial permeability and post-delivery flow reduction by balloon occlusion. Thus it may be possible to reach mucous secreting cells of the bronchial luminal epithelium and the submucosal glands in an increased and homogenous fashion. Testing This combination of techniques to the best of our knowledge has not previously been investigated, and may enable us to overcome some of the current limitations to gene therapy for Cystic Fibrosis.

  16. Gene therapy prospects--intranasal delivery of therapeutic genes. (United States)

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


    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.

  17. Magnetic targeting strategies in gene delivery. (United States)

    Delyagina, Evgenya; Li, Wenzhong; Ma, Nan; Steinhoff, Gustav


    Gene delivery is a process of the insertion of transgenes into cells with the purpose to obtain the expression of encoded protein. The therapeutic application of this process is termed gene therapy, which is becoming a promising instrument to treat genetic and acquired diseases. Although numerous methods of gene transfer have already been developed, including biological, physical and chemical approaches, the optimal strategy has to be discovered. Importantly, it should be effective, selective and safe to be translated to the clinic. Magnetic targeting has been demonstrated as an effective strategy to decrease side effects of gene transfer, while increasing the selectivity and efficiency of the applied vector. This article will focus on the latest progress in the development of different magnetic vectors, based on both viral and nonviral gene delivery agents. It will also include a description of magnetic targeting applications in stem cells and in vivo, which has gained interest in recent years due to the rapid development of technology.

  18. Bioreducible polymers for gene silencing and delivery. (United States)

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


    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

  19. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

    Directory of Open Access Journals (Sweden)

    Shyamal Kumar Kundu


    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.

  20. Future prospects for gene delivery systems. (United States)

    Kuşcu, Lale; Sezer, Ali Demir


    Gene therapy is the challenging area of biotechnology. Despite its promise for critical diseases, it has serious safety and efficiency issues, particularly with regards to gene transfer systems. Areas covered: We examined the current situation with gene transfer systems and addressed problems this technology. We then searched patent applications about in the area from the Patentscope online system, the international patent database. We analyzed the data obtained to get a general idea about gene delivery systems designed for future use and assessed approaches for more efficient, safer and valid delivery systems. Expert opinion: When quality assurance terms are fulfilled, some of these issues (genetic changes, mutations) could be minimized during the production process. Modification of vectors for improving their efficiency and safety or development of alternative transfer systems could be the solutions for these problems. Gene transfer technologies are important for gene therapy and should demonstrate effective, target-specific and acceptable safety profiles. For this reason, searching for alternatives to current systems is a necessity.

  1. Novel Polymeric Nanoparticles for Pulmonary Gene Delivery (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

  2. Recent progress in polymer-based gene delivery vectors

    Institute of Scientific and Technical Information of China (English)

    HUANG Shiwen; ZHUO Renxi


    The gene delivery system is one of the three components of a gene medicine, which is the bottle neck of current gene therapy. Nonviral vectors offer advantages over the viral system of safety, ease of manufacturing, etc. As important nonviral vectors, polymer gene delivery systems have gained increasing attention and have begun to show increasing promising. In this review, the fundamental and recent progress of polymer-based gene delivery vectors is reviewed.

  3. An intestinal Trojan horse for gene delivery (United States)

    Peng, Haisheng; Wang, Chao; Xu, Xiaoyang; Yu, Chenxu; Wang, Qun


    The intestinal epithelium forms an essential element of the mucosal barrier and plays a critical role in the pathophysiological response to different enteric disorders and diseases. As a major enteric dysfunction of the intestinal tract, inflammatory bowel disease is a genetic disease which results from the inappropriate and exaggerated mucosal immune response to the normal constituents in the mucosal microbiota environment. An intestine targeted drug delivery system has unique advantages in the treatment of inflammatory bowel disease. As a new concept in drug delivery, the Trojan horse system with the synergy of nanotechnology and host cells can achieve better therapeutic efficacy in specific diseases. Here, we demonstrated the feasibility of encapsulating DNA-functionalized gold nanoparticles into primary isolated intestinal stem cells to form an intestinal Trojan horse for gene regulation therapy of inflammatory bowel disease. This proof-of-concept intestinal Trojan horse will have a wide variety of applications in the diagnosis and therapy of enteric disorders and diseases.

  4. Retrotransposon vectors for gene delivery in plants

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    Hou Yi


    Full Text Available Abstract Background Retrotransposons are abundant components of plant genomes, and although some plant retrotransposons have been used as insertional mutagens, these mobile genetic elements have not been widely exploited for plant genome manipulation. In vertebrates and yeast, retrotransposons and retroviruses are routinely altered to carry additional genes that are copied into complementary (cDNA through reverse transcription. Integration of cDNA results in gene delivery; recombination of cDNA with homologous chromosomal sequences can create targeted gene modifications. Plant retrotransposon-based vectors, therefore, may provide new opportunities for plant genome engineering. Results A retrotransposon vector system was developed for gene delivery in plants based on the Tnt1 element from Nicotiana tabacum. Mini-Tnt1 transfer vectors were constructed that lack coding sequences yet retain the 5' and 3' long terminal repeats (LTRs and adjacent cis sequences required for reverse transcription. The internal coding region of Tnt1 was replaced with a neomycin phosphotransferase gene to monitor replication by reverse transcription. Two different mini-Tnt1 s were developed: one with the native 5' LTR and the other with a chimeric 5' LTR that had the first 233 bp replaced by the CaMV 35 S promoter. After transfer into tobacco protoplasts, both vectors undergo retrotransposition using GAG and POL proteins provided in trans by endogenous Tnt1 elements. The transposition frequencies of mini-Tnt1 vectors are comparable with native Tnt1 elements, and like the native elements, insertion sites are within or near coding sequences. In this paper, we provide evidence that template switching occurs during mini-Tnt1 reverse transcription, indicating that multiple copies of Tnt1 mRNA are packaged into virus-like particles. Conclusions Our data demonstrate that mini-Tnt1 vectors can replicate efficiently in tobacco cells using GAG and POL proteins provided in trans by

  5. Lipid Nanoparticles for Ocular Gene Delivery

    Directory of Open Access Journals (Sweden)

    Yuhong Wang


    Full Text Available Lipids contain hydrocarbons and are the building blocks of cells. Lipids can naturally form themselves into nano-films and nano-structures, micelles, reverse micelles, and liposomes. Micelles or reverse micelles are monolayer structures, whereas liposomes are bilayer structures. Liposomes have been recognized as carriers for drug delivery. Solid lipid nanoparticles and lipoplex (liposome-polycation-DNA complex, also called lipid nanoparticles, are currently used to deliver drugs and genes to ocular tissues. A solid lipid nanoparticle (SLN is typically spherical, and possesses a solid lipid core matrix that can solubilize lipophilic molecules. The lipid nanoparticle, called the liposome protamine/DNA lipoplex (LPD, is electrostatically assembled from cationic liposomes and an anionic protamine-DNA complex. The LPD nanoparticles contain a highly condensed DNA core surrounded by lipid bilayers. SLNs are extensively used to deliver drugs to the cornea. LPD nanoparticles are used to target the retina. Age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy are the most common retinal diseases in humans. There have also been promising results achieved recently with LPD nanoparticles to deliver functional genes and micro RNA to treat retinal diseases. Here, we review recent advances in ocular drug and gene delivery employing lipid nanoparticles.

  6. Synthetic virology: engineering viruses for gene delivery. (United States)

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


    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.

  7. Liposomes for Use in Gene Delivery

    Directory of Open Access Journals (Sweden)

    Daniel A. Balazs


    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.

  8. [Developments in gene delivery vectors for ocular gene therapy]. (United States)

    Khabou, Hanen; Dalkara, Deniz


    Gene therapy is quickly becoming a reality applicable in the clinic for inherited retinal diseases. Its remarkable success in safety and efficacy, in clinical trials for Leber's congenital amaurosis (LCA) type II generated significant interest and opened up possibilities for a new era of retinal gene therapies. Success in these clinical trials was mainly due to the favorable characteristics of the retina as a target organ. The eye offers several advantages as it is readily accessible and has some degree of immune privilege making it suitable for application of viral vectors. The viral vectors most frequently used for retinal gene delivery are lentivirus, adenovirus and adeno-associated virus (AAV). Here we will discuss the use of these viral vectors in retinal gene delivery with a strong focus on favorable properties of AAV. Thanks to its small size, AAV diffuses well in the inter-neural matrix making it suitable for applications in neural retina. Building on this initial clinical success with LCA II, we have now many opportunities to extend this proof-of-concept to other retinal diseases using AAV as a vector. This article will discuss what are some of the most imminent cellular targets for such therapies and the AAV toolkit that has been built to target these cells successfully. We will also discuss some of the challenges that we face in translating AAV-based gene therapies to the clinic. © 2015 médecine/sciences – Inserm.

  9. Microfluidic methods for non-viral gene delivery. (United States)

    Lai, Wing-Fu


    Microfluidics is a compelling technology that shows considerable promise in applications ranging from gene expression profiling to cell-based assays. Owing to its capacity to enable generation of single droplets and multiple droplet arrays with precisely controlled composition and a narrow size distribution, recently microfluidics has been exploited for delivery of genes. This article provides an overview of recent advances in microfluidic gene delivery, and speculates the prospects for further research. The objectives of this article are to illustrate the potential roles played by microfluidics in gene delivery research, and to shed new light on strategies to enhance the efficiency of gene therapy.

  10. Microneedles as a Delivery System for Gene Therapy

    Directory of Open Access Journals (Sweden)

    Wei eChen


    Full Text Available Gene delivery systems can be divided to two major types: vector-based (either viral vector or non-viral vector and physical delivery technologies. Many physical carriers, such as electroporation, gene gun, ultrasound start to be proved to have the potential to enable gene therapy. A relatively new physical delivery technology for gene delivery consists of microneedles (MNs, which has been studied in many fields and for many molecule types and indications. Microneedles can penetrate the stratum corneum, which is the main barrier for drug delivery through the skin with ease of administration and without significant pain. Many different kinds of MNs, such as metal MNs, coated MNs, dissolving MNs have turned out to be promising in gene delivery. In this review, we discussed the potential as well as the challenges of utilizing MNs to deliver nucleic acids for gene therapy. We also proposed that a combination of MNs and other gene delivery approaches may lead to a better delivery system for gene therapy.

  11. Bioreducible polymers for efficient gene and siRNA delivery

    Energy Technology Data Exchange (ETDEWEB)

    Jere, Dhananjay; Arote, Rohidas; Jiang Hulin; Kim, You-Kyoung; Cho, Chong-Su [Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921 (Korea, Republic of); Cho, Myung-Haing, E-mail: [College of Veterinary Medicines, Seoul National University, Seoul 151-742 (Korea, Republic of)


    Bioreducible disulfide linkage-employing drug conjugate has already been approved for drug delivery application, and also has shown immense potential in gene and siRNA transfection. This paper will focus on the recent developments in bioreducible polymeric systems for gene and siRNA delivery application, and will discuss the advantages and challenges associated with reducible polymeric carriers.

  12. Gene delivery in tissue engineering and regenerative medicine. (United States)

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


    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.

  13. Investigation of a thiolated polymer in gene delivery (United States)

    Bacalocostantis, Irene

    Thiol-containing bioreducible polymers show significant potential as delivery vectors in gene therapy, a rapidly growing field which seeks to treat genetic-based disorders by delivering functional synthetic genes to diseased cells. Studies have shown that thiolated polymers exhibit improved biodegradability and prolonged in vivo circulation times over non-thiolated polymers. However, the extent to which thiol concentrations impact the carrier's delivery potential has not been well explored. The aim of this dissertation is to investigate how relative concentrations of free thiols and disulfide crosslinks impact a polymeric carriers delivery performance with respect to DNA packaging, complex stability, cargo protection, gene release, internalization efficiency and cytotoxicity. To accomplish this goal, several fluorescent polymers containing varying concentrations of thiol groups were synthesized by conjugating thiol-pendant chains onto the primary amines of cationic poly(allylamine). In vitro delivery assays and characterization techniques were employed to assess the effect of thiols in gene delivery.

  14. Recent progresses in gene delivery-based bone tissue engineering. (United States)

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen


    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.

  15. State-of-the-art human gene therapy: part I. Gene delivery technologies. (United States)

    Wang, Dan; Gao, Guangping


    Safe and effective gene delivery is a prerequisite for successful gene therapy. In the early age of human gene therapy, setbacks due to problematic gene delivery vehicles plagued the exciting therapeutic outcome. However, gene delivery technologies rapidly evolved ever since. With the advancement of gene delivery techniques, gene therapy clinical trials surged during the past decade. As the first gene therapy product (Glybera) has obtained regulatory approval and reached clinic, human gene therapy finally realized the promise that genes can be medicines. The diverse gene delivery techniques available today have laid the foundation for gene therapy applications in treating a wide range of human diseases. Some of the most urgent unmet medical needs, such as cancer and pandemic infectious diseases, have been tackled by gene therapy strategies with promising results. Furthermore, combining gene transfer with other breakthroughs in biomedical research and novel biotechnologies opened new avenues for gene therapy. Such innovative therapeutic strategies are unthinkable until now, and are expected to be revolutionary. In part I of this review, we introduced recent development of non-viral and viral gene delivery technology platforms. As cell-based gene therapy blossomed, we also summarized the diverse types of cells and vectors employed in ex vivo gene transfer. Finally, challenges in current gene delivery technologies for human use were discussed.

  16. The Effect of Growth Differentiation Factor-5 on the Mandibular Condylar Cartilage of Developing Temporomandibular Joint%GDF5在大鼠颞下颌关节发育早期髁状突软骨中作用的研究

    Institute of Scientific and Technical Information of China (English)

    陈建中; 李松; 罗应伟; 杨春; 沈丽宁


    目的 通过观察生长分化因子-5(GDF-5)在大鼠颞下颌关节发育早期髁状突软骨中表达的时空变化特点,探讨GDF-5在髁状突软骨发育中的意义.方法 建立大鼠胚胎(E)13、15、17、19、21 d的颞下颌关节发育模型,应用免疫组化法检测GDF-5在颞下颌关节早期发育的不同时期髁状突软骨中的表达.结果 免疫组织化学检测GDF-5在髁状突软骨的发育过程中具有时空特异性,GDF-5在E13 d的髁状突细胞凝聚区中开始表达,在E15 d的增殖层及成软骨细胞层表达较强,之后表达减弱;GOF-5在肥大细胞层表达由弱至强又减弱;GDF-5在髁状突软骨不同发育时间、不同分化阶段的表达水平不同、结论 GDF-5参与调控髁状突软骨细胞增殖、分化及成熟的过程.

  17. Gene delivery in peritoneal dialysis related peritoneal fibrosis research

    Institute of Scientific and Technical Information of China (English)

    LI Xie-jia; SUN Lin; XIAO Li; LIU Fu-you


    Objective To summarize the development of gene delivery vectors in peritoneal fibrosis research and discuss the feasibility and superiority of lentiviral vectors.Data sources The data in this article were collected from PubMed database with relevant English articles published from 1995 to 2011.Study selection Articles regarding the gene therapy in peritoneal fibrosis research using non-viral vectors,adenoviral vectors,ratroviral vectors,and lentiviral vectors were selected.Data were mainly extracted from 60 articles,which are listed in the reference section of this review.Results Non-viral vector-mediated gene delivery (including naked DNA for ex vivo,oligonucleotides,ultrasoundcontrast agent mediated naked gene delivery,etc.) and viral vector-mediated gene delivery (including adenovirus,helper-dependant adenovirus,and retrovirus vectors) have been successfully applied both in the mechanistic investigation and the potential prevention and treatment of peritoneal fibrosis.Conclusions Peritoneal fibrosis is a major complication of peritoneal dialysis (PD).Recently,the wide use of the gene delivery technique made it possible to access and further research peritoneal fibrosis.The use of lentiviral vector is expected to be widely used in PD research in the future due to its advantages in gene delivery.

  18. Advance in polyamidoamine dendrimers as gene delivery agents

    Institute of Scientific and Technical Information of China (English)


    @@ Gene therapy recently has become an important area of research as a new therapeutic method. In vivo and in vitro gene therapies require efficient delivery of genetic material into a cell and preferably high levels of expression of transferred gene. Traditionally, gene delivery systems are classified as viral vector-mediated systems and nonviral vector-mediated systems. Viral vectors, which have been demonstrated as systems with high transfection efficiency, however, are limited due to adverse effects such as immunogenicity, toxicity, limited DNA carrying capacity and mutagenesis caused by cell-infected viruses[1].

  19. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery (United States)

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


    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.

  20. The evolution of heart gene delivery vectors


    Wasala, Nalinda B.; Shin, Jin-Hong; Duan, Dongsheng


    Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic m...

  1. Magnetic nanoparticles for gene and drug delivery


    Dobson, J


    Stuart C McBain, Humphrey HP Yiu, Jon DobsonInstitute of Science and Technology in Medicine, Keele University, Thornburrow Drive, Hartshill, Stoke-on-Trent, Staffordshire, ST4 7QB, U.K.Abstract: Investigations of magnetic micro- and nanoparticles for targeted drug delivery began over 30 years ago. Since that time, major progress has been made in particle design and synthesis techniques, however, very few clinical trials have taken place. Here we review advances in magnetic nanoparticle design...

  2. Therapeutic globin gene delivery using lentiviral vectors. (United States)

    Rivella, Stefano; Sadelain, Michel


    The severe hemoglobinopathies, including beta-thalassemia major and sickle cell anemia, are candidate diseases for a genetic treatment based on the transfer of a regulated globin gene in autologous hematopoietic stem cells. Two years ago, May et al reported that an optimized beta-globin transcription unit containing multiple proximal and distal regulatory elements harbored by a recombinant lentiviral vector could efficiently integrate into murine hematopoietic stem cells and express therapeutic levels of the human beta-globin gene. Here, we review the advantages afforded by lentivirus-mediated globin gene transfer and recent studies based on this strategy.

  3. Micelles and nanoparticles for ultrasonic drug and gene delivery. (United States)

    Husseini, Ghaleb A; Pitt, William G


    Drug delivery research employing micelles and nanoparticles has expanded in recent years. Of particular interest is the use of these nanovehicles that deliver high concentrations of cytotoxic drugs to diseased tissues selectively, thus reducing the agent's side effects on the rest of the body. Ultrasound, traditionally used in diagnostic medicine, is finding a place in drug delivery in connection with these nanoparticles. In addition to their non-invasive nature and the fact that they can be focused on targeted tissues, acoustic waves have been credited with releasing pharmacological agents from nanocarriers, as well as rendering cell membranes more permeable. In this article, we summarize new technologies that combine the use of nanoparticles with acoustic power both in drug and gene delivery. Ultrasonic drug delivery from micelles usually employs polyether block copolymers and has been found effective in vivo for treating tumors. Ultrasound releases drug from micelles, most probably via shear stress and shock waves from the collapse of cavitation bubbles. Liquid emulsions and solid nanoparticles are used with ultrasound to deliver genes in vitro and in vivo. The small packaging allows nanoparticles to extravasate into tumor tissues. Ultrasonic drug and gene delivery from nanocarriers has tremendous potential because of the wide variety of drugs and genes that could be delivered to targeted tissues by fairly non-invasive means.

  4. Chitosan for gene delivery and orthopedic tissue engineering applications. (United States)

    Raftery, Rosanne; O'Brien, Fergal J; Cryan, Sally-Ann


    Gene therapy involves the introduction of foreign genetic material into cells in order exert a therapeutic effect. The application of gene therapy to the field of orthopaedic tissue engineering is extremely promising as the controlled release of therapeutic proteins such as bone morphogenetic proteins have been shown to stimulate bone repair. However, there are a number of drawbacks associated with viral and synthetic non-viral gene delivery approaches. One natural polymer which has generated interest as a gene delivery vector is chitosan. Chitosan is biodegradable, biocompatible and non-toxic. Much of the appeal of chitosan is due to the presence of primary amine groups in its repeating units which become protonated in acidic conditions. This property makes it a promising candidate for non-viral gene delivery. Chitosan-based vectors have been shown to transfect a number of cell types including human embryonic kidney cells (HEK293) and human cervical cancer cells (HeLa). Aside from its use in gene delivery, chitosan possesses a range of properties that show promise in tissue engineering applications; it is biodegradable, biocompatible, has anti-bacterial activity, and, its cationic nature allows for electrostatic interaction with glycosaminoglycans and other proteoglycans. It can be used to make nano- and microparticles, sponges, gels, membranes and porous scaffolds. Chitosan has also been shown to enhance mineral deposition during osteogenic differentiation of MSCs in vitro. The purpose of this review is to critically discuss the use of chitosan as a gene delivery vector with emphasis on its application in orthopedic tissue engineering.

  5. A Novel Gene Delivery System Targeting Urokinase Receptor

    Institute of Scientific and Technical Information of China (English)

    Xing-Hui SUN; Li TAN; Chun-Yang LI; Chang TONG; Jin FAN; Ping LI; Yun-Song ZHU


    Recombinant proteins that combine different functions required for cell targeting and intracellular delivery of DNA present an attractive approach for the development of nonviral gene delivery vectors. Here, we described a novel protein termed ATF-lys10 which facilitated cell-specific gene transfer via receptor-mediated endocytosis. ATF-lys 10 was composed of the amino-terminal fragment of urokinase and ten lysines at the carboxyl terminus. Bacterially expressed ATF-lys 10 protein existed in soluble form, and had antigenicity of human urokinase. Purified ATF-lys 10 specifically bound to uPAR-expressing cells and formed protein-DNA complexes with plasmid pGL3-control. After neutralization of excess negative charge with poly-L-lysine, these complexes served as a specific gene delivery vector for uPAR-expressing cells. Lysosomotropic compounds, such as chloroquine, drastically increased the ATF-lysl0 mediated gene delivery efficiency. Our results suggest that the recombinant protein ATF-lys 10 with the properties of DNA binding and tumor cell targeting represents a promising method for gene transfer and expression in tumor cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fillat, Cristina, E-mail:; 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)


    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.

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

    LENUS (Irish Health Repository)

    Rajendran, Simon


    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.

  8. Physical non-viral gene delivery methods for tissue engineering. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Qiu-Lan Zhou


    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.

  10. AAV vectors as gene delivery vehicles in the central nervous system

    NARCIS (Netherlands)

    Broekman, M.L.D.


    Recombinant gene delivery vehicles based on the replication-defective AAV have gained a preeminent position in the field of gene delivery to the brain. Efficient global gene delivery to the CNS is beneficial for the study of gene products is the entire CNS as well as for introducing and expressing g

  11. In vivo electroporation mediated gene delivery to the beating heart.

    Directory of Open Access Journals (Sweden)

    Erick L Ayuni

    Full Text Available Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase and TNT (Troponin T were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.

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

    Directory of Open Access Journals (Sweden)

    Yu Kuang


    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.

  13. Gene gun delivery systems for cancer vaccine approaches. (United States)

    Aravindaram, Kandan; Yang, Ning Sun


    Gene-based immunization with transgenic DNA vectors expressing tumor-associated antigens (TAA), cytokines, or chemokines, alone or in combination, provides an attractive approach to increase the cytotoxic T cell immunity against various cancer diseases. With this consideration, particle-mediated or gene gun technology has been developed as a nonviral method for gene transfer into various mammalian tissues. It has been shown to induce both humoral and cell-mediated immune responses in both small and large experimental animals. A broad range of somatic cell types, including primary cultures and established cell lines, has been successfully transfected ex vivo or in vitro by gene gun technology, either as suspension or adherent cultures. Here, we show that protocols and techniques for use in gene gun-mediated transgene delivery system for skin vaccination against melanoma using tumor-associated antigen (TAA) human gpl00 and reporter gene assays as experimental systems.

  14. Nonviral gene delivery: principle, limitations, and recent progress. (United States)

    Al-Dosari, Mohammed S; Gao, Xiang


    Gene therapy is becoming a promising therapeutic modality for the treatment of genetic and acquired disorders. Nonviral approaches as alternative gene transfer vehicles to the popular viral vectors have received significant attention because of their favorable properties, including lack of immunogenicity, low toxicity, and potential for tissue specificity. Such approaches have been tested in preclinical studies and human clinical trials over the last decade. Although therapeutic benefit has been demonstrated in animal models, gene delivery efficiency of the nonviral approaches remains to be a key obstacle for clinical applications. This review focuses on existing and emerging concepts of chemical and physical methods for delivery of therapeutic nucleic acid molecules in vivo. The emphasis is placed on discussion about problems associated with current nonviral methods and recent efforts toward refinement of nonviral approaches.

  15. Baculovirus-mediated Gene Delivery and RNAi Applications

    Directory of Open Access Journals (Sweden)

    Kaisa-Emilia Makkonen


    Full Text Available Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism.

  16. Reducible, dibromomaleimide-linked polymers for gene delivery. (United States)

    Tan, James-Kevin Y; Choi, Jennifer L; Wei, Hua; Schellinger, Joan G; Pun, Suzie H


    Polycations have been successfully used as gene transfer vehicles both in vitro and in vivo; however, their cytotoxicity has been associated with increasing molecular weight. Polymers that can be rapidly degraded after internalization are typically better tolerated by mammalian cells compared to their non-degradable counterparts. Here, we report the use of a dibromomaleimide-alkyne (DBM-alkyne) linking agent to reversibly bridge cationic polymer segments for gene delivery and to provide site-specific functionalization by azide-alkyne cycloaddition chemistry. A panel of reducible and non-reducible, statistical copolymers of (2-dimethylamino)ethyl methacrylate (DMAEMA) and oligo(ethylene glycol)methyl ether methacrylate (OEGMA) were synthesized and evaluated. When complexed with plasmid DNA, the reducible and non-reducible polymers had comparable DNA condensation properties, sizes, and transfection efficiencies. When comparing cytotoxicity, the DBM-linked, reducible polymers were significantly less toxic than the non-reducible polymers. To demonstrate polymer functionalization by click chemistry, the DBM-linked polymers were tagged with an azide-fluorophore and were used to monitor cellular uptake. Overall, this polymer system introduces the use of a reversible linker, DBM-alkyne, to the area of gene delivery and allows for facile, orthogonal, and site-specific functionalization of gene delivery vehicles.

  17. Progress in non-viral gene delivery systems fabricated via supramolecular assembly

    Institute of Scientific and Technical Information of China (English)

    WANG Youxiang; SHEN Jiacong


    Gene delivery systems are one of key issues that limit the development of gene therapy. The novel non-viral gene delivery systems fabricated via supramolecular assembly have begun to show increasing promising and applications in gene therapy due to its suitable nanometric size, controllable structure and excellent biocompatibility. In this review, the fundamental and recent progress of non-viral gene supramolecular assembly is reviewed. Artificial viruses--the future direction of non-viral gene delivery systems are also described.


    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL


    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  19. 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: [Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan (China)


    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.

  20. Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery

    Directory of Open Access Journals (Sweden)

    Teerapong Yata


    Full Text Available Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage, viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

  1. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems (United States)

    Fillat, Cristina; Jose, Anabel; Ros, Xavier Bofill-De; Mato-Berciano, Ana; Maliandi, Maria Victoria; Sobrevals, Luciano


    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. PMID:24212620

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

    Directory of Open Access Journals (Sweden)

    Maria Victoria Maliandi


    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.

  3. Polyphosphazenes: Multifunctional, Biodegradable Vehicles for Drug and Gene Delivery

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    Ian Teasdale


    Full Text Available Poly[(organophosphazenes] are a unique class of extremely versatile polymers with a range of applications including tissue engineering and drug delivery, as hydrogels, shape memory polymers and as stimuli responsive materials. This review aims to divulge the basic principles of designing polyphosphazenes for drug and gene delivery and portray the huge potential of these extremely versatile materials for such applications. Polyphosphazenes offer a number of distinct advantages as carriers for bioconjugates; alongside their completely degradable backbone, to non-toxic degradation products, they possess an inherently and uniquely high functionality and, thanks to recent advances in their polymer chemistry, can be prepared with controlled molecular weights and narrow polydispersities, as well as self-assembled supra-molecular structures. Importantly, the rate of degradation/hydrolysis of the polymers can be carefully tuned to suit the desired application. In this review we detail the recent developments in the chemistry of polyphosphazenes, relevant to drug and gene delivery and describe recent investigations into their application in this field.

  4. Mannan-Modified PLGA Nanoparticles for Targeted Gene Delivery

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    Fansheng Kong


    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.

  5. Hydrogels: a journey from diapers to gene delivery. (United States)

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


    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.

  6. Nanoparticle-mediated delivery of suicide genes in cancer therapy. (United States)

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


    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.

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

  8. Gene Delivery into Plant Cells for Recombinant Protein Production

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    Qiang Chen


    Full Text Available Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

  9. Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers. (United States)

    Raad, Markus de; Teunissen, Erik A; Mastrobattista, Enrico


    The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

  10. Polyamine-DNA interactions and development of gene delivery vehicles. (United States)

    Thomas, T J; Tajmir-Riahi, H A; Thomas, Thresia


    Polyamines are positively charged organic cations under physiologic ionic and pH conditions and hence they interact with negatively charged macromolecules such as DNA and RNA. Although electrostatic interaction is the predominant mode of polyamine-nucleic acid interactions, site- and structure-specific binding has also been recognized. A major consequence of polyamine-DNA interaction is the collapse of DNA to nanoparticles of approximately 100 nm diameter. Electron and atomic force microscopic studies have shown that these nanoparticles are spheroids, toroids and rods. DNA transport to cells for gene therapy applications requires the condensation of DNA to nanoparticles and hence the study of polyamines and related compounds with nucleic acids has received technological importance. In addition to natural and synthetic polyamines, several amine-terminated or polyamine-substituted agents are under intense investigation for non-viral gene delivery vehicles.

  11. Polyethylenimine-grafted polycarbonates as biodegradable polycations for gene delivery. (United States)

    Wang, Chang-Fang; Lin, Yan-Xin; Jiang, Tao; He, Feng; Zhuo, Ren-Xi


    Polycations as one of non-viral vectors have gained increasing attentions. In this paper, polyethylenimine(PEI)-grafted polycarbonates (PMAC-g-PEIx) were synthesized as a kind of biodegradable polycations for gene delivery. Backbone polymer, poly(5-methyl-5-allyloxycarbonyl-trimethylene carbonate) (PMAC), was synthesized in bulk catalyzed by immobilized porcine pancreas lipase (IPPL). Then, PMAC-O, the allyl epoxidation product of PMAC, was further modified by PEIx with low molecular weight (x = 423, 800 and 1800). The MWs of PMAC-g-PEIx, measured by GPC-MALLS, were 81,900, 179,900 and 200,600 g/mol with polydispersities of 1.2, 1.4 and 1.7, respectively. PMAC-g-PEIx could form positively charged nano-sized particles (30-90 nm) with pDNA, and all the three PAMC-g-PEIx/DNA polyplexes had similar buffer capabilities. In vitro experiments demonstrated that the PAMC-g-PEIx showed much low cytotoxicity and enhanced transfection efficiency could be found in comparison with PEI25K in 293T cells. Furthermore, pre-incubation of PMAC-g-PEI1800 showed a weakening binding capacity with DNA. The biodegradability of PMAC-g-PEIx can facilitate the efficient release of pDNA from polyplexes and reduce cell cytotoxicity. These results suggested that PMAC-g-PEIx would be a promising non-viral biodegradable vector for gene delivery system.

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

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    Kitae Ryu


    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.

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

    NARCIS (Netherlands)

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


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

  14. Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy. (United States)

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


    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.

  15. Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery. (United States)

    Jang, J-H; Koerber, J T; Gujraty, K; Bethi, S R; Kane, R S; Schaffer, D V


    Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

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

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda


    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.

  17. Immune Activities of Polycationic Vectors for Gene Delivery

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    Xiaotian Zhao


    Full Text Available Polycationic vectors are used widely in the field of gene delivery, while currently their immune activities in vivo are poorly understood. In this comprehensive review, we aim to present an overview of existing mechanisms of adverse immune responses induced by the polycation/gene complexes, which includes the polycations themselves, the gene sequences and the ROS produced by them. These causes can induce pro-inflammatory cytokines, hypersensitivity as well as the activation of toll-like receptors, and finally the immunostimulation occur. In addition, we introduce some different opinions and research results on the immunogenicity of classical polycations such as polylysine (PLL, polyethyleneimine (PEI, polyamidoamine dendrimers (PAMAM, chitosan and gelatin, most of which have immunogenicity and can induce immunoreactions in vivo. The methods now used to adjust their immunogenicity are shown in the final part of this review. Nowadays, there is still no accurate conclusion on immunogenicity of polycations, which confuses researchers seriously in in vivo test. We conclude that further research is needed in order to skillfully utilize or inhibit the immunogenicity of these polycationic vectors.

  18. In vivo gene delivery with L-tyrosine polyphosphate nanoparticles. (United States)

    Ditto, Andrew J; Reho, John J; Shah, Kush N; Smolen, Justin A; Holda, James H; Ramirez, Rolando J; Yun, Yang H


    The concept of gene therapy is promising; however, the perceived risks and side effects associated with this technology have severely dampened the researchers' enthusiasm. Thus, the development of a nonviral gene vector without immunological effects and with high transfection efficiency is necessary. Currently, most nonviral vectors have failed to achieve the in vivo transfection efficiencies of viral vectors due to their toxicity, rapid clearance, and/or inappropriate release rates. Although our previous studies have successfully demonstrated the controlled-release of plasmid DNA (pDNA) polyplexes encapsulated into nanoparticles formulated with l-tyrosine polyphosphate (LTP-pDNA nanoparticles), the in vivo transfection capabilities and immunogenicity of this delivery system have yet to be examined. Thus, we evaluate LTP-pDNA nanoparticles in an in vivo setting via injection into rodent uterine tissue. Our results demonstrate through X-gal staining and immunohistochemistry of uterine tissue that transfection has successfully occurred after a nine-day incubation. In contrast, the results for the control nanoparticles show results similar to those of shams. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) from the injected tissues confirms the transfection in vivo. To examine the immunogenicity, the l-tyrosine polyphosphate (LTP) nanoparticles have been evaluated in a mouse model. No significant differences in the activation of the innate immune system are observed. These data provide the first report for the potential use of controlled-release nanoparticles formulated from an amino acid based polymer as an in vivo nonviral vector for gene therapy.

  19. A novel gene delivery system for mammalian cells. (United States)

    Gibson, Brian; Duffy, Angela M; Gould Fogerite, Susan; Krause-Elsmore, Sara; Lu, Ruying; Shang, Gaofeng; Chen, Zi-Wei; Mannino, Raphael J; Bouchier-Hayes, David J; Harmey, Judith H


    Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Non-viral efficacy of delivery remains quite poor. We are investigating the feasibility of a novel lipid-based delivery system, cochleates, to deliver transgenes to mammalian cells. Rhodamine-labelled empty cochleates were incubated with two cell-lines (4T1 adenocarcinoma and H36.12 macrophage hybridoma) and primary macrophages in vitro and in vivo. Cochleates containing green fluorescent protein (GFP) expression plasmid were incubated with 4T1 adenocarcinoma cells. Cellular uptake of labelled cochleates or transgene GFP expression were visualised with fluorescence microscopy. 4T1 and H36.12 lines showed 39% and 23.1% uptake of rhodamine-cochleates, respectively. Human monocyte-derived macrophages and mouse peritoneal macrophages had 48+/-5.38% and 51.46+/-15.6% uptake of rhodamine-cochleates in vitro. In vivo 25.69+/-0.127% of peritoneal macrophages were rhodamine-positive after intra-peritoneal injection of rhodamine-cochleates. 19.49+/-10.12% of 4T1 cells expressed GFP. Cochleates may therefore be an effective, non-toxic and non-immunogenic method to introduce transgenes in vitro and in vivo.

  20. 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:


    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.

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

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    Roya Cheraghi


    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.

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

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    Huang XZ


    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

  3. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing. (United States)

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


    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.

  4. Solid Lipid Nanoparticles as Efficient Drug and Gene Delivery Systems: Recent Breakthroughs

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    Jafar Ezzati Nazhad Dolatabadi


    Full Text Available In recent years, nanomaterials have been widely applied as advanced drug and gene delivery nanosystems. Among them, solid lipid nanoparticles (SLNs have attracted great attention as colloidal drug delivery systems for incorporating hydrophilic or lipophilic drugs and various macromolecules as well as proteins and nucleic acids. Therefore, SLNs offer great promise for controlled and site specific drug and gene delivery. This article includes general information about SLN structures and properties, production procedures, characterization. In addition, recent progress on development of drug and gene delivery systems using SLNs was reviewed.

  5. In vivo characteristics of cationic liposomes as delivery vectors for gene therapy

    NARCIS (Netherlands)

    Audouy, SAL; de Leij, LFMH; Hoekstra, D; Molema, G


    After a decade of clinical trials, gene therapy seems to have found its place between excessive ambitions and feasible aims, with encouraging results obtained in recent years. Intracellular delivery of genetic material is the key step in gene therapy. Optimization of delivery vectors is of major imp

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

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    Marxa Figueiredo


    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.

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

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    Sante Di Gioia


    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

  8. Intrinsic Bio-Signature of Gene Delivery Nanocarriers May Impair Gene Therapy Goals

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    Jaleh Barar


    Full Text Available Non-viral lipid/polymeric vectors have widely been used as nanocarriers (NCs for gene delivery. They possess large surface area to volume ratio and are able to interact with biomolecules through functional moieties, resulting in inadvertent biological impacts, in particular at genomic level. Thus, their genomic bio-signature needs to be investigated prior to use in vivo. Using high-throughput microarray and qPCR gene expression profiling techniques, we have reported the genomic impacts of lipid/polymeric NCs. Given the fact that the ultimate objectives of gene therapy may inevitably be impaired by nonspecific intrinsic genomic impacts of these NCs, here, we highlight their nonspecific genomic bio-signature. We envision that better understanding on the genotoxicity of gene delivery NCs, as guiding premise, will help us to develop much safer NCs and also to accelerate their translation into clinical use and to provide pivotal information on safety liabilities early in discovery and developments process prior to its inevitable consequences in vivo.

  9. Design of Dendrimer Modified Carbon Nanotubes for Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    PAN Bi-feng; BAO Chen-chen; GAO Feng; HE Rong; SHU Meng-jun; MA Yong-jie; CUI Da-xiang; XU Ping; CHEN Hao; LIU Feng-tao; LI Qing; HUANG Tuo; YOU Xiao-gang; SHAO Jun


    Objective: To investigate the efficiency of polyamidoamine dendrimer grafted carbon nanotube (dendrimer-CNT) mediated entrance of anti-survivin oligonucleotide into MCF-7 cells, and its effects on the growth of MCF-7 cells. Methods: Antisense survivin oligonucleotide was anchored onto polyamidoamine dendrimer grafted carbon nanotubes to form dendrimer-CNT-asODN complex and the complex was characterized by Zeta potential, AFM, TEM, and 1% agarose gel electrophoresis analysis. Dendrimer-CNT-asODN complexes were added into the medium and incubated with MCF-7 cells. MTT method was used to detect the effects of asODN and dendrimer-CNT-asODN on the growth of MCF-7 cells. TEM was used to observe the distribution of dendrimer-CNT-asODN complex within MCF-7 cells. Results: Successful synthesis of dendrimer-CNT-asODN complexes was proved by TEM, AFM and agarose gel electrophoresis. TEM showed that the complexes were located in the cytoplasm, endosome, and lysosome within MCF-7 cells. When dendrimer-CNT-asODN (1.0 μmol/L) and asODN (1.0 μmol/L) were used for 120 h incubation, the inhibitory rates of MCF-7 cells were (28.22±3.5)% for dendrimer-CNT-asODN complex group, (9.23±0.56)% for only asODN group, and (3.44±0.25)% for dendrimer-CNT group. Dendrimer-CNT-asODN complex at 3.0 μmol/L inhibited MCF-7 cells by (30.30±10.62)%, and the inhibitory effects were in a time- and concentration- dependent manner. Conclusion: Dendrimer-CNT nanoparticles may serve as a gene delivery vector with high efficiency, which can bring foreign gene into cancer cells, inhibiting cancer cell proliferation and markedly enhancing the cancer therapy effects.

  10. Nonviral gene delivery systems by the combination of bubble liposomes and ultrasound. (United States)

    Omata, Daiki; Negishi, Yoichi; Suzuki, Ryo; Oda, Yusuke; Endo-Takahashi, Yoko; Maruyama, Kazuo


    The combination of therapeutic ultrasound (US) and nano/microbubbles is an important system for establishing a novel and noninvasive gene delivery system. Genes are delivered more efficiently using this system compared with a conventional nonviral vector system such as the lipofection method, resulting in higher gene expression. This higher efficiency is due to the gene being delivered into the cytosol and bypassing the endocytosis pathway. Many in vivo studies have demonstrated US-mediated gene delivery with nano/microbubbles, and several gene therapy feasibility studies for various diseases have been reported. In addition, nano/microbubbles can deliver genes site specifically by the control of US exposure site. In the present review, we summarize the gene delivery systems by the combination of nano/microbubbles and US, describe their properties, and assess applications and challenges of US theranostics.


    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL


    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

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

    Directory of Open Access Journals (Sweden)

    Kenya Kamimura


    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.

  13. Baculovirus as a gene delivery vector for cartilage and bone tissue engineering. (United States)

    Lin, Chin-Yu; Lu, Chia-Hsin; Luo, Wen-Yi; Chang, Yu-Han; Sung, Li-Yu; Chiu, Hsin-Yi; Hu, Yu-Chen


    Baculovirus is an effective vector for gene delivery into various mammalian cells, including chondrocytes and mesenchymal stem cells, and has been employed for diverse applications. By gene delivery and expression of the growth factor, recombinant baculovirus has been shown to modulate the differentiation state of the cells and stimulates the production of extracellular matrix and tissue formation, hence repairing the damaged cartilage and bone in vivo. This article reviews the studies pertaining to the applications of baculovirus-mediated gene delivery in cartilage and bone tissue engineering and discusses recent progress, future applications and potential hurdles.

  14. Gene expression and antitumor effect following imelectroporation delivery of human interferon α2 gene

    Institute of Scientific and Technical Information of China (English)

    ZHANGGuo-Hua; TANXiao-Fan; SHENDong; ZHAOShu-Yuan; SHIYan-Yi; JINCai-Ke; SUNWei-Gu; GUOYan-Hong; CHENKuang-Hueih; TANGJian


    AIM: To investigate the gene expression and antitumor effect following im electroporation delivery of humaninterferon α2 (hlFN-α2) gene. METHODS: The pcD2/hIFN-α2 was injected into the middle of the quadricepsmuscle of female BALB/c mice or the leukemia-bearing female BALB/c nude mice, and then electroporation wasgiven to the injection site. Optimal electrical parameters and the efficiency of gene transfer was studied with hlFNα2 ELISA kit. The HL-60 tumor model in BALB/c nude mice was used to investigate therapeutic effects of imelectroporation delivery of pcD2/hlFN-α2. RESULTS: The optimal conditions for the electric pulses were asfollows: voltage at 200 V/cm; pulse duration at 40 ms per pulse; number of pulse at 6 pulses and frequency at 1 Hz.Under optimal conditions, the serum hlFN-α2 levels in electroporation group (160μg/L±31 μg/L) were 45-foldhigher than those of nonelectroporation group (3.6μg/L±1.6μg/L, P<0.01). The growth of leukemia was inhibitedmore obviously and the survival time of the leukemia-bearing nude mice was prolonged after im electroporationdelivery of pcD2/hlFN-α2 100μg or 200μg. CONCLUSION: Electroporation was an efficient method for thedelivery of plasmid DNA and im electroporation delivery of pcDz/hlFN-α2 was effective in treating leukemia.

  15. In Vivo Delivery of CRISPR/Cas9 for Therapeutic Gene Editing: Progress and Challenges. (United States)

    Mout, Rubul; Ray, Moumita; Lee, Yi-Wei; Scaletti, Federica; Rotello, Vincent M


    The successful use of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based gene editing for therapeutics requires efficient in vivo delivery of the CRISPR components. There are, however, major challenges on the delivery front. In this Topical Review, we will highlight recent developments in CRISPR delivery, and we will present hurdles that still need to be overcome to achieve effective in vivo editing.

  16. Complexation of oppositely charged polyelectrolytes in gene delivery and biology (United States)

    Shklovskii, Boris


    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 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.

  17. Advanced drug and gene delivery systems based on functional biodegradable polycarbonates and copolymers

    NARCIS (Netherlands)

    Chen, Wei; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z.


    Biodegradable polymeric nanocarriers are one of the most promising systems for targeted and controlled drug and gene delivery. They have shown several unique advantages such as excellent biocompatibility, prolonged circulation time, passive tumor targeting via the enhanced permeability and retention

  18. 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)


    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.

  19. Evaluation and optimization of chitosan derivatives-based gene delivery system via kidney epithelial cells

    Directory of Open Access Journals (Sweden)

    S. Safari


    Full Text Available Purpose: Non-viral vectors have been widely proposed as safer alternatives to viral vectors, and cationic polymers have gained increasing attention because they can form self-assembly with DNA. Chitosan is also considered to be a good candidate for gene delivery systems, since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic potential. However, low solubility and transfection efficiency need to be overcome prior to clinical trial. In this work, we focus on alkyl modified chitosan which might be useful in DNA condensing and efficient gene delivery. Methods: N, N- Diethyl N- Methyl (DEMC and N- Triethyl Chitosan (TEC were synthesized from chitosan polymer. In order to optimize the polymers for gene delivery, we used FITC-dextran (FD. Then the optimized polymer concentrations were used for gene delivery. Fluorescent microscope was used, in order to evaluate the polymers’ efficiency for gene delivery to human embryonic kidney epithelial cells (HEK 293T. Results: This modification increased chitosan’s positive charge, thus these chitosan derivatives spontaneously formed complexes with FD, green fluorescence protein plasmid DNA (pEGFP, red fluorescence protein plasmid DNA (pJred and fluorescent labeled miRNA. Results gained from fluorescent microscope showed that TEC and DEMC were able to transfer FD, DNA and miRNA (micro RNA to HEK cell line. Conclusion: We conclude that these chitosan derivatives present suitable characteristics to be used as non-viral gene delivery vectors to epithelial cells.

  20. Electroporation-mediated delivery of genes in rodent models of lung contusion. (United States)

    Machado-Aranda, David; Raghavendran, Krishnan


    Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated gene delivery to the lung. Electroporation is a highly efficient and easily reproducible technique that allows circumvention of several of lung gene delivery challenges and safety issues present with other forms of lung gene therapy.

  1. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers. (United States)

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin


    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  2. Parameters Affecting Image-guided, Hydrodynamic Gene Delivery to Swine Liver

    Directory of Open Access Journals (Sweden)

    Kenya Kamimura


    Full Text Available Development of a safe and effective method for gene delivery to hepatocytes is a critical step toward gene therapy for liver diseases. Here, we assessed the parameters for gene delivery to the livers of large animals (pigs, 40–65 kg using an image-guided hydrodynamics-based procedure that involves image-guided catheter insertion into the lobular hepatic vein and hydrodynamic injection of reporter plasmids using a computer-controlled injector. We demonstrated that injection parameters (relative position of the catheter in the hepatic vasculature, intravascular pressure upon injection, and injection volume are directly related to the safety and efficiency of the procedure. By optimizing these parameters, we explored for the first time, the advantage of the procedure for sequential injections to multiple lobes in human-sized pigs. The optimized procedure resulted in sustained expression of the human α-1 antitrypsin gene in livers for more than 2 months after gene delivery. In addition, repeated hydrodynamic gene delivery was safely conducted and no adverse events were seen in the entire period of the study. Our results support the clinical applicability of the image-guided hydrodynamic gene delivery method for the treatment of liver diseases.

  3. Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness. (United States)

    Keeney, Michael; Onyiah, Sheila; Zhang, Zhe; Tong, Xinming; Han, Li-Hsin; Yang, Fan


    Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.

  4. Gene delivery with cationic lipids : fundamentals and potential applications

    NARCIS (Netherlands)

    Wasungu, Luc Bakomma


    Principle of gene therapy. Although the objectives and principles of gene therapy have been well-defined over the last decades, its application as a versatile, therapeutically successful approach has not yet met expectations. At the onset, the primary goal of gene therapy was to replace a deficient

  5. Gene delivery with cationic lipids : fundamentals and potential applications

    NARCIS (Netherlands)

    Wasungu, Luc Bakomma


    Principle of gene therapy. Although the objectives and principles of gene therapy have been well-defined over the last decades, its application as a versatile, therapeutically successful approach has not yet met expectations. At the onset, the primary goal of gene therapy was to replace a deficient

  6. Gene delivery with cationic lipids : fundamentals and potential applications

    NARCIS (Netherlands)

    Wasungu, L.B.


    Principle of gene therapy.Although the objectives and principles of gene therapy have been well-defined over the last decades, its application as a versatile, therapeutically successful approach has not yet met expectations. At the onset, the primary goal of gene therapy was to replace a deficient g

  7. Electroporation for drug and gene delivery in the clinic: doctors go electric

    DEFF Research Database (Denmark)

    Gehl, J.


    Electroporation is a unique system for drug and gene delivery, as it is possible to very specifically target certain tissues within the body with whatever drug, gene, isotope, or other product is desired in a specific situation. An increasing number of clinical trials are being launched, and soph...

  8. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing. (United States)

    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin


    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  9. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  11. Self-assembled pentablock copolymers for selective and sustained gene delivery (United States)

    Zhang, Bingqi

    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. Gene transfer to hemophilia A mice via oral delivery of FVIII-chitosan nanoparticles. (United States)

    Bowman, Katherine; Sarkar, Rita; Raut, Sanj; Leong, Kam W


    Effective oral delivery of a non-viral gene carrier would represent a novel and attractive strategy for therapeutic gene transfer. To evaluate the potential of this approach, we studied the oral gene delivery efficacy of DNA polyplexes composed of chitosan and Factor VIII DNA. Transgene DNA was detected in both local and systemic tissues following oral administration of the chitosan nanoparticles to hemophilia A mice. Functional factor VIII protein was detected in plasma by chromogenic and thrombin generation assays, reaching a peak level of 2-4% FVIII at day 22 after delivery. In addition, a bleeding challenge one month after DNA administration resulted in phenotypic correction in 13/20 mice given 250-600 microg of FVIII DNA in chitosan nanoparticles, compared to 1/13 mice given naked FVIII DNA and 0/6 untreated mice. While further optimization would be required to render this type of delivery system practical for hemophilia A gene therapy, the findings suggest the feasibility of oral, non-viral delivery for gene medicine applications.

  13. DOPC-Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery. (United States)

    Pierrat, Philippe; Casset, Anne; Kereselidze, Dimitri; Lux, Marie; Pons, Françoise; Lebeau, Luc


    Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments.

  14. DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy. (United States)

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


    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.

  15. Lipid Phases Eye View to Lipofection. Cationic Phosphatidylcholine Derivatives as Efficient DNA Carriers for Gene Delivery

    Directory of Open Access Journals (Sweden)

    Rumiana Koynova


    Full Text Available Efficient delivery of genetic material to cells is needed for tasks of utmost importance in laboratory and clinic, such as gene transfection and gene silencing. Synthetic cationic lipids can be used as delivery vehicles for nucleic acids and are now considered the most promising non-viral gene carriers. They form complexes (lipoplexes with the polyanionic nucleic acids. A critical obstacle for clinical application of the lipid-mediated DNA delivery (lipofection is its unsatisfactory efficiency for many cell types. Understanding the mechanism of lipid-mediated DNA delivery is essential for their successful application, as well as for rational design and synthesis of novel cationic lipoid compounds for enhanced gene delivery. According to the current understanding, the critical factor in lipid-mediated transfection is the structural evolution of lipoplexes within the cell, upon interacting and mixing with cellular lipids. In particular, recent studies with cationic phospha- tidylcholine derivatives showed that the phase evolution of lipoplex lipids upon interaction and mixing with membrane lipids appears to be decisive for transfection success: specifically, lamellar lipoplex formulations, which were readily susceptible to undergoing lamellar-nonlamellar (precisely lamellar-cubic phase transition upon mixing with cellular lipids, were found rather consistently associated with superior transfection potency, presumably as a result of facilitated DNA release subsequent to lipoplex fusion with the cellular membranes. Further, hydrophobic moiety of the cationic phospholipids was found able to strongly modulate liposomal gene delivery into primary human umbilical artery endothelial cells; superior activity was found for cationic phosphatidylcholine derivatives with two 14-carbon atom monounsaturated hydrocarbon chains, able to induce formation of cubic phase in membranes. Thus, understanding the lipoplex structure and the phase changes upon interacting

  16. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing. (United States)

    Liu, Jia; Shui, Sai-Lan


    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.

  17. A sight on protein-based nanoparticles as drug/gene delivery systems. (United States)

    Salatin, Sara; Jelvehgari, Mitra; Maleki-Dizaj, Solmaz; Adibkia, Khosro


    Polymeric nanomaterials have extensively been applied for the preparation of targeted and controlled release drug/gene delivery systems. However, problems involved in the formulation of synthetic polymers such as using of the toxic solvents and surfactants have limited their desirable applications. In this regard, natural biomolecules including proteins and polysaccharide are suitable alternatives due to their safety. According to literature, protein-based nanoparticles possess many advantages for drug and gene delivery such as biocompatibility, biodegradability and ability to functionalize with targeting ligands. This review provides a general sight on the application of biodegradable protein-based nanoparticles in drug/gene delivery based on their origins. Their unique physicochemical properties that help them to be formulated as pharmaceutical carriers are also discussed.

  18. Structure-function investigations of DNA condensing agents with application to gene delivery (United States)

    Evans, Heather Marie

    Lipid-based systems are notoriously poor for gene delivery, and their use has been primarily empirical. In order to improve these systems, it is imperative to obtain a greater understanding of molecular interactions between DNA and positively charged molecules. A variety of cationic molecules have been studied with DNA, in an attempt to correlate structural properties of these assemblies (using x-ray diffraction) with their efficiency as DNA carriers for gene delivery (using a luciferase assay). Several systems have been studied, some of which use the same charged amine moieties presented in three distinct morphologies: the multivalent salts spermine and spermidine, dendrimers, and dendrimeric lipids. The dendrimers somewhat approximate the properties of histories, cylindrical proteins that condense intracellular DNA. Structural studies of histone and DNA have also been conducted in order to better understand these interactions and their possible relevance to the gene delivery pathway. In addition, empirical evidence suggests that for successful in vivo gene delivery, cholesterol should be used as a helper lipid. The delivery efficiency and structural behavior of cholesterol and other sterol molecules have been studied in ternary lipid mixtures.

  19. An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway

    Directory of Open Access Journals (Sweden)

    Luo J


    Full Text Available Jing Luo,1,2,* Caixia Li,3,* Jianlin Chen,1,2 Gang Wang,2 Rong Gao,1 Zhongwei Gu2 1Key Laboratory for Bio-Resource and Eco-Environment of Ministry of Education, Key Laboratory for Animal Disease Prevention and Food Safety of Sichuan Province, College of Life Science, Sichuan University, Chengdu, People’s Republic of China; 2National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, People’s Republic of China; 3Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, People’s Republic of China *These authors contributed equally to this work Abstract: Transfection efficiency was the primary goal for in vitro gene delivery mediated by nonviral gene carriers. Here, we report a modified gene transfection method that could greatly increase the efficiency of, and accelerate the process mediated by, 25 kDa branched polyethyleneimine and Lipofectamine™ 2000 in a broad range of cell strains, including tumor, normal, primary, and embryonic stem cells. In this method, the combination of transfection procedure with optimized complexation volume had a determinant effect on gene delivery result. The superiorities of the method were found to be related to the change of cellular endocytosis pathway and decrease of particle size. The efficient and simple method established in this study can be widely used for in vitro gene delivery into cultured cells. We think it may also be applicable for many more nonviral gene delivery materials than polyethyleneimine and liposome. Keywords: gene delivery, gene expression, endocytosis, polyethyleneimine, Lipofectamine™ 2000

  20. Advances in liver-directed gene therapy for hepatocellular carcinoma by non-viral delivery systems. (United States)

    Ding, Buyun; Li, Tao; Zhang, Jian; Zhao, Lixia; Zhai, Guangxi


    Hepatocellular carcinoma (HCC) is a malignancy with a high mortality. Gene therapy provides a promising way for the treatment of HCC. Efficient gene delivery system, suitable gene target and appropriate way of administration together determine the effect of gene therapy for HCC. In recent years, employing non-viral gene delivery systems in gene therapy for HCC has attracted a lot of attention. Compared with viral vectors, non-viral gene delivery systems are nearly non-immunogenic, relatively safer, less expensive to produce and can carry a good many of genetic materials. But the transfection efficiency of these vectors still needs to be improved. And the liver targeting is another problem that needs to be solved. Attaching ligands to the non-viral vectors to enhance the targeting ability to the specific receptor and targeting to molecular targets of HCC are the effective strategies. Adopting suitable ways of administration is also a factor that plays an important role to achieve liver targeting. This review introduced the advances in liver-targeted gene therapy by non-viral vectors including the efforts to overcome the low transfection efficiency and enhance the liver targeting effect.

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

  2. Structure-activity relationship of dendrimers engineered with twenty common amino acids in gene delivery. (United States)

    Wang, Fei; Hu, Ke; Cheng, Yiyun


    Systematic explorations on the structure-activity relationship of surface-engineered dendrimers are essential to design high efficient and safe gene vectors. The chemical diversity of residues in naturally occurring amino acids allows us to generate a library of dendrimers with various surface properties. Here, we synthesized a total number of 40 dendrimers engineered with the twenty common amino acids and investigated their performances in gene delivery. The results show that gene transfection efficacy of the synthesized materials depends on both the type of amino acid and the conjugation ratio. Dendrimers engineered with cationic and hydrophobic amino acids possess relatively higher transfection efficacies. Engineering dendrimers with cationic amino acids such as arginine and lysine facilitates polyplex formation and cellular uptake, with histidine improves endosomal escape of the polyplexes, and with hydrophobic amino acids such as tyrosine and phenylalanine modulates the balance between hydrophobicity and hydrophilicity on dendrimer surface, which is beneficial for efficient cellular internalization. Dendrimers engineered with anionic or hydrophilic amino acids show limited transfection efficacy due to poor DNA binding capacity and/or limited cellular uptake. In the aspect of cytotoxicity, dendrimers engineered with arginine, lysine, tyrosine, phenylalanine and tryptophan show much higher cytotoxicity than other engineered dendrimers. These results are helpful for us to tailor the surface chemistry of dendrimers for efficient gene delivery. Cationic polymers such as dendrimers were widely used as gene vectors but are limited by relatively low delivery efficacy and high toxicity. To achieve efficient and low toxic gene delivery, the polymers were modified with various ligands. However, these ligand-modified polymers in gene delivery are reported by independent researchers using different polymer scaffolds and cell lines. It is hard to provide structure

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

    LENUS (Irish Health Repository)

    Rajendran, Simon


    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

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

    LENUS (Irish Health Repository)

    Rajendran, Simon


    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

  5. Non-viral gene delivery strategies for gene therapy: a "ménage à trois" among nucleic acids, materials, and the biological environment. Stimuli-responsive gene delivery vectors (United States)

    Pezzoli, Daniele; Candiani, Gabriele


    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.

  6. DNA Ministrings: Highly Safe and Effective Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Nafiseh Nafissi


    Full Text Available Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA—mini linear covalently closed DNA vectors—that are devoid of unwanted bacterial sequences and encode only the gene(s of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.

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

    KAUST Repository

    Alsaiari, Shahad K.


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

    Directory of Open Access Journals (Sweden)

    Hoda Y. Henein


    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.

  9. Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems. (United States)

    Bakhshinejad, Babak; Sadeghizadeh, Majid


    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.

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

    NARCIS (Netherlands)

    van Gaal, E.V.B.|info:eu-repo/dai/nl/30483629X


    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

  11. A novel receptor-targeted gene delivery system for cancer gene therapy

    Institute of Scientific and Technical Information of China (English)

    田培坤; 任圣俊; 任常春; 滕青山; 曲淑敏; 姚明; 顾健人


    Some growth factor receptors, such as insulin like growth factor Ⅰ and Ⅱ receptor (IGF Ⅰ R, IGF Ⅱ R) and epidermal growth factor receptor (EGF R), have been proved to be over-expressed in a variety of human cancers derived from different tissue origins. Based on this molecular alteration, a polypeptide conjugate gene delivery system was designed and synthesized. It contains three essential moieties: a ligand oligopeptide (LOP) for receptor recognition, a polycationic polypeptide (PCP) such as protamine (PA) or poly-L-lysine (PL) as a backbone for DNA binding and an endosome-releasing oligopeptide (EROP) such as influenza baenagglutinin oligopeptide (HA20) for endosomolysis. These components are covalently conjugated as LOP-PCP-HA20 or in the form of a mixture of LOP-PCP and HA20-PCP. A 14 amino acid E5 was designed and synthesized as LOP for IGF Ⅰ R and IGF Ⅱ R, and a 16 amino acid GE7 as LOP for EGF R. Both E5 and GE7 systems could form stable complex with the plasmid DNA as E5-PCP/DNA/PCP-HA20 a

  12. Poly(ethylene glycol)-block-cationic polylactide nanocomplexes of differing charge density for gene delivery. (United States)

    Chen, Chih-Kuang; Jones, Charles H; Mistriotis, Panagiotis; Yu, Yun; Ma, Xiaoni; Ravikrishnan, Anitha; Jiang, Ming; Andreadis, Stelios T; Pfeifer, Blaine A; Cheng, Chong


    Representing a new type of biodegradable cationic block copolymer, well-defined poly(ethylene glycol)-block-cationic polylactides (PEG-b-CPLAs) with tertiary amine-based cationic groups were synthesized by thiol-ene functionalization of an allyl-functionalized diblock precursor. Subsequently the application of PEG-b-CPLAs as biodegradable vectors for the delivery of plasmid DNAs (pDNAs) was investigated. Via the formation of PEG-b-CPLA:pDNA nanocomplexes by spontaneous electrostatic interaction, pDNAs encoding luciferase or enhanced green fluorescent protein were successfully delivered to four physiologically distinct cell lines (including macrophage, fibroblast, epithelial, and stem cell). Formulated nanocomplexes demonstrated high levels of transfection with low levels of cytotoxicity and hemolysis when compared to a positive control. Biophysical characterization of charge densities of nanocomplexes at various polymer:pDNA weight ratios revealed a positive correlation between surface charge and gene delivery. Nanocomplexes with high surface charge densities were utilized in an in vitro serum gene delivery inhibition assay, and effective gene delivery was observed despite high levels of serum. Overall, these results help to elucidate the influence of charge, size, and PEGylation of nanocomplexes upon the delivery of nucleic acids in physiologically relevant conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Self-Assembled Fluorodendrimers Combine the Features of Lipid and Polymeric Vectors in Gene Delivery. (United States)

    Wang, Hui; Wang, Yitong; Wang, Yu; Hu, Jingjing; Li, Tianfu; Liu, Hongmei; Zhang, Qiang; Cheng, Yiyun


    An ideal vector in gene therapy should exhibit high serum stability, excellent biocompatibility, a desired transfection efficacy and permeability into targeted tissues. Here, we describe a class of low-molecular-weight fluorodendrimers for efficient gene delivery. These materials self-assemble into uniform nanospheres and allow for efficient transfection at low charge ratios and very low DNA doses with minimal cytotoxicity. Our results demonstrate that these vectors combine the features of synthetic gene vectors such as liposomes and cationic polymers and present promising potential for clinical gene therapy.

  14. A novel gene delivery system targeting cells expressing VEGF receptors

    Institute of Scientific and Technical Information of China (English)



    Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.

  15. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers

    Energy Technology Data Exchange (ETDEWEB)

    Igor I. Slowing; Juan L. Viveo-Escoto; Chia-Wen Wu; Victor S. Y. Lin


    In this review, we highlight the recent research developments of a series of surface-functionalized mesoporous silica nanoparticle (MSN) materials as efficient drug delivery carriers. The synthesis of this type of MSN materials is described along with the current methods for controlling the structural properties and chemical functionalization for biotechnological and biomedical applications. We summarized the advantages of using MSN for several drug delivery applications. The recent investigations of the biocompatibility of MSN in vitro are discussed. We also describe the exciting progress on using MSN to penetrate various cell membranes in animal and plant cells. The novel concept of gatekeeping is introduced and applied to the design of a variety of stimuli-responsive nanodevices. We envision that these MSN-based systems have a great potential for a variety of drug delivery applications, such as the site-specific delivery and intracellular controlled release of drugs, genes, and other therapeutic agents.

  16. Lipopolyplex for Therapeutic Gene Delivery and Its Application for the Treatment of Parkinson's Disease. (United States)

    Chen, Wei; Li, Hui; Liu, Zhenguo; Yuan, Weien


    Lipopolyplex is a core-shell structure composed of nucleic acid, polycation and lipid. As a non-viral gene delivery vector, lipopolyplex combining the advantages of polyplex and lipoplex has shown superior colloidal stability, reduced cytotoxicity, extremely high gene transfection efficiency. Following intravenous administration, there are many strategies based on lipopolyplex to overcome the complex biological barriers in systemic gene delivery including condensation of nucleic acids into nanoparticles, long circulation, cell targeting, endosomal escape, release to cytoplasm and entry into cell nucleus. Parkinson's disease (PD) is the second most common neurodegenerative disorder and severely influences the patients' life quality. Current gene therapy clinical trials for PD employing viral vectors didn't achieve satisfactory efficacy. However, lipopolyplex may become a promising alternative approach owing to its stability in blood, ability to cross the blood-brain barrier (BBB) and specific targeting to diseased brain cells.

  17. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation. (United States)

    Rincon, Melvin Y; VandenDriessche, Thierry; Chuah, Marinee K


    Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality.

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

    Directory of Open Access Journals (Sweden)

    Mathieu eBOURDENX


    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. Ultrasound-Mediated Drug/Gene Delivery in Solid Tumor Treatment

    Directory of Open Access Journals (Sweden)

    Yufeng Zhou


    Full Text Available Ultrasound is an emerging modality for drug delivery in chemotherapy. This paper reviews this novel technology by first introducing the designs and characteristics of three classes of drug/gene vehicles, microbubble (including nanoemulsion, liposomes, and micelles. In comparison to conventional free drug, the targeted drug-release and delivery through vessel wall and interstitial space to cancerous cells can be activated and enhanced under certain sonication conditions. In the acoustic field, there are several reactions of these drug vehicles, including hyperthermia, bubble cavitation, sonoporation, and sonodynamics, whose physical properties are illustrated for better understanding of this approach. In vitro and in vivo results are summarized, and future directions are discussed. Altogether, ultrasound-mediated drug/gene delivery under imaging guidance provides a promising option in cancer treatment with enhanced agent release and site specificity and reduced toxicity.

  20. 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.

  1. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5 (United States)

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad


    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. PMID:20927353

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

    NARCIS (Netherlands)

    Martens, T.F.; Remaut, K.; Deschout, H.; Engbersen, Johannes F.J.; Hennink, W.E.; van Steenbergen, M.J.; Demeester, J.; de Smedt, S.C.; Braeckmans, K.


    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

  3. Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA

    NARCIS (Netherlands)

    Eefting, D.; Grimbergen, J.M.; Vries, M.R. de; Weel, V. van; Kaijzel, E.L.; Que, I.; Moon, R.T.; Löwik, C.W.; Bockel, J.H. van; Quax, P.H.A.


    For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a pl

  4. Cationic surface modification of PLG nanoparticles offers sustained gene delivery to pulmonary epithelial cells. (United States)

    Baoum, Abdulgader; Dhillon, Navneet; Buch, Shilpa; Berkland, Cory


    Biodegradable polymeric nanoparticles are currently being explored as a nonviral gene delivery system; however, many obstacles impede the translation of these nanomaterials. For example, nanoparticles delivered systemically are inherently prone to adsorbing serum proteins and agglomerating as a result of their large surface/volume ratio. What is desired is a simple procedure to prepare nanoparticles that may be delivered locally and exhibit minimal toxicity while improving entry into cells for effectively delivering DNA. The objective of this study was to optimize the formulation of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles for gene delivery performance to a model of the pulmonary epithelium. Using a simple solvent diffusion technique, the chemistry of the particle surface was varied by using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (approximately 200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for 2 weeks. In A549 alveolar lung epithelial cells, high levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least 2 weeks. In contrast, PEI gene expression ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium.

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

    DEFF Research Database (Denmark)

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


    Electroporation designates the use of electric pulses to transiently permeabilize the cell membrane. It has been shown that DNA can be transferred to cells through a combined effect of electric pulses causing (1) permeabilization of the cell membrane and (2) an electrophoretic effect on DNA...... 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. CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene Delivery and Promotion of Angiogenesis in HUVECs in Vivo. (United States)

    Yang, Jing; Hao, Xuefang; Li, Qian; Akpanyung, Mary; Nejjari, Abdelilah; Neve, Agnaldo Luis; Ren, Xiangkui; Guo, Jintang; Feng, Yakai; Shi, Changcan; Zhang, Wencheng


    Gene therapy is a promising strategy for angiogenesis, but developing gene carriers with low cytotoxicity and high gene delivery efficiency in vivo is a key issue. In the present study, we synthesized the CAGW peptide- and poly(ethylene glycol) (PEG)-modified amphiphilic copolymers. CAGW peptide serves as a targeting ligand for endothelial cells (ECs). Different amounts of CAGW peptide were effectively conjugated to the amphiphilic copolymer via heterofunctional poly(ethylene glycol). These CAG- and PEG-modified copolymers could form nanoparticles (NPs) by self-assembly method and were used as gene carriers for the pEGFP-ZNF580 (pZNF580) plasmid. CAGW and PEG modification coordinately improved the hemocompatibility and cytocompatibility of NPs. The results of cellular uptake showed significantly enhanced internalization efficiency of pZNF580 after CAGW modification. Gene expression at mRNA and protein levels demonstrated that EC-targeted NPs possessed high gene delivery efficiency, especially the NPs with higher content of CAGW peptide (1.16 wt %). Furthermore, in vitro and in vivo vascularization assays also showed outstanding vascularization ability of human umbilical vein endothelial cells treated by the NP/pZNF580 complexes. This study demonstrates that the CAGW peptide-modified NP is a promising candidate for gene therapy in angiogenesis.

  7. Lipid nanoparticles as drug/gene delivery systems to the retina. (United States)

    del Pozo-Rodríguez, Ana; Delgado, Diego; Gascón, Alicia R; Solinís, Maria Ángeles


    This review highlights the application of lipid nanoparticles (Solid Lipid Nanoparticles, Nanostructured Lipid Carriers, or Lipid Drug Conjugates) as effective drug/gene delivery systems for retinal diseases. Most drug products for ocular disease treatment are marketed as eye drop formulations but, due to ocular barriers, the drug concentration in the retina hardly ever turns out to be effective. Up to this date, several delivery systems have been designed to deliver drugs to the retina. Drug delivery strategies may be classified into 3 groups: noninvasive techniques, implants, and colloidal carriers. The best known systems for drug delivery to the posterior eye are intravitreal implants; in fact, some of them are being clinically used. However, their long-term accumulation might impact the patient's vision. On the contrary, colloidal drug delivery systems (microparticles, liposomes, or nanoparticles) can be easily administered in a liquid form. Nanoparticular systems diffuse rapidly and are better internalized in ocular tissues than microparticles. In comparison with liposomes, nanoparticles have a higher loading capacity and are more stable in biological fluids and during storage. In addition, their capacity to adhere to the ocular surface and interact with the endothelium makes these drug delivery systems interesting as new therapeutic tools in ophthalmology. Within the group of nanoparticles, those composed of lipids (Solid Lipid Nanoparticles, Nanostructred Lipid Carriers, and Lipid Drug Conjugates) are more biocompatible, easy to produce at large scale, and they may be autoclaved or sterilized. The present review summarizes scientific results that evidence the potential application of lipid nanoparticles as drug delivery systems for the retina and also as nonviral vectors in gene therapy of retina disorders, although much more effort is still needed before these lipidic systems could be available in the market.

  8. [Melanoma: surface markers as the first point of targeted delivery of therapeutic genes in multilevel gene therapy]. (United States)

    Pleshkan, V V; Zinov'eva, M V; Sverdlov, E D


    Melanoma is one of the most malignant tumors, aggressively metastasizing by lymphatic and hematogenous routes. Due to the resistance of melanoma cells to many types of chemotherapy, this disease causes high mortality rate. High hopes are pinned on gene therapeutic approaches to melanoma treatment. At present, one of the main problems of the efficient use of the post-genomic generation therapeutic means is the lack of optimal techniques of delivery of foreign genetic material to the patient's target cells. Surface specific markers of melanoma cells can be considered as promising therapeutic targets. This review describes currently known melanoma specific receptors and its stem cells, as well as contains data on melanoma antigens presented on the cell surface by major histocompatibility complex proteins. The ability of surface proteins to internalize might be successfully used for the development of methods of targeted delivery of gene therapeutic constructs. In conclusion, a concept of multilevel gene therapy and the possible role therein of surface determinants as targets of gene systems delivery to the tumor are discussed.

  9. Macrophage mannose receptor-specific gene delivery vehicle for macrophage engineering. (United States)

    Ruan, Gui-Xin; Chen, Yu-Zhe; Yao, Xing-Lei; Du, Anariwa; Tang, Gu-Ping; Shen, You-Qing; Tabata, Yasuhiko; Gao, Jian-Qing


    Macrophages are the most plastic cells in the hematopoietic system and they exhibit great functional diversity. They have been extensively applied in anti-inflammatory, anti-fibrotic and anti-cancer therapies. However, the application of macrophages is limited by the efficiency of their engineering. The macrophage mannose receptor (MMR, CD206), a C-type lectin receptor, is ubiquitously expressed on macrophages and has a high affinity for mannose oligosaccharides. In the present study, we developed a novel non-viral vehicle with specific affinity for MMR. Mannan was cationized with spermine at a grafted ratio of ∼12% to deliver DNA and was characterized as a stable system for delivery. This spermine-mannan (SM)-based delivery system was evaluated as a biocompatible vehicle with superior transfection efficiency on murine macrophages, up to 28.5-fold higher than spermine-pullulan, 11.5-fold higher than polyethylenimine and 3.0-fold higher than Lipofectamine™ 2000. We confirmed that the SM-based delivery system for macrophages transfection was MMR-specific and we described the intracellular transport of the delivery system. To our knowledge, this is the first study using SM to demonstrate a mannose receptor-specific gene delivery system, thereby highlighting the potential of a novel specific non-viral delivery vehicle for macrophage engineering.

  10. Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation. (United States)

    Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei


    Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

  11. 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.

  12. Current strategies in modification of PLGA-based gene delivery system. (United States)

    Ramezani, Mohammad; Ebrahimian, Mahboubeh; Hashemi, Maryam


    The successful gene therapy has been limited by safe and efficient delivery of nucleic acid to the target cells. Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) are able to deliver drugs and gene efficiently. This formulation has several advantages in comparison with other formulations including improvement of solubility, stability, controlling of degradation and release of the entrapped agents. For application of PLGA as gene carrier, there exist many challenges. PLGA nanoparticles could protect the encapsulated DNA from in vivo degradation but the DNA release is slowl and their negative charge acts as a barrier to DNA incorporation and delivery. Also, during the preparation process, DNA could be exposed to high shear stress and organic solvents which could result in its inactivation. Moreover, PLGA NPs could be modified with different agents to reduce its cytotoxicity, to enhance the delivery efficiency and to target it to specific tissues/cells. This review summarizes different methods used for the preparation of PLGA NPs as gene carriers and recent strategies for modification of PLGA particles applied in gene therapy.

  13. PLGA-Chitosan nanoparticle-mediated gene delivery for oral cancer treatment: A brief review (United States)

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


    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.

  14. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA. (United States)

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O


    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  15. Multifunctional spider silk polymers for gene delivery to human mesenchymal stem cells. (United States)

    Tokareva, Olena S; Glettig, Dean L; Abbott, Rosalyn D; Kaplan, David L


    Non-viral gene delivery systems are important transport vehicles that can be safe and effective alternatives to currently available viral systems. A new family of multifunctional spider silk-based gene carriers was bioengineered and found capable of targeting human mesenchymal stem cells (hMSCs). These carriers successfully delivered DNA to the nucleus of these mammalian cells. The presence of specific functional sequences in the recombinant proteins, such as a nuclear localization sequence (NLS) of the large tumor (T) antigen of the Simian virus 40 (SV40 ), an hMSC high affinity binding peptide (HAB), and a translocation motif (TLM) of the hepatitis-B virus surface protein (PreS2), and their roles in mitigation and enhancement of gene transfection efficiency towards hMSCs were characterized. The results demonstrate that these bioengineered spider silk proteins serve as effective carriers, without the well-known complications associated with viral delivery systems. © 2014 Wiley Periodicals, Inc.

  16. Split vector systems for ultra-targeted gene delivery: a contrivance to achieve ethical assurance of somatic gene therapy in vivo. (United States)

    Tolmachov, Oleg E


    Tightly controlled spatial localisation of therapeutic gene delivery is essential to maximize the benefits of somatic gene therapy in vivo and to reduce its undesired effects on the 'bystander' cell populations, most importantly germline cells. Indeed, complete ethical assurance of somatic gene therapy can only be achieved with ultra-targeted gene delivery, which excludes the risk of inadvertent germline gene transfer. Thus, it is desired to supplement existing strategies of physical focusing and biological (cell-specific) targeting of gene delivery with an additional principle for the rigid control over spread of gene transfer within the body. In this paper I advance the concept of 'combinatorial' targeting of therapeutic gene transfer in vivo. I hypothesize that it is possible to engineer complex gene delivery vector systems consisting of several components, each one of them capable of independent spread within the human body but incapable of independent facilitation of gene transfer. As the gene delivery augmented by such split vector systems would be reliant on the simultaneous availability of all the vector system components at a predetermined body site, it is envisaged that higher order reaction kinetics required for the assembly of the functional gene transfer configuration would sharpen spatial localisation of gene transfer via curtailing the blurring effect of the vector spread within the body. A particular implementation of such split vector system could be obtained through supplementing a viral therapeutic gene vector with a separate auxiliary vector carrying a non-integrative and non-replicative form of a gene (e.g., mRNA) coding for a cellular receptor of the therapeutic vector component. Gene-transfer-enabling components of the vector system, which would be delivered separately from the vector component loaded with the therapeutic gene cargo, could also be cell-membrane-insertion-proficient receptors, elements of artificial transmembrane channels

  17. Implementation of BacMam virus gene delivery technology in a drug discovery setting. (United States)

    Kost, Thomas A; Condreay, J Patrick; Ames, Robert S; Rees, Stephen; Romanos, Michael A


    Membrane protein targets constitute a key segment of drug discovery portfolios and significant effort has gone into increasing the speed and efficiency of pursuing these targets. However, issues still exist in routine gene expression and stable cell-based assay development for membrane proteins, which are often multimeric or toxic to host cells. To enhance cell-based assay capabilities, modified baculovirus (BacMam virus) gene delivery technology has been successfully applied to the transient expression of target proteins in mammalian cells. Here, we review the development, full implementation and benefits of this platform-based gene expression technology in support of SAR and HTS assays across GlaxoSmithKline.

  18. Functionalized layered double hydroxide nanoparticles conjugated with disulfide-linked polycation brushes for advanced gene delivery. (United States)

    Hu, H; Xiu, K M; Xu, S L; Yang, W T; Xu, F J


    Layered double hydroxides (LDHs) have aroused great attention as potential nanosized drug delivery carriers, but independent inorganic LDH wrapped with DNA shows very low transfection efficiency. To manipulate and control the surface properties of LDH nanoparticles is of crucial importance in the designing of LDH-based drug carriers. In this work, surface-initiated atom transfer radical polymerization (ATRP) of 2-(dimethylamino)ethyl methacrylate (DMAEMA) is employed to tailor the functionality of LDH surfaces in a well-controlled manner and produce a series of well-defined novel gene delivery vectors (termed as LDH-PDs), where a flexible three-step method was first developed to introduce the ATRP initiation sites containing disulfide bonds onto LDH surfaces. In comparison the pristine LDH particles, the resultant LDH-PDs exhibited better ability to condense plasmid DNA (pDNA) and much higher levels to delivery genes in different cell lines including COS7 and HepG2 cell lines. Moreover, the LDH-PDs also could largely enhance cellular uptake. This present study demonstrates that functionalization of bioinorganic LDH with flexible polycation brushes is an effective means to produce new LDH-based gene delivery systems.

  19. Versatile types of polysaccharide-based supramolecular polycation/pDNA nanoplexes for gene delivery (United States)

    Hu, Yang; Zhao, Nana; Yu, Bingran; Liu, Fusheng; Xu, Fu-Jian


    Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations with adamantane-functionalized polysaccharides is an effective strategy for the production of new nanoplex delivery systems.Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations

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

    KAUST Repository

    Kuwahara, Hiroyuki


    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.

  1. Cyclen-based cationic lipids for highly efficient gene delivery towards tumor cells.

    Directory of Open Access Journals (Sweden)

    Qing-Dong Huang

    Full Text Available BACKGROUND: Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. METHODS: In this report, we designed and synthesized three amphiphilic molecules (L1-L3 with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen, imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. RESULTS: Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1-L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. CONCLUSION: Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen.

  2. Gene delivery in conjunction with gold nanoparticle and tumor treating electric field (United States)

    Tiwari, Pawan K.; Soo Lee, Yeon


    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.

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

    Directory of Open Access Journals (Sweden)

    Yoichi Negishi


    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.

  4. IONP-PLL: a novel non-viral vector for efficient gene delivery. (United States)

    Xiang, Juan-Juan; Tang, Jing-Qun; Zhu, Shi-Guo; Nie, Xin-Min; Lu, Hong-Bin; Shen, Shou-Rong; Li, Xiao-Ling; Tang, Ke; Zhou, Ming; Li, Gui-Yuan


    Non-viral methods of gene delivery have been an attractive alternative to virus-based gene therapy. However, the vectors that are currently available have drawbacks limiting their therapeutic application. We have developed a self-assembled non-viral gene carrier, poly-L-lysine modified iron oxide nanoparticles (IONP-PLL), which is formed by modifying poly-L-lysine to the surface of iron oxide nanoparticles. The ability of IONP-PLL to bind DNA was determined by ratio-dependent retardation of DNA in the agarose gel and co-sedimentation assay. In vitro cytotoxic effects were quantified by MTT assay. The transfection efficiency in vitro was evaluated by delivering exogenous DNA to different cell lines using IONP-PLL. Intravenous injection of IONP-PLL/DNA complexes into mice was evaluated as a gene delivery system for gene therapy. The PGL2-control gene encoding firefly luciferase and the EGFP-C2 gene encoding green fluorescent protein were used as marker genes. IONP-PLL could bind and protect DNA. In contrast to PLL and cationic liposomes, IONP-PLL described here was less cytotoxic in a broad range of concentrations. In the current study, we have demonstrated that IONP-PLL can deliver exogenous gene to cells in vitro and in vivo. After intravenous injection, IONP-PLL transferred reporter gene EGFP-C2 to lung, brain, spleen and kidney. Furthermore, we have demonstrated that IONP-PLL transferred exogenous DNA across the blood-brain barrier to the glial cells and neuron of brain. IONP-PLL, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy in vitro and in vivo, especially for gene therapy of CNS disease. Copyright 2003 John Wiley & Sons, Ltd.

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

    LENUS (Irish Health Repository)

    van Pijkeren, Jan Peter


    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.

  6. Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension (United States)

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


    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

  7. New development and application of ultrasound targeted microbubble destruction in gene therapy and drug delivery. (United States)

    Chen, Zhi-Yi; Yang, Feng; Lin, Yan; Zhang, Jin-Shan; Qiu, Ri-Xiang; Jiang, Lan; Zhou, Xing-Xing; Yu, Jiang-Xiu


    Ultrasound is a common used technique for clinical imaging. In recent years, with the advances in preparation technology of microbubbles and the innovations in ultrasound imaging, ultrasound is no longer confined to detection of tissue perfusion, but extends to specific ultrasound molecular imaging and target therapy gradually. With the development of research, ultrasound molecular imaging and target therapy have made great progresses. Targeted microbubbles for molecular imaging are achieved by binding target molecules, specific antibody or ligand to the surface of microbubbles to obtain specific imaging by attaching to target tissues. Meanwhile, it can also achieve targeting gene therapy or drug delivery by ultrasound targeted microbubble destruction (UTMD) mediating genes or drugs to specific target sites. UTMD has a number of advantages, such as target-specific, highly effective, non-invasivity, relatively low-cost and no radiation, and has broad application prospects, which is regarded as one hot spot in medical studies. We reviewed the new development and application of UTMD in gene therapy and drug delivery in this paper. With further development of technology and research, the gene or drug delivery system and related methods will be widely used in application and researches.

  8. Factorial Design and Development of Solid Lipid Nanoparticles (SLN) for Gene Delivery. (United States)

    Radaic, Allan; de Paula, Eneida; de Jesus, Marcelo Bispo


    Several scientific hurdles still have to be overcome before gene therapy becomes a reality. One of them is the development of safe and efficient gene delivery system. Here, we have employed factorial design to optimize the production of solid lipid nanoparticles (SLN) for gene delivery. A 2 x 3 full-factorial experimental design was used for the optimization of SLNs formulations. The variables were defined by the components of the formulation: concentration of stearic acid, DOTAP, and Pluronic F68 at two levels (-1, 1) and 3 central points (0). Different SNL formulations were prepared by varying the amount of components and several properties were tested, including their capacity to accommodate DNA and protection against DNase degradation, colloidal stability, in vitro cytotoxicity, and transfection efficiency in prostate cancer cells. Finally, response Surface Methodology was used to select the most effective formulation for gene delivery to prostate cancer cells in vitro. In conclusion, this study revealed that stearic acid and Pluronic F68 were determinant to SLN size and stability, respectively, while small amounts of DOTAP are essential for a successful transfection.

  9. Evaluation of Jeffamine®-cored PAMAM dendrimers as an efficient in vitro gene delivery system. (United States)

    Aydin, Zeynep; Akbas, Fahri; Senel, Mehmet; Koc, S Naci


    In this study, we investigated gene delivery properties of Jeffamine-cored polyamidoamine (PAMAM) dendrimers (JCPDs). The effects of dendrimer concentration, generation, and core size on the gene delivery have been analyzed. The experimental results showed that the JCPD effectively delivered plasmid DNA inside the HeLa cells, and the transfection efficiency improved considerably as the number of generation increased. The cytotoxicity of JCPD in different concentration was tested for HeLa cell line. JCPD was complexed with a lacZ gene carrying plasmid and tested for transfection efficiency using quantitative β-galactosidase expression assay. Additionally, confocal microscopy results revealed that JCPD effectively delivered green fluorescent protein-expressing plasmid into HeLa cells and produced fluorescent signal with satisfactory efficiency. The highest transfection efficiency was obtained from JCPDs G4 and G5, which mixed with expression plasmid vectors at a 10/1 weight ratio. These results indicated that under optimized conditions, JCPD can be considered as an efficient transfection reagent and can be effectively used for gene delivery applications.

  10. 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;


    delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers......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...... 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....

  11. Structure-Function Assessment of Mannosylated Poly(β-amino esters) upon Targeted Antigen Presenting Cell Gene Delivery. (United States)

    Jones, Charles H; Chen, Mingfu; Gollakota, Akhila; Ravikrishnan, Anitha; Zhang, Guojian; Lin, Sharon; Tan, Myles; Cheng, Chong; Lin, Haiqing; Pfeifer, Blaine A


    Antigen presenting cell (APC) gene delivery is a promising avenue for modulating immunological outcomes toward a desired state. Recently, our group developed a delivery methodology to elicit targeted and elevated levels of APC-mediated gene delivery. During these initial studies, we observed APC-specific structure-function relationships with the vectors used during gene delivery that differ from current non-APC cell lines, thus, emphasizing a need to re-evaluate vector-associated parameters in the context of APC gene transfer. Thus, we describe the synthesis and characterization of a second-generation mannosylated poly(β-amino ester) library stratified by molecular weight. To better understand the APC-specific structure-function relationships governing polymeric gene delivery, the library was systematically characterized by (1) polymer molecular weight, (2) relative mannose content, (3) polyplex biophysical properties, and (4) gene delivery efficacy. In this library, polymers with the lowest molecular weight and highest relative mannose content possessed gene delivery transfection efficiencies as good as or better than commercial controls. Among this group, the most effective polymers formed the smallest polymer-plasmid DNA complexes (∼300 nm) with moderate charge densities (structure and polyplex biophysical properties suggests a unique mode of action and provides a framework within which future APC-targeting polymers can be designed.

  12. Real-time fluorescence tracking of gene delivery via multifunctional nanocomposites. (United States)

    Bai, Min; Bai, Xilin; Wang, Leyu


    Fluorescence imaging of transduced cells and tissues is valuable in the development of gene vectors and the evaluation of gene therapy efficacy. We report here the simple and rational design of multifunctional nanocomposites (NCs) for simultaneous gene delivery and fluorescence tracking based on ZnS:Mn(2+) quantum dots (QDs) and positively charged polymer coating. The positively charged imidazole in the as-synthesized amphiphilic copolymer can be used for gene loading via electrostatic interaction. While the introduced poly(ethylene glycol) (PEG) can be used to reduce the binding of plasma proteins to nanovectors and minimize clearance by the reticuloendothelial system after intravenous administration. Most importantly, these multifunctional nanovectors showed much lower cellular toxicity than the commercial polyethylenimine (PEI) transfection vectors. On the basis of the red fluorescence of QDs, we can real-time track the gene delivery in cells, and the transfection efficacy of pDNA encoding enhanced green fluorescence protein (pEGFP) was monitored via the green fluorescence of the GFP expressed by the pDNA delivered into the nuclei. Fluorescence imaging analysis confirmed that the QDs-based nanovectors delivered pDNA into HepG2 cells efficiently. These new insights and capabilities pave a new way toward nanocomposite engineering for fluorescence imaging tracking of gene therapy.

  13. Avidin-biotin interaction mediated peptide assemblies as efficient gene delivery vectors for cancer therapy. (United States)

    Qu, Wei; Chen, Wei-Hai; Kuang, Ying; Zeng, Xuan; Cheng, Si-Xue; Zhou, Xiang; Zhuo, Ren-Xi; Zhang, Xian-Zheng


    Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin-biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.

  14. The potential of adeno-associated viral vectors for gene delivery to muscle tissue. (United States)

    Wang, Dan; Zhong, Li; Nahid, M Abu; Gao, Guangping


    Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8(+) T-cell response against AAV capsid in humans. In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.

  15. Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

    Institute of Scientific and Technical Information of China (English)


    A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

  16. 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:; Du, Libo, E-mail: [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China)


    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.

  17. Synthesis of a novel multivalent galactoside with high hepatocyte targeting for gene delivery

    Institute of Scientific and Technical Information of China (English)

    Qing Lin Jiang; Li Hai; Lei Chen; Jiao Lu; Zhi Rong Zhang; Yong Wu


    A novel bifunctional glycolipid which carried a cluster of thiogalactosides as the bepatocyte targeting ligand for gene delivery was prepared.Hexa-antennary alcohol 1 was used as the core scaffold to attach a cholesterol molecule by a poly(ethylene glycol)chain,while its remaining branches were linked with five acetylgalactosides,which would be deacetylated later to produce pentaantennary galaetoside.Liposome containing the galactoside showed high affinity and transfection activity in hepatoma cells HepG2.

  18. Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry. (United States)

    Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J


    Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi

  19. Local gene delivery via endovascular stents coated with dodecylated chitosan–plasmid DNA nanoparticles

    Directory of Open Access Journals (Sweden)

    Dunwan Zhu


    Full Text Available Dunwan Zhu1*, Xu Jin2*, Xigang Leng1, Hai Wang1, Junbo Bao1, Wenguang Liu3, Kangde Yao3, Cunxian Song11Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; 2Department of Anesthesia and Pain Therapy, Capital Medical University Affiliated Beijing Tiantan Hospital, Beijing, China; 3Research Institute of Polymeric Materials, Tianjin University, Tianjin, China; *Both investigators contributed equally to this work and are senior authors.Abstract: Development of efficacious therapeutic strategies to prevent and inhibit the occurrences of restenosis after percutaneous transluminal coronary angioplasty is critical for the treatment of cardiovascular diseases. In this study, the feasibility and efficiency of stents coated with dodecylated chitosan–plasmid DNA nanoparticles (DCDNPs were evaluated as scaffolds for localized and prolonged delivery of reporter genes into the diseased blood vessel wall. Dodecylated chitosan–plasmid DNA complexes formed stable positive charged nanospheres with mean diameter of approximately 90–180 nm and zeta potential of +28 ± 3 mV. As prepared DCDNPs were spray-coated on stents, a thin layer of dense DCDNPs was successfully distributed onto the metal struts of the endovascular stents as demonstrated by scanning electron microscopy. The DCDNP stents were characterized for the release kinetics of plasmid DNA, and further evaluated for gene delivery and expression both in vitro and in vivo. In cell culture, DCDNP stents containing plasmid EGFP-C1 exhibited high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, reporter gene activity was observed in the region of the artery in contact with the DCDNP stents, but not in adjacent arterial segments or distal organs. The DCDNP stent provides a very promising strategy for cardiovascular gene therapy

  20. Integrating mitosis, toxicity, and transgene expression in a telecommunications packet-switched network model of lipoplex-mediated gene delivery. (United States)

    Martin, Timothy M; Wysocki, Beata J; Beyersdorf, Jared P; Wysocki, Tadeusz A; Pannier, Angela K


    Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms,including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet-switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels.

  1. Novel therapeutic approaches for various cancer types using a modified sleeping beauty-based gene delivery system. (United States)

    Hong, In-Sun; Lee, Hwa-Yong; Kim, Hyun-Pyo


    Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the host's chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the non-viral SB-based gene delivery system still has limited therapeutic efficacy due to the lack of long-lasting gene expression potential and tumor cell specific gene transfer ability. These limitations could be overcome by modifying the SB system through the introduction of the hTERT promoter and the SV40 enhancer. In this study, a modified SB delivery system, under control of the hTERT promoter in conjunction with the SV40 enhancer, was able to successfully transfer the suicide gene (HSV-TK) into multiple types of cancer cells. The modified SB transfected cancer cells exhibited a significantly increased cancer cell specific death rate. These data suggest that our modified SB-based gene delivery system can be used as a safe and efficient tool for cancer cell specific therapeutic gene transfer and stable long-term expression.

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

    Directory of Open Access Journals (Sweden)

    Yoon JY


    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

  3. A translatable, closed recirculation system for AAV6 vector-mediated myocardial gene delivery in the large animal. (United States)

    Swain, JaBaris D; Katz, Michael G; White, Jennifer D; Thesier, Danielle M; Henderson, Armen; Stedman, Hansell H; Bridges, Charles R


    Current strategies for managing congestive heart failure are limited, validating the search for an alternative treatment modality. Gene therapy holds tremendous promise as both a practical and translatable technology platform. Its effectiveness is evidenced by the improvements in cardiac function observed in vector-mediated therapeutic transgene delivery to the murine myocardium. A large animal model validating these results is the likely segue into clinical application. However, controversy still exists regarding a suitable method of vector-mediated cardiac gene delivery that provides for efficient, global gene transfer to the large animal myocardium that is also clinically translatable and practical. Intramyocardial injection and catheter-based coronary delivery techniques are attractive alternatives with respect to their clinical applicability; yet, they are fraught with numerous challenges, including concerns regarding collateral gene expression in other organs, low efficiency of vector delivery to the myocardium, inhomogeneous expression, and untoward immune response secondary to gene delivery. Cardiopulmonary bypass (CPB) delivery with dual systemic and isolated cardiac circuitry precludes these drawbacks and has the added advantage of allowing for control of the pharmacological milieu, multiple pass recirculation through the coronary circulation, the selective addition of endothelial permeabilizing agents, and an increase in vector residence time. Collectively, these mechanics significantly improve the efficiency of global, vector-mediated cardiac gene delivery to the large animal myocardium, highlighting a potential therapeutic strategy to be extended to some heart failure patients.

  4. Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery (United States)

    Yang, Xinchao; Zhao, Nana; Xu, Fu-Jian


    Graphene oxide (GO) has been proven to be promising in many biomedical fields due to its biocompatibility, unique conjugated structure, easily tunable surface functionalization and facile synthesis. In this work, a flexible two-step method was first developed to introduce the atom transfer radical polymerization (ATRP) initiation sites containing disulfide bonds onto GO surfaces. Surface-initiated ATRP of (2-dimethyl amino)ethyl methacrylate (DMAEMA) was then employed to tailor the GO surfaces in a well-controlled manner, producing a series of organic-inorganic hybrids (termed as SS-GPDs) for highly efficient gene delivery. Under reducible conditions, the PDMAEMA side chains can be readily cleavable from the GO backbones, benefiting the resultant gene delivery process. Moreover, due to the conjugated structure of the graphene basal plane, SS-GPD can attach and absorb aromatic, water insoluble drugs, such as 10-hydroxycamptothecin (CPT), producing SS-GPD-CPT. The MTT assay and the simultaneous double-staining procedure revealed that SS-GPD-CPT possessed a high potency of killing cancer cells in vitro. With a high aqueous solubility and coulombic interaction with cell membrane, SS-GPDs may have great potential in gene/drug delivery fields.

  5. Stability studies of chitosan-DNA-FAP-B nanoparticles for gene delivery to lung epithelial cells. (United States)

    Mohammadi, Zohreh; Dorkoosh, Farid Abedin; Hosseinkhani, Saman; Amini, Tina; Rahimi, Amir Abbas; Najafabadi, Abdolhossein Rouholamini; Tehrani, Morteza Rafiee


    A successful gene delivery system requires efficiency and stability during storage. Stability studies are imperative for nanomedicines containing biotechnological products such as plasmids and targeting peptides. Chitosan-DNA-FAP-B nanoparticles are novel non-viral vectors for specific gene delivery to the lung epithelial cells. In this study, the storage stability of chitosan-DNA-FAP-B nanoparticles at -20, 5 and 24 °C was examined. Size, zeta potential and transfection efficiency of these nano-particles in storage were also evaluated. Stability studies showed that chitosan-DNA-FAP-B nanoparticles were stable after 1 month when stored at -20 °C and retained their initial size, zeta potential and transfection efficiency. However, their stability was not desirable at 5 and 24 °C. Based on these results, it can be concluded that chitosan-DNA-FAP-B nanoparticles can be a promising candidate for gene delivery to lung epithelial cells with good storage stability at -20 °C during 1 month.

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

    Directory of Open Access Journals (Sweden)

    Keiji Numata


    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.

  7. Protein antigen delivery by gene gun-mediated epidermal antigen incorporation (EAI). (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Michael R. Strand


    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.

  9. Overview of gene delivery into cells using HSV-1-based vectors. (United States)

    Neve, Rachael L


    This overview describes the considerations involved in the preparation and use of a herpes simplex virus type 1 (HSV-1) amplicon as a vector for gene transfer into neurons. Strategies for gene delivery into neurons, either to study the molecular biology of brain function or for gene therapy, must utilize vectors that persist stably in postmitotic cells and that can be targeted both spatially and temporally in the nervous system in vivo. This unit describes the biology of HSV-1 along with a discussion covering development of amplicon and genomic HSV-1 vectors. Advantages and disadvantages of current HSV-1 vectors are presented, and HSV-1 vectors are compared with other vectors for gene transfer into neurons.

  10. 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.

  11. 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


    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.

  12. Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases. (United States)

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


    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.

  13. Rational design of didodecyldimethylammonium bromide-based nanoassemblies for gene delivery. (United States)

    Jin, Yiguang; Wang, Shuangmiao; Tong, Li; Du, Lina


    Nonviral gene vectors are a hot topic for gene delivery. High cost and low transfection efficiency hinder the application of them. The aim of this study was to find out the optimal gene vectors with lower cost and more effective gene delivery than commonly used gene vectors. A cheap cationic lipid, didodecyldimethylammonium bromide (DDAB) was the basic component and the other components included oleic acid (OA), cholesterol (Chol), cholesteryl succinyl poly(ethylene glycol) 1500 (CHS-PEG), poly(D,L-lactide-co-glycolide)-methoxy-poly(ethylene glycol) (PLGA-PEG). The combinations of DDAB/OA/Chol, DDAB/OA/CHS-PEG and DDAB/PLGA-PEG were adopted to prepare the nanoassemblies named CNA, CPNA and PPNA, respectively. The optimal component ratios were screened out according to their Langmuir monolayer behavior. The optimal preparation method of nanoassemblies involved firstly compressing DNA or siRNA with the cationic lipid (DDAB) and secondly being coated with the helper lipids (OA and CHS-PEG) or the helper polymer (PLGA-PEG). The complexes of genes and cationic lipids were encapsulated into the core of CPNA and PPNA. The optimal gene vectors (CPNA and PPNA) with small sizes, low negative surface charges and non-exposure of cationic lipids were achieved. They had the advantages of no cytotoxicity, high transfection efficiency and low cost. More importantly, CPNA and PPNA were not sensitive to serum and showed the similar or higher transfection efficiency of pDNA and siRNA compared to Lipofectamine 2000. CPNA could mainly enter cell plasma based on endocytosis. The rational design method is useful for the design and optimization of DDAB-based gene carriers and other cationic lipid-based carriers.

  14. A Novel Approach of Low-frequency Ultrasonic Naked Plasmid Gene Delivery and Its Assessment

    Institute of Scientific and Technical Information of China (English)



    Objective To deliver the naked genes into cells through the bioeffects of cell membrane porous produced by low-frequency ultrasound (US) and to investigate the safety by determining the threshold of cell damage and membrane permeability. Methods The suspension of red cells from chickens, rabbits, rats, and S180 cells was exposed to calibrated US field with different parameters in still and flowing state. Laser scanning confocal microscopy, fluorescent microscopy, scanning electron microscopy, flow cytometry and spectrophotometry were used to examine cell morphology, membrane permeability, enzymes, free radicals, naked gene expression efficiency, threshold of cell damage and cell viability. Results The plasmid of green fluorescent protein (GFP) as a reporter gene was delivered into S180 cells under optimal conditions without cell damage and cytotoxicity. The transfection rate was (35.83±2.53)% (n=6) in viable cells, and the cell viability was (90.17±1.47)% (n=6). Also, malondialdehyde, hydroxyl free radical, alkaline phosphatase, and acid phosphatase showed a S-shaped growth model (r=0.98±0.01) in response to the permeability change and alteration of cell morphology. The constant E of energy accumulation in US delivery at 90% cell viability was an optimal control factor, and at 80% cell viability was the damage threshold. Conclusion US under optimal conditions is a versatile gene therapy tool. The intensity of GFP expression in US group has a higher fluorescent peak than that in AVV-GFP group and control group (P<0.001). The optimal gene uptakes, expression of gene and safety depend on E, which can be applied to control gene delivery efficiency in combination with other parameters. The results are helpful for development of a novel clinical naked gene therapeutic system and non-hyperthermia cancer therapeutic system.

  15. Stable Somatic Gene Expression in Mouse Lungs Following Electroporation-mediated Tol2 Transposon Delivery. (United States)

    Muliawan, Hary Sakti; Nakayama, Kazuhiko; Yagi, Keiko; Ikeda, Koji; Yagita, Kazuhiro; Hirata, Ken-ichi; Emoto, Noriaki


    Gene delivery to the lung has rapidly progressed as an important method for studying various chronic lung diseases. Viral vectors, albeit highly efficient, are limited by the host immune response. Electroporation, a well-known non-viral method, can efficiently deliver genes to the lung, but is unable to induce stable gene expression. The Tol2 transposon is another non-viral method that can induce stable gene expression by reinserting its genes into the host genome. In this study, we combined electroporation and Tol2 transposons to obtain stable, high-level gene expression in the mouse lung. Tol2 transposon plasmids (pT2A-EGFP; Tol2, pCAGGS-TP; transposase) were optimized in vitro, and the electroporation procedure (pCAG-EGFP) was optimized in mouse lungs. After optimization, a combination of electroporation plus the Tol2 transposon was used in a comparative analysis with electroporation plus pCAG-EGFP. GFP expression levels were quantified and visualized on days 4 and 7 post-electroporation. We successfully reproduced the Tol2 transposon system in vitro and the electroporation procedure in vivo. We observed sustainable GFP expression using electroporation plus the Tol2 transposon on days 4 and 7, while electroporation plus pCAG-EGFP resulted in decreased GFP expression on day 7. We were able to induce high-level, stable gene expression in mouse lungs using a combination of electroporation and the Tol2 transposon. This represents a safer method for lung gene delivery that can be used as an alternative to viral vectors.

  16. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector (United States)

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


    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.

  17. Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers

    National Research Council Canada - National Science Library

    Köping-Höggård, M; Vårum, K M; Issa, M; Danielsen, S; Christensen, B E; Stokke, B T; Artursson, P


    Nonviral gene delivery systems based on conventional high-molecular-weight chitosans are efficient after lung administration in vivo, but have poor physical properties such as aggregated shapes, low...

  18. Association with amino acids does not enhance efficacy of polymerized liposomes as a system for lung gene delivery


    Elga eBernardo Bandeira De Melo; Miquéias eLopes-Pacheco; Nadia eChiaramoni; Débora eFerreira; Maria Julieta eFernandez-Ruocco; Maria Jimena ePrieto; Tatiana eMaron-Gutierrez; Perrotta, Ramiro M.; Hugo C Castro-Faria-Neto; Patricia Rieken Macedo Rocco; Silvia del Valle Alonso; Marcelo Marcos Morales


    Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study,...

  19. Non-viral delivery of genome-editing nucleases for gene therapy. (United States)

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


    Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.Gene Therapy advance online publication, 1 December 2016; doi:10.1038/gt.2016.72.

  20. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    Directory of Open Access Journals (Sweden)

    Ming Yan

    Full Text Available Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

  1. Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. (United States)

    LaFountaine, Justin S; Fathe, Kristin; Smyth, Hugh D C


    In recent years, several new genome editing technologies have been developed. Of these the zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system are the most widely described. Each of these technologies utilizes restriction enzymes to introduce a DNA double stranded break at a targeted location with the guide of homologous binding proteins or RNA. Such targeting is viewed as a significant advancement compared to current gene therapy methods that lack such specificity. Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials. Careful consideration and investigation of delivery strategies will be required so that the therapeutic potential for gene editing is achieved. In this review, the mechanisms of each of these gene editing technologies and evidence of therapeutic potential will be briefly described and a comprehensive list of past studies will be provided. The pharmaceutical approaches of each of these technologies are discussed along with the current delivery obstacles. The topics and information reviewed herein provide an outline of the groundbreaking research that is being performed, but also highlights the potential for progress yet to be made using these gene editing technologies.

  2. Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration. (United States)

    Belmadi, Nawal; Midoux, Patrick; Loyer, Pascal; Passirani, Catherine; Pichon, Chantal; Le Gall, Tony; Jaffres, Paul-Alain; Lehn, Pierre; Montier, Tristan


    Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein; the replacement of a missing or inactive gene; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.

  3. Polydioxanone-based bio-materials for tissue engineering and drug/gene delivery applications. (United States)

    Goonoo, Nowsheen; Jeetah, Roubeena; Bhaw-Luximon, Archana; Jhurry, Dhanjay


    Since the commercialization of polydioxanone (PDX) as a biodegradable monofilament suture by Ethicon in 1981, the polymer has received only limited interest until recently. The limitations of polylactide-co-glycolide (PLGA) coupled with the growing need for materials with enhanced features and the advent of new fabrication techniques such as electrospinning have revived interest for PDX in medical devices, tissue engineering and drug delivery applications. Electrospun PDX mats show comparable mechanical properties as the major structural components of native vascular extracellular matrix (ECM) i.e. collagen and elastin. In addition, PDX's unique shape memory property provides rebound and kink resistance when fabricated into vascular conduits. The synthesis of methyl dioxanone (MeDX) monomer and copolymers of dioxanone (DX) and MeDX have opened up new perspectives for poly(ester-ether)s, enabling the design of the next generation of tissue engineering scaffolds for application in regenerating such tissues as arteries, peripheral nerve and bone. Tailoring of polymer properties and their formulation as nanoparticles, nanomicelles or nanofibers have brought along important developments in the area of controlled drug or gene delivery. This paper reviews the synthesis of PDX and its copolymers and provides for the first time an exhaustive account of its applications in the (bio)medical field with focus on tissue engineering and drug/gene delivery.

  4. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. (United States)

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels


    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  5. Identifying Intracellular pDNA Losses From a Model of Nonviral Gene Delivery. (United States)

    Martin, Timothy Michael; Wysocki, Beata Joanna; Wysocki, Tadeusz Antoni; Pannier, Angela K


    Nonviral gene delivery systems are a type of nanocommunication system that transmit plasmid packets (i.e., pDNA packets) that are programmed at the nanoscale to biological systems at the microscopic cellular level. This engineered nanocommunication system suffers large pDNA losses during transmission of the genetically encoded information, preventing its use in biotechnological and medical applications. The pDNA losses largely remain uncharacterized, and the ramifications of reducing pDNA loss from newly designed gene delivery systems remain difficult to predict. Here, the pDNA losses during primary and secondary transmission chains were identified utilizing a MATLAB model employing queuing theory simulating delivery of pEGFPLuc transgene to HeLa cells carried by Lipofectamine 2000 nonviral DNA carrier. Minimizing pDNA loss during endosomal escape of the primary transmission process results in increased number of pDNA in the nucleus with increased transfection, but with increased probability of cell death. The number of pDNA copies in the nucleus and the amount of time the pDNAs are in the nucleus directly correlates to improved transfection efficiency. During secondary transmission, pDNAs are degraded during distribution to daughter cells. Reducing pDNA losses improves transfection, but a balance in quantity of nuclear pDNA, mitosis, and toxicity must be considered in order to achieve therapeutically relevant transfection levels.

  6. 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 expressi

  7. Protection of Mice from Lethal Endotoxemia by Chimeric Human BPI-Fcγ1 Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    Chen Li; Jing Li; Zhe Lv; Xinghua Guo; Qinghua Chen; Qingli Kong; Yunqing An


    To evaluate the potentiality of applying gene therapy to endotoxemia in high-risk patients, we investigated the effects of transferring an adeno-associated virus serotype 2 (AAV2)-mediated BPI-Fcγ1 gene on protecting mice from challenge of lethal endotoxin. The chimeric BPI-Fcγ1 gene consists of two parts, one encods functional N-terminus (1 to 199 amino acidic residues) of human BPI, which is a bactericidal/permeability-increasing protein,and the other encodes Fc segment of human immunoglobulin G1 (Fcγ1). Our results indicated that the target protein could be expressed and secreted into the serum of the gene-transferred mice. After lethal endotoxin challenge, the levels of endotoxin and TNF-α in the gene-transferred mice were decreased. The survival rate of the BPI-Fcγ1 gene-transferred mice was markedly increased. Our data suggest that AAV2-mediated chimeric BPI-Fcγ1 gene delivery can potentially be used clinically for the protection and treatment of endotoxemia and endotoxic shock in high-risk individuals.

  8. Human artificial chromosome vectors meet stem cells: new prospects for gene delivery. (United States)

    Ren, Xianying; Tahimic, Candice Ginn T; Katoh, Motonobu; Kurimasa, Akihiro; Inoue, Toshiaki; Oshimura, Mitsuo


    The recent emergence of stem cell-based tissue engineering has now opened up new venues for gene therapy. The task now is to develop safe and effective vectors that can deliver therapeutic genes into specific stem cell lines and maintain long-term regulated expression of these genes. Human artificial chromosomes (HACs) possess several characteristics that require gene therapy vectors, including a stable episomal maintenance, and the capacity for large gene inserts. HACs can also carry genomic loci with regulatory elements, thus allowing for the expression of transgenes in a genetic environment similar to the chromosome. Currently, HACs are constructed by a two prone approaches. Using a top-down strategy, HACs can be generated from fragmenting endogenous chromosomes. By a bottom-up strategy, HACs can be created de novo from cloned chromosomal components using chromosome engineering. This review describes the current advances in developing HACs, with the main focus on their applications and potential value in gene delivery, such as HAC-mediated gene expression in embryonic, adult stem cells, and transgenic animals.

  9. Spermine-modified Antheraea pernyi silk fibroin as a gene delivery carrier (United States)

    Yu, Yanni; Hu, Yongpei; Li, Xiufang; Liu, Yu; Li, Mingzhong; Yang, Jicheng; Sheng, Weihua


    The development of a novel cationized polymer used as a gene delivery carrier that can conveniently and effectively transfect cells resulting in a stably expressed target gene remains a challenge. Antheraea pernyi silk fibroin (ASF) is a cytocompatible and biodegradable natural polymer, and it possesses Arg–Gly–Asp sequences but a negative charge. In order to render ASF amenable to packaging plasmid DNA (pDNA), spermine was used to modify ASF to synthesize cationized ASF (CASF), which was used as a gene delivery carrier. CASF was characterized using trinitrobenzene sulfonic acid assay, the zeta potential determination, and a Fourier transform infrared analysis, and the results of these characterizations indicated that the –NH2 in spermine effectively reacts with the –COOH in the side chains of ASF. Spermine grafted to the side chains of ASF resulted in the conversion of the negative charge of ASF to a positive charge. CASF packaged pDNA and formed CASF/pDNA complexes, which exhibited spherical morphology with average particle sizes of 215–281 nm and zeta potential of approximately +3.0 mV to +3.2 mV. The results of the MTT assay, confocal laser scanning microscopy, and flow cytometry analysis in a human endothelial cell line revealed that CASF/pDNA complexes exhibited lower cytotoxicity and higher transfection efficiency compared to the pDNA complexes of polyethyleneimine. These results indicate that our synthesized CASF, a cationized polymer, is a potential gene delivery carrier with the advantages of biodegradability and low cytotoxicity. PMID:27042056

  10. Delivery of antioxidant enzyme genes to protect against ischemia/reperfusion-induced injury to retinal microvasculature. (United States)

    Chen, Baihua; Caballero, Sergio; Seo, Soojung; Grant, Maria B; Lewin, Alfred S


    Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice. I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated. Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration. Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.

  11. Real-Time Imaging of Gene Delivery and Expression with DNA Nanoparticle Technologies (United States)

    Sun, Wenchao; Ziady, Assem G.

    The construction of safe, efficient, and modifiable synthetic DNA nanoparticles is an emerging technology that has achieved important milestones of success in the past 5 years. Advances in chemical conjugation, purification, and controlled synthesis have allowed researchers to produce uniform and stable particles, whose physical characteristics can be well characterized and monitored. As a result of these improvements, DNA nanoparticles have now been cleared for clinical testing, and show good potential for human gene therapy. A very important recent development in the study of DNA nanoparticles is the use of small-animal imaging. Real-time imaging has become a valuable technique for tracking particle biodistribution and gene transfer efficacy. In this chapter, we discuss how bioluminescent, positron emission tomography, and magnetic resonance imaging can be used separately or in concert to study particle delivery, localization, and magnitude of gene expression in vivo.

  12. 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


    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...... delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers...

  13. 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.


    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).

  14. Spermine-modified Antheraea pernyi silk fibroin as a gene delivery carrier

    Directory of Open Access Journals (Sweden)

    Yu Y


    Full Text Available Yanni Yu,1 Yongpei Hu,1 Xiufang Li,1 Yu Liu,1 Mingzhong Li,1 Jicheng Yang,2 Weihua Sheng2 1National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, 2Cell and Molecular Biology Institute, College of Medicine, Soochow University, Suzhou, People’s Republic of China Abstract: The development of a novel cationized polymer used as a gene delivery carrier that can conveniently and effectively transfect cells resulting in a stably expressed target gene remains a challenge. Antheraea pernyi silk fibroin (ASF is a cytocompatible and biodegradable natural polymer, and it possesses Arg–Gly–Asp sequences but a negative charge. In order to render ASF amenable to packaging plasmid DNA (pDNA, spermine was used to modify ASF to synthesize cationized ASF (CASF, which was used as a gene delivery carrier. CASF was characterized using trinitrobenzene sulfonic acid assay, the zeta potential determination, and a Fourier transform infrared analysis, and the results of these characterizations indicated that the –NH2 in spermine effectively reacts with the –COOH in the side chains of ASF. Spermine grafted to the side chains of ASF resulted in the conversion of the negative charge of ASF to a positive charge. CASF packaged pDNA and formed CASF/pDNA complexes, which exhibited spherical morphology with average particle sizes of 215–281 nm and zeta potential of approximately +3.0 mV to +3.2 mV. The results of the MTT assay, confocal laser scanning microscopy, and flow cytometry analysis in a human endothelial cell line revealed that CASF/pDNA complexes exhibited lower cytotoxicity and higher transfection efficiency compared to the pDNA complexes of polyethyleneimine. These results indicate that our synthesized CASF, a cationized polymer, is a potential gene delivery carrier with the advantages of biodegradability and low cytotoxicity. Keywords: silk fibroin, spermine, cationized polymer, gene delivery

  15. Macrophages in gene therapy: cellular delivery vehicles and in vivo targets. (United States)

    Burke, B; Sumner, S; Maitland, N; Lewis, C E


    The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumors, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we critically review the efficacy of various gene transfer methods (viral, bacterial, protozoan, and various chemical and physical methods) in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles. Finally, we discuss the use of various viral and nonviral methods to transfer genes to macrophages in vivo. As will be seen, definitive evidence for the use of macrophages as gene transfer vehicles has yet to be provided and awaits detailed trafficking studies in vivo. Moreover, although methods for transfecting macrophages have improved considerably in efficiency in recent years, targeting of gene transfer specifically to macrophages in vivo remains a problem. However, possible solutions to this include placing transgenes under the control of macrophage-specific promoters to limit expression to macrophages or stably transfecting CD34(+) precursors of monocytes/macrophages and then differentiating these cells into monocytes/macrophages ex vivo. The latter approach could conceivably lead to the bone marrow precursor cells of patients with inherited genetic disorders being permanently fortified or even replaced with genetically modified cells.

  16. A top-down approach for construction of hybrid polymer-virus gene delivery vectors. (United States)

    Ramsey, Joshua D; Vu, Halong N; Pack, Daniel W


    Safe and efficient delivery of therapeutic nucleic acids remains the primary hurdle for human gene therapy. While many researchers have attempted to re-engineer viruses to be suited for gene delivery, others have sought to develop non-viral alternatives. We have developed a complementary approach in which viral and synthetic components are combined to form hybrid nanoparticulate vectors. In particular, we complexed non-infectious retrovirus-like particles lacking a viral envelope protein, from Moloney murine leukemia virus (M-VLP) or human immunodeficiency virus (H-VLP), with poly-L-lysine (PLL) or polyethylenimine (PEI) over a range of polymer/VLP ratios. At appropriate stoichiometry (75-250 microg polymer/10(6) VLP), the polymers replace the function of the viral envelope protein and interact with the target cell membrane, initiate cellular uptake and facilitate escape from endocytic vesicles. The viral particle, once in the cytosol, efficiently completes its normal infection process including integration of viral genes with the host genome as demonstrated by long-term (at least 5 weeks) transgene expression. In addition, hybrid vectors comprising H-VLP were shown to be capable of infecting non-dividing cells.

  17. Effects of hydrophobic and hydrophilic modifications on gene delivery of amphiphilic chitosan based nanocarriers. (United States)

    Wang, Bingqing; He, Chunbai; Tang, Cui; Yin, Chunhua


    The structure-activity relationships between hydrophobic and hydrophilic modification on chitosan and resultant physicochemical properties along with performances in dealing with critical gene delivery barriers were investigated through amphiphilic linoleic acid(LA) and poly (β-malic acid) (PMLA) double grafted chitosan (LMC)/plasmid DNA (pDNA) nanocomplexes. LMC polymers with various LA and PMLA substitution degrees were synthesized and their hydrophilicity/hydrophobicity was characterized. Compared to chitosan, LMC nanoparticles retained the pDNA binding ability at pH 5.5 when they formed nanocomplexes with pDNA encoding enhanced green fluorescence protein (pEGFP) and the resultant complexes showed diameters below 300 nm. Hydrophobic LA and hydrophilic PMLA substitution contributed to suppressed non-specific adsorption, reduced interactions inside LMC/pDNA nanocomplexes, and enhanced pDNA dissociation. However, enzymatic degradation resistance, cell adsorption, and cellular uptake through clathrin-mediated pathway were promoted by hydrophobic LA grafting while being inhibited by hydrophilic PMLA substitution. In vitro transfection assay suggested the optimal LMC/pEGFP nanocomplexes mediated an 8.0-fold improved transfection compared to chitosan/pEGFP nanocomplexes. The 4.2-fold and 2.2-fold higher intramuscular gene expression in mice compared to chitosan/pEGFP and polyethyleneimine (PEI)/pEGFP nanocomplexes further demonstrated the superiority of LMC/pDNA nanocomplexes. Therefore, amphiphilic chitosan derivates with appropriate combination of hydrophobic and hydrophilic modification would be promising gene delivery nanocarriers.

  18. Development of TMTP-1 targeted designer biopolymers for gene delivery to prostate cancer. (United States)

    McBride, John W; Massey, Ashley S; McCaffrey, J; McCrudden, Cian M; Coulter, Jonathan A; Dunne, Nicholas J; Robson, Tracy; McCarthy, Helen O


    Designer biopolymers (DBPs) represent state of the art genetically engineered biomacromolecules designed to condense plasmid DNA, and overcome intra- and extra- cellular barriers to gene delivery. Three DBPs were synthesized, each with the tumor molecular targeting peptide-1 (TMTP-1) motif to specifically target metastases. Each DBP was complexed with a pEGFP-N1 reporter plasmid to permit physiochemical and biological assay analysis. Results indicated that two of the biopolymers (RMHT and RM3GT) effectively condensed pEGFP-N1 into cationic nanoparticles prostate cancer cells. Conversely the anionic RMGT DBP nanoparticles could not transfect PC-3 cells. RMHT and RM3GT nanoparticles were stable in the presence of serum and protected the cargo from degradation. Additionally it was concluded that cell viability could recover post-transfection with these DBPs, which were less toxic than the commercially available transfection reagent Lipofectamine(®) 2000. With both DBPs, a higher transfection efficacy was observed in PC-3 cells than in the moderately metastatic, DU145, and normal, PNT2-C2, cell lines. Blocking of the TMTP-1 receptors inhibited gene transfer indicating internalization via this receptor. In conclusion RMHT and RM3GT are fully functional DBPs that address major obstacles to gene delivery and target metastatic cells expressing the TMTP-1 receptor.

  19. Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery. (United States)

    Zaharoff, D A; Barr, R C; Li, C-Y; Yuan, F


    Interstitial transport is a crucial step in plasmid DNA-based gene therapy. However, interstitial diffusion of large nucleic acids is prohibitively slow. Therefore, we proposed to facilitate interstitial transport of DNA via pulsed electric fields. To test the feasibility of this approach to gene delivery, we developed an ex vivo technique to quantify the magnitude of DNA movement due to pulsed electric fields in two tumor tissues: B16.F10 (a mouse melanoma) and 4T1 (a mouse mammary carcinoma). When the pulse duration and strength were 50 ms and 233 V/cm, respectively, we found that the average plasmid DNA movements per 10 pulses were 1.47 microm and 0.35 microm in B16.F10 and 4T1 tumors, respectively. The average plasmid DNA movements could be approximately tripled, ie to reach 3.69 microm and 1.01 microm, respectively, when the pulse strength was increased to 465 V/cm. The plasmid DNA mobility was correlated with the tumor collagen content, which was approximately eight times greater in 4T1 than in B16.F10 tumors. These data suggest that electric field can be a powerful driving force for improving interstitial transport of DNA during gene delivery.

  20. Close-field electroporation gene delivery using the cochlear implant electrode array enhances the bionic ear. (United States)

    Pinyon, Jeremy L; Tadros, Sherif F; Froud, Kristina E; Y Wong, Ann C; Tompson, Isabella T; Crawford, Edward N; Ko, Myungseo; Morris, Renée; Klugmann, Matthias; Housley, Gary D


    The cochlear implant is the most successful bionic prosthesis and has transformed the lives of people with profound hearing loss. However, the performance of the "bionic ear" is still largely constrained by the neural interface itself. Current spread inherent to broad monopolar stimulation of the spiral ganglion neuron somata obviates the intrinsic tonotopic mapping of the cochlear nerve. We show in the guinea pig that neurotrophin gene therapy integrated into the cochlear implant improves its performance by stimulating spiral ganglion neurite regeneration. We used the cochlear implant electrode array for novel "close-field" electroporation to transduce mesenchymal cells lining the cochlear perilymphatic canals with a naked complementary DNA gene construct driving expression of brain-derived neurotrophic factor (BDNF) and a green fluorescent protein (GFP) reporter. The focusing of electric fields by particular cochlear implant electrode configurations led to surprisingly efficient gene delivery to adjacent mesenchymal cells. The resulting BDNF expression stimulated regeneration of spiral ganglion neurites, which had atrophied 2 weeks after ototoxic treatment, in a bilateral sensorineural deafness model. In this model, delivery of a control GFP-only vector failed to restore neuron structure, with atrophied neurons indistinguishable from unimplanted cochleae. With BDNF therapy, the regenerated spiral ganglion neurites extended close to the cochlear implant electrodes, with localized ectopic branching. This neural remodeling enabled bipolar stimulation via the cochlear implant array, with low stimulus thresholds and expanded dynamic range of the cochlear nerve, determined via electrically evoked auditory brainstem responses. This development may broadly improve neural interfaces and extend molecular medicine applications.

  1. 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: [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)


    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.

  2. Peptide GE11-Polyethylene Glycol-Polyethylenimine for targeted gene delivery in laryngeal cancer. (United States)

    Ren, Henglei; Zhou, Liang; Liu, Min; Lu, Weiyue; Gao, Chunli


    The objective of this study was to evaluate the possibility of using GE11-polyethylene glycol-polyethylenimine (GE11-PEG-PEI) for targeted gene delivery to treat epidermal growth factor receptor (EGFR)-overexpressing laryngeal cancer. This study described the design, characterization, and in vitro and in vivo study of the nanocarrier GE11-PEG-PEI for gene delivery to treat laryngeal cancer. Analysis of the sizes and zeta potentials indicated that the formation of PEGylated complexes was dependent on the N/P ratio, and these complexes were capable of binding plasmid DNA and condensing DNA into small positively charged nanoparticles. The results also revealed that GE11-PEG-PEI had a weaker effect on cell survival in vitro. Gene transfection was performed on human laryngeal cancer Hep-2 cells in vitro and in vivo. Both the in vitro and in vivo results demonstrated that GE11-PEG-PEI had greater transfection efficiency than mPEG-PEI. Compared with mPEG-PEI/pORF-hTRAIL and saline, GE11-PEG-PEI/pORFh-TRAIL significantly (p < 0.05) reduced tumor growth in nude mice with laryngeal cancer. Moreover, the GE11-PEG-PEI/pORF-hTRAIL-treated groups showed more apoptosis than the mPEG-PEI/pORF-hTRAIL-treated groups. Therefore, our results showed that the peptide GE11 conjugated to PEG-PEI delivered significantly more genes to EGFR-overexpressing laryngeal cancer cells in vivo, indicating that GE11-PEG-PEI may be a suitable gene vector for treating EGFR-overexpressing laryngeal cancer.

  3. Translational Advancement of Somatostatin Gene Delivery for Disease Modification and Cognitive Sparing in Intractable Epilepsy (United States)


    human clinical trials, and could provide a new, safe, and effective way to interfere with this evolution , associated loss of brain tissue from... brain . Our initial tests demonstrated that intracranial somatostatin gene delivery prevented the evolution to high-level seizures in 70% of rats...variables, effects on seizure­stimulated  brain  stem cell  division or differentiation, or obvious  brain  pathology.  Kindling increased new cell

  4. Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors. (United States)

    Miao, Carol H


    Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable

  5. Degradable terpolymers with alkyl side chains demonstrate enhanced gene delivery potency and nanoparticle stability. (United States)

    Eltoukhy, Ahmed A; Chen, Delai; Alabi, Christopher A; Langer, Robert; Anderson, Daniel G


    Degradable, cationic poly(β-amino ester)s (PBAEs) with alkyl side chains are developed for non-viral gene delivery. Nanoparticles formed from these PBAE terpolymers exhibit significantly enhanced DNA transfection potency and resistance to aggregation. These hydrophobic PBAE terpolymers, but not PBAEs lacking alkyl side chains, support interaction with PEG-lipid conjugates, facilitating their functionalization with shielding and targeting moieties and accelerating the in vivo translation of these materials. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Gene Silencing in Adult Aedes aegypti Mosquitoes Through Oral Delivery of Double-Stranded RNA (United States)


    OR I GI N AL C ONTR I BUTI O N Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double-stranded RNA M. R. Coy1, N. D...we tested whether such an approach could be used in the yellow fever mosquito, Aedes aegypti . Using a non-specific dsRNA construct, we found Ae. aegypti ingested dsRNA through this method and that the ingested dsRNA can be recovered from the mosquitoes post-feeding. Through the feeding of

  7. Non-viral gene delivery strategies for gene therapy: a 'menage a trois' among nucleic acids, materials, and the biological environment

    Energy Technology Data Exchange (ETDEWEB)

    Pezzoli, Daniele; Candiani, Gabriele, E-mail: [INSTM (National Interuniversity Consortium of Materials Science and Technology), Research Unit Milano Politecnico (Italy)


    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.

  8. Delivery of surface-mediated non-viral gene nanoparticles from ultrathin layer-by-layer multilayers

    Institute of Scientific and Technical Information of China (English)


    An efficient and safe gene delivery system remains a challenge in the development of gene therapy.Polycation-based gene nanoparticles are a typical non-viral gene delivery system,which are able to transfect cells in vitro and in vivo.This paper reported a facile method for constructing biodegradable multilayers via layer-by-layer self-assembly,in which the polycation-based gene nanoparticles were loaded.Through this surface-mediated delivery system,adherent cells on the multilayer could be transfected in situ.Gene nanoparticles-loaded multilayers transfect cells with higher efficiency than naked DNA-loaded multilayers because of the complex configuration of the DNA.DNA nanoparticles/PGA multilayers constructed on the scaffold surface could also realize in situ transfection on the adherent cells.The well-structured,easy-processed multilayers may provide a novel approach to precisely controlled delivery of gene nanoparticles,which may have potential applications for gene therapy in tissue engineering and medical implants.

  9. Biodegradable particle formation for drug and gene delivery using supercritical fluid and dense gas. (United States)

    Mishima, Kenji


    Recent developments in biodegradable particle formation using supercritical fluids and dense gases have been reviewed with an emphasis on studies of micronizing and encapsulating poorly-soluble pharmaceuticals and gene. General review articles published in previous years have then been provided. A brief description of the operating principles of some types of particle formation processes is given. These include the rapid expansion of supercritical solutions (RESS), the particles from gas-saturated solution (PGSS) processes, the gas antisolvent process (GAS), and the supercritical antisolvent process (SAS). The papers have been reviewed under two groups, one involving the production of particles from pure biodegradable substances, and the other involving coating, capsule, and impregnation that contain active components, especially those that relate to pharmaceuticals. This review is a comprehensive review specifically focused on the formation of biodegradable particles for drug and gene delivery system using supercritical fluid and dense gas.

  10. Charge-reversal Lipids, Peptide-based Lipids, and Nucleoside-based Lipids for Gene Delivery (United States)

    LaManna, Caroline M.; Lusic, Hrvoje; Camplo, Michel; McIntosh, Thomas J.; Barthélémy, Philippe; Grinstaff, Mark W.


    Conspectus Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy’s appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to a myriad of diseases by tailoring the treatment to a specific individual’s genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when utilizing non-viral vectors have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: 1) that releasing the nucleic acid from the carrier will increase gene transfection; 2) that utilizing biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery; and 3) that mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will give unique supramolecular assembles with high transfection efficiency. The results presented in this Account demonstrate that cellular uptake and transfection efficacy can be improved by engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate change of the lipid from positive to negative net charge during the transfection process improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, and aromatic) between the cationic headgroup and the hydrophobic chains, lipids can be tailored to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. Introduction of a peptide

  11. Delivery of cationic polymer-siRNA nanoparticles for gene therapies in neural regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Yanran [Department of Neurology, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, No. 107, West Yanjiang Road, Guangzhou 510120, People' s Republic of China (China); Liu, Zhonglin, E-mail: [Department of Neurology, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, No. 107, West Yanjiang Road, Guangzhou 510120, People' s Republic of China (China); Shuai, Xintao; Wang, Weiwei [School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, People' s Republic of China (China); Liu, Jun [Department of Neurology, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, No. 107, West Yanjiang Road, Guangzhou 510120, People' s Republic of China (China); Bi, Wei [Department of Neurology, The First Affiliated Hospital of Jinan University, No. 613, West Huangpu Road, Guangzhou 510630, People' s Republic of China (China); Wang, Chuanming; Jing, Xiuna; Liu, Yunyun [Department of Neurology, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, No. 107, West Yanjiang Road, Guangzhou 510120, People' s Republic of China (China); Tao, Enxiang, E-mail: [Department of Neurology, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, No. 107, West Yanjiang Road, Guangzhou 510120, People' s Republic of China (China)


    Highlights: Black-Right-Pointing-Pointer Nogo receptor can inhibit growth of injured axons, thus affecting neural regeneration. Black-Right-Pointing-Pointer The delivery of siRNA is crucial to inhibit NgR expression in NSCs. Black-Right-Pointing-Pointer Non-viral vector PEG-PEI condensed siRNA targeting NgR into nanoscale particles. Black-Right-Pointing-Pointer PEG-PEI/siRNA at N/P = 15 displayed high transfection efficiency and low cytotoxicity. Black-Right-Pointing-Pointer PEG-PEI has great potential in carrying siRNA to diminish the gene expression in NSCs. -- Abstract: The therapeutic applications of neural stem cells (NSCs) have potential to promote recovery in many obstinate diseases in central nervous system. Regulation of certain gene expressions using siRNA may have significant influence on the fate of NSC. To achieve the optimum gene silencing effect of siRNA, non-viral vector polyethylene glycol-polyethyleneimine (PEG-PEI) was investigated in the delivery of siRNA to NSCs. The characteristics of PEG-PEI/siRNA polyplexes were detected by scanning electron microscopy (SEM). The effects of nanoparticles on cell viability were measured via CCK-8 assay. In addition, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry, and real-time PCR and Western Blot were employed to detect the gene inhibition effect of siRNA delivered by PEG-PEI. The SEM micrographs showed that PEG-PEI could condense siRNA to form diffuse and spherical nanoparticles. The cytotoxicity of PEG-PEI/siRNA nanocomplexes (N/P = 15) was significantly lower when compared with that of Lipofectamine 2000/siRNA (P < 0.05). Moreover, the highest transfection efficiency of PEG-PEI/siRNA nanoparticles was obtained at an N/P ratio of 15, which was better than that achieved in the transfection using Lipofectamine 2000 (P < 0.05). Finally, the gene knockdown effect of PEG-PEI/siRNA nanoparticles was verified at the levels of mRNA and protein. These results suggest that


    Directory of Open Access Journals (Sweden)

    Erhan Süleymanoglu,


    Full Text Available Human gene therapy research is currently discouraging due to the lack of suitable delivery vehicles for nucleic acid transfer to affected cell types. There is an urgent need for optimized gene delivery tools capable of protecting the polynucleotide from degradation through its route from site of administration to gene expression. Besides difficulties arising during the preparation of the currently employed cationic lipids, their cytotoxicity has been an unavoidable hurdle. Some energetics issues related to preparation and use of self-assemblies formed between neutral lipid and polynucleotides with various conformation and size are presented. The divalent metal cation-governed adsorption, aggregation and adhesion between single- and double-stranded polynucleotides with multilamellar and unilamellar phosphatidylcholine vesicles was followed turbidimetrically. Thermotropic phase transitions of zwitterionic liposomes and their complexes with polynucleotides and calf thymus DNA with Ca2+ and Mg2+ is presented and compared to the previous data for various electrostatic lipid - nucleic acid complexes. Differential scanning microcalorimetric measurements of synthetic phosphatidylcholine vesicles and polynucleotides and their ternary complexes with inorganic cations were used to build the thermodynamic model of their structural transitions. The increased thermal stability of the phospholipid bilayers is achieved by affecting their melting transition temperature by nucleic acid induced electrostatic charge screening. Thermodynamic measurements give evidence for the stabilization of polynucleotide helices upon their association with liposomes in presence of divalent metal cations. It is thus possible to suggest this self-assembly as an improved formulation with further potential in gene therapy trials. Although the pharmacodynamical features of the zwitterionic lipid-metal ion-DNA nanocondensates remain to be tested in further transfection experiments, at

  13. Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery. (United States)

    Nunes, Antonio; Amsharov, Nadja; Guo, Chang; Van den Bossche, Jeroen; Santhosh, Padmanabhan; Karachalios, Theodoros K; Nitodas, Stephanos F; Burghard, Marko; Kostarelos, Kostas; Al-Jamal, Khuloud T


    Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-βGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. β-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.

  14. 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


    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.

  15. Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection (United States)

    Chang, Chun-Chi; Wang, Liangli; Yuan, Fan


    A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA. PMID:28182739

  16. Nonviral gene-delivery by highly fluorinated gemini bispyridinium surfactant-based DNA nanoparticles. (United States)

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


    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.

  17. Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery. (United States)

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


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

  18. Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery (United States)

    Gaspar, V. M.; Marques, J. G.; Sousa, F.; Louro, R. O.; Queiroz, J. A.; Correia, I. J.


    Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan-histidine-arginine (CH-H-R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy.

  19. Niemann-Pick C1 affects the gene delivery efficacy of degradable polymeric nanoparticles. (United States)

    Eltoukhy, Ahmed A; Sahay, Gaurav; Cunningham, James M; Anderson, Daniel G


    Despite intensive research effort, the rational design of improved nanoparticulate drug carriers remains challenging, in part due to a limited understanding of the determinants of nanoparticle entry and transport in target cells. Recent studies have shown that Niemann-Pick C1 (NPC1), the lysosome membrane protein that mediates trafficking of cholesterol in cells, is involved in the endosomal escape and subsequent infection caused by filoviruses, and that its absence promotes the retention and efficacy of lipid nanoparticles encapsulating siRNA. Here, we report that NPC1 deficiency results in dramatic reduction in internalization and transfection efficiency mediated by degradable cationic gene delivery polymers, poly(β-amino ester)s (PBAEs). PBAEs utilized cholesterol and dynamin-dependent endocytosis pathways, and these were found to be heavily compromised in NPC1-deficient cells. In contrast, the absence of NPC1 had minor effects on DNA uptake mediated by polyethylenimine or Lipofectamine 2000. Strikingly, stable overexpression of human NPC1 in chinese hamster ovary cells was associated with enhanced gene uptake (3-fold) and transfection (10-fold) by PBAEs. These findings reveal a role of NPC1 in the regulation of endocytic mechanisms affecting nanoparticle trafficking. We hypothesize that in-depth understanding sites of entry and endosomal escape may lead to highly efficient nanotechnologies for drug delivery.

  20. Molecular engineering of dendritic polymers and their application as drug and gene delivery systems. (United States)

    Paleos, Constantinos M; Tsiourvas, Dimitris; Sideratou, Zili


    This review discusses the development of functional and multifunctional dendrimeric and hyperbranched polymers, collectively called dendritic polymers, with the objective of being applied as drug and gene delivery systems. In particular, using as starting materials known and well-characterized basic dendritic polymers, the review deals with the type of structural modifications to which these dendritic polymers were subjected for the development of drug carriers with low toxicity, high encapsulating capacity, a specificity for certain biological cells, and the ability to be transported through their membranes. Proceeding from functional to multifunctional dendritic polymers, one is able to prepare products that fulfill one or more of these requirements, which an effective drug carrier should exhibit. A common feature of the dendritic polymers is the exhibition of polyvalent interactions, while for multifunctional derivatives, a number of targeting ligands determine specificity, another type of group secures stability in biological milieu and prolonged circulation, while others facilitate their transport through cell membranes. Furthermore, dendritic polymers employed for gene delivery should be or become cationic in the biological environment for the formation of complexes with the negatively charged genetic material.

  1. A peptide-mediated targeting gene delivery system for malignant glioma cells

    Directory of Open Access Journals (Sweden)

    Wang C


    Full Text Available Chuanwei Wang,1,2,* Liping Ning,3,* Hongwei Wang,1,2,* Zaijun Lu,4 Xingang Li,1,2 Xiaoyong Fan,5 Xuping Wang,6 Yuguang Liu1,2 1Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, People's Republic of China; 2Brain Science Research Institute of Shandong University, Jinan, People's Republic of China; 3Department of Rehabilitation, Provincial Hospital Affiliated to Shandong University, Jinan, People's Republic of China; 4School of Chemistry and Chemical Engineering of Shandong University, Jinan, People's Republic of China; 5Department of Neurosurgery, Shandong Qianfoshan Hospital Affiliated to Shandong University, Jinan, People's Republic of China; 6Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan, People's Republic of China *These authors contributed equally to this work Abstract: Glioblastoma multiforme (GBM is the most common and malignant glioma. Although there has been considerable progress in treatment strategies, the prognosis of many patients with GBM remains poor. In this work, polyethylenimine (PEI and the VTWTPQAWFQWV (VTW peptide were modified and synthesized into GBM-targeting nanoparticles. The transfection efficiency of U-87 (human glioblastoma cells was evaluated using fluorescence microscopy and flow cytometry. Cell internalization was investigated to verify the nanoparticle delivery into the cytoplasm. Results showed that the methods of polymer conjugation and the amount of VTW peptide were important factors to polymer synthesis and transfection. The PEI-VTW20 nanoparticles increased the transfection efficiency significantly. This report describes the use of VTW peptide-based PEI nanoparticles for intracellular gene delivery in a GBM cell-specific manner. Keywords: glioblastoma, polyethylenimine, nanoparticles, drug-delivery systems, gene transfer techniques

  2. Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity.

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    Osamu Kakinohana

    Full Text Available 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 (GABA(B 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

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

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    J Barar


    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.

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

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    Michael S. Detamore


    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.

  5. Intracranial gene delivery of LV-NAGLU vector corrects neuropathology in murine MPS IIIB. (United States)

    Di Domenico, Carmela; Villani, Guglielmo R D; Di Napoli, Daniele; Nusco, Edoardo; Calì, Gaetano; Nitsch, Lucio; Di Natale, Paola


    Mucopolysacccharidosis (MPS) IIIB is an inherited lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase (NAGLU). The disease is characterized by mild somatic features and severe neurological involvement with high mortality. Although several therapeutic approaches have been applied to the murine model of the disease, no effective therapy is available for patients. In this study, we used the lentiviral-NAGLU vector to deliver the functional human NAGLU gene into the brain of young adult MPS IIIB mice. We report the restoration of active enzyme with a sustained expression throughout a large portion of the brain, and a significantly improved behavioral performance of treated animals. Moreover, we analyzed the effect of therapy on the expression profile of some genes related to neurotrophic signaling molecules and inflammatory cytokines previously found altered in MPS IIIB mice. At 1 month from treatment, the level of cerebellin 1 (Cbln1) was decreased while the brain-derived neurotrophic factor (Bdnf) expression was increased, both reaching normal values. At 6 months from treatment a significant reduction in the expression of all the inflammation- and oxidative stress-related genes was observed, as well as the maintenance of the correction of the Bdnf gene expression. These results indicate that NAGLU delivery from intracerebral sources has the capacity to alleviate most disease manifestations in MPS IIIB mice; furthermore, Bdnf might be a response-to-therapy biomarker for MPS IIIB.

  6. Efficient gene delivery and silencing of mouse and human pancreatic islets

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    Moerman Ericka


    Full Text Available Abstract Background In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells. Results This study describes a new method of gene and siRNA delivery into human pancreatic islets by microporation technology. We demonstrated that mild islet distention with accutase greatly enhanced the transfection efficiency without compromising in vitro function (secretion, apoptosis and viability. As an example, the recently identified gene involved in type 2 diabetes, ZnT8, can be over-expressed or silenced by RNA interference using this technology. Microporation can also be used on rodent islets. Conclusions Taken together, our results demonstrate that microporation technology can be used to modify gene expression in whole rodent and human islets without altering their in vitro function and will be key to the elucidation of the factors responsible for proper islet function.

  7. Photoluminescent hyperbranched poly(amido amine) containing β-cyclodextrin as a nonviral gene delivery vector. (United States)

    Chen, Yan; Zhou, Linzhu; Pang, Yan; Huang, Wei; Qiu, Feng; Jiang, Xulin; Zhu, Xinyuan; Yan, Deyue; Chen, Qun


    Hyperbranched poly(amido amine)s (HPAAs) containing different amounts of β-cyclodextrin (β-CD) (HPAA-CDs) were synthesized in one-pot by Michael addition copolymerization of N,N'-methylene bisacrylamide, 1-(2-aminoethyl)piperazine, and mono-6-deoxy-6-ethylenediamino-β-CD. In comparison to pure HPAA, the fluorescence intensity of HPAA-CDs was enhanced significantly while the cytotoxicity became lower. Ascribed to plenty of amino groups and strong photoluminescence, HPAA-CDs could be used as nonviral gene delivery vectors, and the corresponding gene transfection was evaluated. The experimental results indicated that HPAA-CDs condensed the plasmid DNA very well. By utilizing the fluorescent properties of HPAA-CDs, the cellular uptake and gene transfection processes were tracked by flow cytometry and confocal laser scanning microscopy without any fluorescent labeling. The transfection efficiencies of HPAA-CDs were similar to that of pure HPAA. In addition, the inner cavities of β-CDs in HPAA-CDs could be used to encapsulate drugs through host--guest interaction. Therefore, the HPAA-CDs may have potential application in the combination of gene therapy and chemotherapy.

  8. Sleeping Beauty Transposon Vectors in Liver-directed Gene Delivery of LDLR and VLDLR for Gene Therapy of Familial Hypercholesterolemia. (United States)

    Turunen, Tytteli A K; Kurkipuro, Jere; Heikura, Tommi; Vuorio, Taina; Hytönen, Elisa; Izsvák, Zsuzsanna; Ylä-Herttuala, Seppo


    Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH.

  9. Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: a strategy to promote cartilage repair. (United States)

    Venkatesan, N; Barré, L; Benani, A; Netter, P; Magdalou, J; Fournel-Gigleux, S; Ouzzine, M


    Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, beta1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1beta down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by (35)S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1beta-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1beta-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1beta. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.

  10. Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice. (United States)

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


    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.

  11. Association with amino acids does not enhance efficacy of polymerized liposomes as a system for lung gene delivery

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    Elga eBernardo Bandeira De Melo


    Full Text Available Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids (1,2-bis-(tricosa-10,12-diynoyl-sn-glycero-3-phosphocholine associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with L-arginine, L-tryptophan, or L-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. L-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases.

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

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    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

  13. Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles. (United States)

    Li, Guang-Feng; Wang, Jing-Cheng; Feng, Xin-Min; Liu, Zhen-Dong; Jiang, Chao-Yong; Yang, Jian-Dong


    The aim of this study was to synthesize a chitosan (CS) derivative, a quaternary ammonium salt crystal called N-2-hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and test a series of HACC and pEGFP-DNA complexes at different weight ratios for their efficiency of gene delivery into human cells. CS was modified with cationic etherifying agent to obtain the CS derivative. Fourier transform infrared spectra were recorded on KBr pellets with a spectrometer. (1)H nuclear magnetic resonance (NMR) spectra of HACC were obtained using a spectrometer. HACC was subsequently used to prepare HACC/DNA complexes at different weight ratios by coacervation method. The resulting particle size and surface charge were assessed by laser light scattering using a zeta potential analyzer. The HACC/DNA complex formation and DNA protection in the nanoparticle complex was investigated by gel mobility shift assay and DNase I protection assay, respectively. The cytotoxicity of HACC and HACC/DNA nanoparticles was evaluated by MTT assay using (mesenchymal stem cell) MSC lines. The nanoscale structure of the particles was obtained by transmission electron microscope (TEM). The FTIR spectrum of HACC showed the characteristic quaternary ammonium group absorption band at 1475 cm(-1), which indicated the presence of quaternary ammonium group. The successful synthesis of HACC was also confirmed by (1)H NMR spectrum. HACC showed good solubility in water and was electropositive. HACC efficiently packed and protected pEGFP-DNA at a weight ratio of 10. With increased weight ratios, the surface charge of the composite particle increased from negative to positive, the average particle size increased, and HACC nanoparticle had a higher carrying efficiency. The nanoparticles released DNA in two distinct phases, and 55 % was released within the first 20 h of solubilization. The nanoparticles under TEM showed circular or oval shapes. The particles exhibited no cytotoxicity against human cells. No

  14. Functional study of dextran-graft-poly((2-dimethyl amino)ethyl methacrylate) gene delivery vector for tumor therapy. (United States)

    Li, Wen-Bin; Yuan, Wei; Xu, Fu-Jian; Zhao, Chen; Ma, Jie; Zhan, Qi-Min


    The obstacle of gene therapy is the shortage of efficient delivery system. The development of the gene delivery system with high transfection efficiency and low toxicity appears to be crucial. Recently, we reported that the dextran-graft-poly((2-dimethyl amino)ethyl methacrylate) (DPD) can be potentially used as efficient gene vector. Herein, DPD was systematically studied for its potential in tumor gene therapy. DPD was synthesized and characterized by agarose gel electrophoresis, particle size and zeta potential. The particle size and zeta potential of the DPD/enhanced green fluorescent protein (pEGFP-C1) plasmid complexes at various N/P ratios were 130-150 nm and about 40 mV, respectively. The results showed that DPD exhibit a higher transfection effect compared with Lipofectamine 2K (Lipo 2K), a commercialized vector. The possibility of DPD in gene therapy was evaluated using p53, a gene that has been wildly applied in the research of cancer gene therapy. DPD/pEGFP-C1-p53 complex was found to be able to inhibit tumor cell proliferation through cell cycle arrest and apoptosis. Moreover, the tumor growth was found to be restrained when DPD/pEGFP-C1-p53 complex was used in a xenograft MCF7 tumor model in vivo. These observations indicated that DPD/pEGFP-C1-p53 complex may be considered to be an efficient delivery system for tumor gene therapy.

  15. A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system

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    Eroglu E


    Full Text Available Erdal Eroglu,1 Pooja M Tiwari,1 Alain B Waffo,1 Michael E Miller,2 Komal Vig,1 Vida A Dennis,1 Shree R Singh1 1Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL, USA; 2Research Instrumentation Facility, Auburn University, AL, USA Abstract: The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs, which consisted of poly (2-hydroxyethyl methacrylate nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV-F gene construct (a model for a DNA vaccine. The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM, fluorescence activated cell sorting (FACS, and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR, we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo. Keywords: pHEMA+chitosan nanoparticles, nonviral vector

  16. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis. (United States)

    Lu, Huading; Dai, Yuhu; Lv, Lulu; Zhao, Huiqing


    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.

  17. CRISPR-Cas9 gene editing: Delivery aspects and therapeutic potential. (United States)

    Oude Blenke, Erik; Evers, Martijn J W; Mastrobattista, Enrico; van der Oost, John


    The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with CRISPR-associated proteins (Cas) are a part of the prokaryotic adaptive immune system and have successfully been repurposed for genome editing in mammalian cells. The CRISPR-Cas9 system has been used to correct genetic mutations and for replacing entire genes, opening up a world of possibilities for the treatment of genetic diseases. In addition, recently some new CRISPR-Cas systems have been discovered with interesting mechanistic variations. Despite these promising developments, many challenges have to be overcome before the system can be applied therapeutically in human patients and enabling delivery technology is one of the key challenges. Furthermore, the relatively high off-target effect of the system in its current form prevents it from being safely applied directly in the human body. In this review, the transformation of the CRISPR-Cas gene editing systems into a therapeutic modality will be discussed and the currently most realistic in vivo applications will be highlighted.

  18. Needle-free gene delivery through the skin: an overview of recent strategies. (United States)

    Elsabahy, Mahmoud; Foldvari, Marianna


    Topical administration is attractive and non-invasive gene delivery approach. It is simple and allows repeated administration. In addition, the skin is active immune surveillance site. Topical gene therapy, although promising for treatment of cancer, dermatological disorders, vaccination and autoimmune disease, has not progressed yet to clinical trials. The inability of nucleic acids to survive the extraand intracellular environment and to permeate through the outermost layer of the skin, the stratum corneum, compromise the therapeutic outcomes of nucleic acids-based therapies. Nanostructured vehicles (e.g. transfersomes, niosomes, nanoemulsions, gemini-lipid nanoparticles and biphasic vesicles) have the ability to partially disrupt and perturb lipids that are found in the skin layers and deliver their nucleic acid cargos to their targeted subcellular compartments. However, the efficiency of these carriers is still inferior to other invasive methods (e.g. epidermal and intradermal injections). The goal of this review is to examine the critical parameters required to enhance the efficiency of the currently available nanostructured vehicles, for example, by combining them with minimally invasive techniques, such as, electroporation, iontophoresis, microneedles, ultrasound, gene gun and femtosecond laser. The recent advances in engineering these nanovectors will be discussed with a focus on their future prospects.

  19. Quaternized chitosan/rectorite intercalative materials for a gene delivery system (United States)

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


    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.

  20. Enzyme-synthesized Poly(amine-co-esters) as Non-viral Vectors for Gene Delivery (United States)

    Liu, Jie; Jiang, Zhaozhong; Zhou, Jiangbing; Zhang, Shengmin; Saltzman, W. Mark


    A family of biodegradable poly(amine-co-esters) was synthesized in one step via enzymatic copolymerization of diesters with amino-substituted diols. Diesters of length C4–C12 (i.e., from succinate to dodecanedioate) were successfully copolymerized with diethanolamines with either an alkyl (methyl, ethyl, n-butyl, t-butyl) or an aryl (phenyl) substituent on the nitrogen. Upon protonation at slightly acidic conditions, these poly(amine-co-esters) readily turned to cationic polyelectrolytes, which were capable of condensing with polyanionic DNA to form nanometer-sized polyplexes. In vitro screening with pLucDNA revealed that two of the copolymers, poly(N-methyldiethyleneamine sebacate) (PMSC) and poly(N-ethyldiethyleneamine sebacate) (PESC), possessed comparable or higher transfection efficiencies compared to Lipofectamine 2000. PMSC/pLucDNA and PESC/pLucDNA nanoparticles had desirable particle sizes (40–70 nm) for cellular uptake and were capable of functioning as proton sponges to facilitate endosomal escape after cellular uptake. These polyplex nanoparticles exhibited extremely low cytotoxicity. Furthermore, in vivo gene transfection experiments revealed that PMSC is a substantially more effective gene carrier than PEI in delivering pLucDNAto cells in tumors in mice. All these properties suggest that poly(amine-co-esters) are promising non-viral vectors for safe and efficient DNA delivery in gene therapy. PMID:21171165

  1. Use of lactobacilli and their pheromone-based regulatory mechanism in gene expression and drug delivery. (United States)

    Diep, D B; Mathiesen, G; Eijsink, V G H; Nes, I F


    Lactobacilli are common microorganisms in diverse vegetables and meat products and several of these are also indigenous inhabitants in the gastro-intestinal (GI) tract of humans and animals where they are believed to have health promoting effects on the host. One of the highly appreciated probiotic effects is their ability to inhibit the growth of pathogens by producing antimicrobial peptides, so-called bacteriocins. Production of some bacteriocins has been shown to be strictly regulated through a quorum-sensing based mechanism mediated by a secreted peptide-pheromone (also called induction peptide; IP), a membrane-located sensor (histidine protein kinase; HPK) and a cytoplasmic response regulator (RR). The interaction between an IP and its sensor, which is highly specific, leads to activation of the cognate RR which in turn binds to regulated promoters and activates gene expression. The HPKs and RRs are built up by conserved modules, and the signalling between them within a network is efficient and directional, and can easily be activated by exogenously added synthetic IPs. Consequently, components from such regulatory networks have successfully been exploited in construction of a number of inducible gene expression systems. In this review, we discuss some well-characterised quorum sensing networks involved in bacteriocin production in lactobacilli, with special focus on the use of the regulatory components in gene expression and on lactobacilli as potential delivery vehicle for therapeutic and vaccine purposes.

  2. Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates. (United States)

    Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise


    Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy.

  3. Novel pH-Sensitive Cationic Lipids with Linear Ortho Ester Linkers for Gene Delivery (United States)

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


    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

  4. Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery. (United States)

    Trubetskoy, V S; Wong, S C; Subbotin, V; Budker, V G; Loomis, A; Hagstrom, J E; Wolff, J A


    The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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. AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease. (United States)

    Bradbury, Allison M; Peterson, Tiffany A; Gross, Amanda L; Wells, Stephen Z; McCurdy, Victoria J; Wolfe, Karen G; Dennis, John C; Brunson, Brandon L; Gray-Edwards, Heather; Randle, Ashley N; Johnson, Aime K; Morrison, Edward E; Cox, Nancy R; Baker, Henry J; Sena-Esteves, Miguel; Martin, Douglas R


    Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Development of a novel gene delivery scaffold utilizing colloidal gold-polyethylenimine conjugates for DNA condensation. (United States)

    Ow Sullivan, M M; Green, J J; Przybycien, T M


    We have developed a novel gene delivery scaffold based on DNA plasmid condensation with colloidal gold/polyethylenimine conjugates. This scaffold system was designed to enable systematic study of the relationships between DNA complex physical properties and transfection efficiency. Using an enhanced green fluorescent protein-coding reporter plasmid and a Chinese hamster ovary cell line, we have measured the transfection efficiencies of our complexes using flow cytometry and their cytotoxicities using the trypan blue assay. We have also assayed complex particle morphologies using atomic force microscopy, photon correlation spectroscopy, and a novel plasmon absorbance peak position analysis. We achieved comparable rates of transfection relative to the commonly used polycationic condensation agents calcium phosphate and LipofectAMINE, with comparably low cytotoxicities. In addition, by manipulating colloidal gold concentration, we could partially decouple complex physical properties including charge ratio, size, DNA loading, and polyethylenimine concentration. Our morphological analyses showed that complexes with a diameter of a few hundred nanometers and a charge ratio of approximately 8 perform best in our transfection efficiency assays. The use of colloidal gold as a component in our delivery system provides a versatile system for manipulating complex properties and morphology as well as a convenient scaffold for planned ligand conjugation studies.

  8. Modular construction of multifunctional bioresponsive cell-targeted nanoparticles for gene delivery. (United States)

    Saeed, Aram O; Magnusson, Johannes P; Moradi, Emilia; Soliman, Mahmoud; Wang, Wenxin; Stolnik, Snow; Thurecht, Kristofer J; Howdle, Steven M; Alexander, Cameron


    Multifunctional and modular block copolymers prepared from biocompatible monomers and linked by a bioreducible disulfide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block copolymer architecture enabled well-defined nanoparticles to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folate-receptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to nonbioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tunable way.

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

    Directory of Open Access Journals (Sweden)

    Abbas Pardakhty


    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. 

  10. Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy. (United States)

    Gori, Jennifer L; Hsu, Patrick D; Maeder, Morgan L; Shen, Shen; Welstead, G Grant; Bumcrot, David


    Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) technology is revolutionizing the study of gene function and likely will give rise to an entire new class of therapeutics for a wide range of diseases. Achieving this goal requires not only characterization of the technology for efficacy and specificity but also optimization of its delivery to the target cells for each disease indication. In this review we survey the various methods by which the CRISPR-Cas9 components have been delivered to cells and highlight some of the more clinically relevant approaches. Additionally, we discuss the methods available for assessing the specificity of Cas9 editing; an important safety consideration for development of the technology.

  11. Gene and Drug delivery system and potential treatment into inner ear for protection and regeneration

    Directory of Open Access Journals (Sweden)

    Sho eKanzaki


    Full Text Available The most common type of hearing loss results from damage to the cochlea including lost hair cells (HCs and spiral ganglion neurons (SGNs. In mammals, cochlear HC loss causes irreversible hearing impairment because this type of sensory cell cannot regenerate. The protection of SGN degeneration has implications for cochlear implant to patients with severe deafness. This review summarizes the several treatments for HC regeneration based on experiments. We discuss how the neurotrophic factor transgene expression can protect SGN degeneration and describe potential new therapeutic interventions to reduce hearing loss.  We also summarized viral vectors and introduced the gene and drug delivery system for cochlear hair cells regeneration and protection. Finally, we introduce the novel endoscopy we developed for local injection into cochlea.

  12. Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation. (United States)

    Tsai, Sen-Wei; Tung, Yu-Tang; Chen, Hsiao-Ling; Yang, Shang-Hsun; Liu, Chia-Yi; Lu, Michelle; Pai, Hui-Jing; Lin, Chi-Chen; Chen, Chuan-Mu


    Muscle atrophy is a common symptom after nerve denervation. Myostatin propeptide, a precursor of myostatin, has been documented to improve muscle growth. However, the mechanism underlying the muscle atrophy attenuation effects of myostatin propeptide in muscles and the changes in gene expression are not well established. We investigated the possible underlying mechanisms associated with myostatin propeptide gene delivery by gene gun in a rat denervation muscle atrophy model, and evaluated gene expression patterns. In a rat botulinum toxin-induced nerve denervation muscle atrophy model, we evaluated the effects of wild-type (MSPP) and mutant-type (MSPPD75A) of myostatin propeptide gene delivery, and observed changes in gene activation associated with the neuromuscular junction, muscle and nerve. Muscle mass and muscle fiber size was moderately increased in myostatin propeptide treated muscles (pmuscle regulatory factors, neurite outgrowth factors (IGF-1, GAP43) and acetylcholine receptors was observed. Our results demonstrate that myostatin propeptide gene delivery, especially the mutant-type of MSPPD75A, attenuates muscle atrophy through myogenic regulatory factors and acetylcholine receptor regulation. Our data concluded that myostatin propeptide gene therapy may be a promising treatment for nerve denervation induced muscle atrophy. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Graphene oxide-cationic polymer conjugates: Synthesis and application as gene delivery vectors. (United States)

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


    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.

  14. Development of genetically flexible mouse models of sarcoma using RCAS-TVA mediated gene delivery.

    Directory of Open Access Journals (Sweden)

    Leah Kabaroff

    Full Text Available Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins KrasG12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively. Disrupting the G1 checkpoint CDKN2 (p16/p19-/- resulted in sarcoma in 30% of p16/p19-/- xN-tva mice with a median latency of 23 weeks (range 8-40 weeks. A similar incidence occurred in p16/p19-/- xBKE mice (32%, however, a shorter median latency (10.4 weeks was observed. p16/p19-/- xAKE mice also developed sarcomas (24% incidence; median 9 weeks yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. KrasG12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research.

  15. 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)


    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Daniela Rodica Radu


    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 Cu{sup 2+} 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 the

  17. 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


    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 a

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


    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. (United States)

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


    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. 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


    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

  1. Stem cell-based delivery of brain-derived neurotrophic factor gene in the rat retina. (United States)

    Park, Hae-Young Lopilly; Kim, Jie Hyun; Sun Kim, Hwa; Park, Chan Kee


    As an alternative to a viral vector, the application of stem cells to transfer specific genes is under investigation in various organs. Using this strategy may provide more effective method to supply neurotrophic factor to the neurodegenerative diseases caused by neurotrophic factor deprivation. This study investigated the possibility and efficacy of stem cell-based delivery of the brain-derived neurotrophic factor (BDNF) gene to rat retina. Rat BDNF cDNA was transduced into rat bone marrow mesenchymal stem cells (rMSCs) using a retroviral vector. Its incorporation into the experimental rat retina and the expression of BDNF after intravitreal injection or subretinal injection were detected by real-time PCR, western blot analysis, and immunohistochemical staining. For the incorporated rMSCs, retinal-specific marker staining was performed to investigate the changes in morphology and the characteristics of the stem cells. Transduction of the rMSCs by retrovirus was effective, and the transduced rMSCs expressed high levels of the BDNF gene and protein. The subretinal injection of rMSCs produced rMSC migration and incorporation into the rat retina (about 15.7% incorporation rate), and retinal BDNF mRNA and protein expression was increased at 4 weeks after transplantation. When subretinal injection of rMSCs was applied to axotomized rat retina, it significantly increased the expression of BDNF until 4 weeks after transplantation. Some of the transplanted rMSCs exhibited morphological changes, but the retinal-specific marker stain was not sufficient to indicate whether neuronal differentiation had occurred. Using mesenchymal stem cells to deliver the BDNF gene to the retina may provide new treatment for glaucoma.

  2. The Synthesis of Cyclic Poly(ethylene imine) and Exact Linear Analogues: An Evaluation of Gene Delivery Comparing Polymer Architectures. (United States)

    Cortez, Mallory A; Godbey, W T; Fang, Yunlan; Payne, Molly E; Cafferty, Brian J; Kosakowska, Karolina A; Grayson, Scott M


    The delivery of genetic material to cells offers the potential to treat many genetic diseases. Cationic polymers, specifically poly(ethylene imine) (PEI), are promising gene delivery vectors due to their inherent ability to condense genetic material and successfully affect its transfection. However, PEI and many other cationic polymers also exhibit high cytotoxicity. To systematically study the effect of polymer architecture on gene delivery efficiency and cell cytotoxicity, a set of cyclic PEIs were prepared for the first time and compared to a set of linear PEIs of the exact same molecular weight. Subsequent in vitro transfection studies determined a higher transfection efficiency for each cyclic PEI sample when compared to its linear PEI analogue in addition to reduced toxicity relative to the branched PEI "gold standard" control. These results highlight the critical role that the architecture of PEI can play in both optimizing transfection and reducing cell toxicity.

  3. Poly(amido amine)s as Gene Delivery Vectors: Effects of Quaternary Nicotinamide Moieties in the Side Chains

    NARCIS (Netherlands)

    Mateos Timoneda, Miguel A.; Lok, Martin C.; Hennink, Wim E.; Feijen, Jan; Engbersen, Johan F.J.


    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

  4. Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery

    NARCIS (Netherlands)

    Cheng, Ru; Feng, Fang; Meng, Fenghua; Deng, Chao; Feijen, Jan; Zhong, Zhiyuan


    The past couple of years have witnessed a tremendous progress in the development of glutathione-responsive nano-vehicles for targeted intracellular drug and gene delivery, as driven by the facts that (i) many therapeutics (e.g. anti-cancer drugs, photosensitizers, and anti-oxidants) and

  5. Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery

    NARCIS (Netherlands)

    Cheng, Ru; Feng, Fang; Meng, Fenghua; Deng, Chao; Feijen, Jan; Zhong, Zhiyuan


    The past couple of years have witnessed a tremendous progress in the development of glutathione-responsive nano-vehicles for targeted intracellular drug and gene delivery, as driven by the facts that (i) many therapeutics (e.g. anti-cancer drugs, photosensitizers, and anti-oxidants) and biotherapeut

  6. Mx1 and IP-10: biomarkers to measure IFN-beta activity in mice following gene-based delivery. (United States)

    Petry, Harald; Cashion, Linda; Szymanski, Paul; Ast, Oliver; Orme, Ann; Gross, Cynthia; Bauzon, Maxine; Brooks, Alan; Schaefer, Caralee; Gibson, Heather; Qian, Husheng; Rubanyi, Gabor M; Harkins, Richard N


    Recombinant interferon-beta (IFN-beta) protein is used successfully for the treatment of multiple sclerosis (MS). Gene therapy might be an alternative approach to overcome drawbacks occurring with IFN-beta protein therapy. A critical issue in developing a new approach is detection of biologically active IFN-beta in preclinical models. The goal of the present study was to determine if Mx1 and IP-10, which are known to be activated after IFN-beta treatment in humans, can be used as biomarkers in mice. In three in vivo experiments, the correlation between different methods of murine IFN-beta (MuIFN-beta) delivery and biomarker induction was studied: (1) bolus protein delivery by intravenous (i.v.) or intramuscular (i.m.) injection, (2) gene-based delivery of IFN- beta by i.m. injection of plasmid DNA, followed by electroporation, and (3) gene-based delivery of IFN-beta by i.m. injection of adenovirus-associated type 1 (AAV1). Short-term induction of Mx1 mRNA and IP-10 was observed after treatment with bolus MuIFN-beta protein. Long-term induction of both biomarkers was observed after IFN-beta plasmid DNA delivery or when AAV1 was used as the vector. The experiments demonstrate that gene-based delivery provides sustained levels of IFN-beta compared with bolus protein injection and that Mx1 RNA and IP-10 can be used to monitor biologically active circulating plasma MuIFN-beta protein in mice.

  7. Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity. (United States)

    Yan, Chen; Jie, Leng; Yongqi, Wang; Weiming, Xiao; Juqun, Xi; Yanbing, Ding; Li, Qian; Xingyuan, Pan; Mingchun, Ji; Weijuan, Gong


    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(+) T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy.

  8. Cross-linked Polyethylenimine as Potential DNA Vector for Gene Delivery with High Efficiency and Low Cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Wei DONG; Guang-Hui JIN; Shu-Feng LI; Qi-Ming SUN; Ding-Yuan MA; Zi-Chun HUA


    Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [ 1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800-4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.

  9. The force analysis for superparamagnetic nanoparticles-based gene delivery in an oscillating magnetic field (United States)

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


    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.

  10. Polyethylene glycol-grafted polyethylenimine used to enhance adenovirus gene delivery. (United States)

    Singarapu, Kumar; Pal, Ivy; Ramsey, Joshua D


    An improved adenoviral-based gene delivery vector was developed by complexing adenovirus (Ad) with a biocompatible, grafted copolymer PEG-g-PEI composed of polyethylene glycol (PEG) and polyethylenimine (PEI). Although an Ad-based gene vector is considered relatively safe, its native tropism, tendency to elicit an immune response, and susceptibility to inactivating antibodies makes the virus less than ideal. The goal of the current study was to determine whether Ad could be complexed with a PEG-g-PEI copolymer that would enable the virus to transduce cells lacking the Ad receptor, while avoiding the issues commonly associated with PEI. A copolymer library was synthesized using 2 kDa PEG and either linear or branched PEI (25 kDa) with a PEG to PEI grafting ratio of 10, 20, or 30. The results of the study indicate that PEG-g-PEI/Ad complexes are indeed able to transduce CAR-negative NIH 3T3 cells. The results also demonstrate that the PEG-g-PEI/Ad complexes are less toxic, less hemolytic, and more appropriately sized than PEI/Ad complexes.

  11. Cationic dialkylarylphosphates: a new family of bio-inspired cationic lipids for gene delivery. (United States)

    Le Corre, Stéphanie S; Belmadi, Nawal; Berchel, Mathieu; Le Gall, Tony; Haelters, Jean-Pierre; Lehn, Pierre; Montier, Tristan; Jaffrès, Paul-Alain


    In this work that aims to synthesize and evaluate new cationic lipids as vectors for gene delivery, we report the synthesis of a series of cationic lipids in which a phosphate functional group acts as a linker to assemble on a molecular scale, two lipid chains and one cationic polar head. The mono or dicationic moiety is connected to the phosphate group by an aryl spacer. In this work, two synthesis strategies were evaluated. The first used the Atherton-Todd coupling reaction to introduce a phenolic derivative to dioleylphosphite. The second strategy used a sequential addition of lipid alcohol and a phenolic derivative on POCl3. The two methods are efficient, but the latter allows larger yields. Different polar head groups were introduced, thus producing amphiphilic compounds possessing either one permanent (N-methyl-imidazolium, pyridinium, trimethylammonium) or two permanent cationic charges. All these cationic lipids were formulated as liposomal solutions and characterized (size and zeta potential). They formed stable liposomal solutions both in water (at pH 7.0) and in a weakly acidic medium (at pH 5.5). Finally, this new generation of cationic lipids was used to deliver DNA into various human-derived epithelial cells cultured in vitro. Compared with Lipofectamine used as a reference commercial lipofection reagent, some cationic dialkylarylphosphates were able to demonstrate potent gene transfer abilities, and noteworthily, monocationic derivatives were much more efficient than dicationic analogues.

  12. Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery. (United States)

    Liu, Ya; Kong, Ming; Cheng, Xiao Jie; Wang, Qian Qian; Jiang, Li Ming; Chen, Xi Guang


    The present work described nanoparticles (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) using coacervation process as novel potential carriers for gene delivery. An N/P ratio of 5 and OCMCS/HA weight ratio of 4 were the optimal conditions leading to the smallest (164.94 nm), positive charged (+14.2 mV) and monodispersed NPs. OCMCS-HA/DNA (OHD) NPs showed higher in vitro DNA release rates and increased cellular uptake by Caco-2 cells due to the HA involved in NPs. The MTT survival assay indicated no significant cytotoxicity. The transfection efficiency of OHD NPs was 5-fold higher than OCMCS/DNA (OD) NPs; however, it decreased significantly in the presence of excess free HA. The results indicated that OHD NPs internalized in Caco-2 cells were mediated by the hyaluronan receptor CD44. The data obtained in the present research gave evidence of the potential of OHD NPs for the targeting and further transfer of genes to the epithelial cells.

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

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    Jeffrey A. Kazzaz


    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.

  14. Effect of a Novel Nonviral Gene Delivery of BMP-2 on Bone Healing

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    P. Schwabe


    Full Text Available Background. Gene therapeutic drug delivery approaches have been introduced to improve the efficiency of growth factors at the site of interest. This study investigated the efficacy and safety of a new nonviral copolymer-protected gene vector (COPROG for the stimulation of bone healing. Methods. In vitro, rat osteoblasts were transfected with COPROG + luciferase plasmid or COPROG + hBMP-2 plasmid. In vivo, rat tibial fractures were intramedullary stabilized with uncoated versus COPROG+hBMP-2-plasmid-coated titanium K-wires. The tibiae were prepared for biomechanical and histological analyses at days 28 and 42 and for transfection/safety study at days 2, 4, 7, 28, and 42. Results. In vitro results showed luciferase expression until day 21, and hBMP-2-protein was measured from day 2 – day 10. In vivo, the local application of hBMP-2-plasmid showed a significantly higher maximum load after 42 days compared to that in the control. The histomorphometric analysis revealed a significantly less mineralized periosteal callus area in the BMP-2 group compared to the control at day 28. The rt-PCR showed no systemic biodistribution of luciferase RNA. Conclusion. A positive effect on fracture healing by nonviral BMP-2 plasmid application from COPROG-coated implants could be shown in this study; however, the effect of the vector may be improved with higher plasmid concentrations. Transfection showed no biodistribution to distant organs and was considered to be safe.

  15. Development of a DNA-liposome complex for gene delivery applications. (United States)

    Rasoulianboroujeni, M; Kupgan, G; Moghadam, F; Tahriri, M; Boughdachi, A; Khoshkenar, P; Ambrose, J J; Kiaie, N; Vashaee, D; Ramsey, J D; Tayebi, L


    The association structures formed by cationic liposomes and DNA (Deoxyribonucleic acid)-liposome have been effectively utilized as gene carriers in transfection assays. In this research study, cationic liposomes were prepared using a modified lipid film hydration method consisting of a lyophilization step for gene delivery applications. The obtained results demonstrated that the mean particle size had no significant change while the polydispersity (PDI) increased after lyophilization. The mean particle size slightly reduced after lyophilization (520±12nm to 464±25nm) while the PDI increased after lyophilization (0.094±0.017 to 0.220±0.004). In addition. The mean particle size of vesicles increases when DNA is incorporated to the liposomes (673±27nm). According to the Scanning Electron Microscopy (SEM) and transmission electron microscopy (TEM) images, the spherical shape of liposomes confirmed their successful preservation and reconstitution from the powder. It was found that liposomal formulation has enhanced transfection considerably compared to the naked DNA as negative control. Finally, liposomal formulation in this research had a better function than Lipofectamine® 2000 as a commercialized product because the cellular activity (cellular protein) was higher in the prepared lipoplex than Lipofectamine® 2000. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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    Duan S


    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

  17. Assessment of cholesterol-derived ionic copolymers as potential vectors for gene delivery. (United States)

    Sevimli, Sema; Sagnella, Sharon; Kavallaris, Maria; Bulmus, Volga; Davis, Thomas P


    A library of cholesterol-derived ionic copolymers were previously synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization as 'smart' gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA)) and cationic poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P(DMAEMA-co-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P(DMAEMA-co-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P(MAA-co-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P(MAA-co-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P(DMAEMA-co-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P(MAA-co-CMA)-DNA) and 20:1 (Q-P(DMAEMA-co-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q

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

    Directory of Open Access Journals (Sweden)

    Liang X


    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

  19. Ternary nanoparticles composed of cationic solid lipid nanoparticles, protamine, and DNA for gene delivery

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    He SN


    Full Text Available Sai-Nan He,1 Yun-Long Li,1,2 Jing-Jing Yan,2 Wei Zhang,2 Yong-Zhong Du,2 He-Yong Yu,1 Fu-Qiang Hu,2 Hong Yuan21Women’s Hospital, 2College of Pharmaceutical Sciences, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of ChinaBackground: The objective of this research was to design an effective gene delivery system composed of cationic solid lipid nanoparticles (SLNs, protamine, and Deoxyribonucleic acid DNA.Methods: Cationic SLNs were prepared using an aqueous solvent diffusion method with octadecylamine as the cationic lipid material. First, protamine was combined with DNA to form binary protamine/DNA nanoparticles, and the ternary nanoparticle gene delivery system was then obtained by combining binary protamine/DNA nanoparticles with cationic SLNs. The size, zeta potential, and ability of the binary and ternary nanoparticles to compact and protect DNA were characterized. The effect of octadecylamine content in SLNs and the SLNS/DNA ratios on transfection efficiency, cellular uptake and cytotoxicity of the ternary nanoparticles were also assessed using HEK293 cells.Results: When the weight ratio of protamine to DNA reached 1.5:1, the plasmid DNA could be effectively compacted and protected. The average hydrodynamic diameter of the ternary nanoparticles when combined with protamine increased from 188.50 ± 0.26 nm to 259.33 ± 3.44 nm, and the zeta potential increased from 25.50 ± 3.30 mV to 33.40 ± 2.80 mV when the weight ratio of SLNs to DNA increased from 16/3 to 80/3. The ternary nanoparticles showed high gene transfection efficiency compared with LipofectamineTM 2000/DNA nanoparticles. Several factors that might affect gene transfection efficiency, such as content and composition of SLNs, post-transfection time, and serum were examined. The ternary nanoparticles composed of SLNs with 15 wt% octadecylamine (50/3 weight ratio of SLNs to DNA showed the best transfection efficiency (26.13% ± 5.22% in the presence of

  20. RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells. (United States)

    Kong, Lingdan; Alves, Carla S; Hou, Wenxiu; Qiu, Jieru; Möhwald, Helmuth; Tomás, Helena; Shi, Xiangyang


    We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

  1. A RGD-Containing Oligopeptide (K)16GRGDSPC: A Novel Vector for Integrin-Mediated Targeted Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    PAN Haitao; ZHENG Qixin; GUO Xiaodong; LIU Yong; LI Changwen; SONG Yulin


    A 23 amino acid, bifunctional integrin-targeted synthetic oligopeptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). Synthesis of the peptide (K)16GRGDSPC was performed on a solid-phase batch peptide synthesizer. BMSCs were transfected with plasmid DNA coding for luciferase by (K)16GRGDSPC and the transfection efficiency was assayed. The influences of chloroquine and polyethyleneimine on the transfection efficiency were also examined. The target specificity of (K)16GRGDSPC to mediate exogenous gene into BMSCs was analyzed using cell attachment test and gene delivery inhibition test. The results showed that the transfection efficiency of the oligopeptide vector was lower than that of Lipofectamine. But in the presence of endosomal buffer chloroquine or endosomal disrupting agent polyethyleneimine, the transfection efficiency of the vector was greatly enhanced. In addition, RGD-containing peptides inhibited BMSCs' attachment to the 96-well plates pretreated with fibronectin or vitronectin and significantly decreased the transfection efficiency of the oligopeptide vector. These studies demonstrated that oligopeptide (K)16GRGDSPC was an ideal novel targeted non-viral gene delivery vector, which was easy to be synthesized, high efficient and low cytotoxicity. The vector could effectively deliver exogenous gene into rat BMSCs.

  2. Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine

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    Wu Chaoqun


    Full Text Available Abstract Background Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB transposon and polyethylenimine (PEI for long-term transgene expression in mouse ovarian tumors. Methods A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk and the monomeric red fluorescent protein (mRFP1 under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Results Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Conclusion Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used

  3. Size effect on transfection and cytotoxicity of nanoscale plasmid DNA/polyethyleneimine complexes for aerosol gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Hoon Byeon, Jeong, E-mail: [Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 (United States); Kim, Jang-Woo, E-mail: [Department of Digital Display Engineering, Hoseo University, Asan 336-795 (Korea, Republic of)


    Nanoscale plasmid DNA (pDNA)/polyethyleneimine (PEI) complexes were fabricated in the aerosol state using a nebulization system consisting of a collison atomizer and a cool-walled diffusion dryer. The aerosol fabricated nanoscale complexes were collected and employed to determine fundamental properties of the complexes, such as size, structure, surface charge, and in vitro gene transfection efficiency and cytotoxicity. The results showed that mass ratio between pDNA and PEI should be optimized to enhance gene transfection efficiency without a significant loss of cell viability. These findings may support practical advancements in the field of nonviral gene delivery.

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

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


    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.

  5. Synthesis and evaluation of cationic nanomicelles for in vitro and in vivo gene delivery (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 (p<0.05) over passive (naked DNA) or positive (FuGENE HD) controls. Additionally, a sustained and efficient expression 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

  6. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles. (United States)

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Chuan, Tjin Swee; Yong, Ken-Tye


    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

  7. Optimizing Cationic and Neutral Lipids for Efficient Gene Delivery at High Serum Content (United States)

    Majzoub, Ramsey N.; Hwu, Yeu-kuang; Liang, Keng S.; Leal, Cecília; Safinya, Cyrus R.


    Background Cationic liposome (CL)-DNA complexes are promising gene delivery vectors with potential applications in gene therapy. A key challenge in creating CL-DNA complexes for applications is that their transfection efficiency (TE) is adversely affected by serum. In particular, little is known about the effects of high serum contents on TE even though this may provide design guidelines for applications in vivo. Methods We prepared CL-DNA complexes in which we varied the neutral lipid (DOPC, 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). Results 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, in particular at high serum content. Conclusions 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 suggest guidelines for optimizing the serum resistance of CL-DNA complexes based on a given cationic lipid. PMID:24753287

  8. Relaxin treatment of solid tumors: effects on electric field-mediated gene delivery. (United States)

    Henshaw, Joshua; Mossop, Brian; Yuan, Fan


    Pulsed electric fields have been shown to enhance interstitial transport of plasmid DNA (pDNA) in solid tumors in vivo. However, the extent of enhancement is still limited partly due to the collagen component in extracellular matrix. To this end, effects of collagen remodeling on interstitial electrophoresis were investigated by pretreatment of tumor-bearing mice with a recombinant human relaxin (rh-Rlx). In the study, two tumor lines (4T1 and B16.F10) were examined and implanted s.c. to establish two murine models: dorsal skin-fold chamber (DSC) and hind leg. Effects of rh-Rlx on pDNA electrophoresis were measured either directly in the DSC model or indirectly in the hind leg model via reporter gene expression. It was observed that rh-Rlx treatment reduced collagen levels in the hind leg tumors but not in the DSC tumors. The observation correlated with the results from electromobility experiments, where rh-Rlx treatment enhanced transgene expression in 4T1 hind leg tumors but did not increase the electromobility of pDNA in the DSC tumors. In addition, it was observed that pDNA binding to collagen could block its diffusion in collagen gel in vitro. These observations showed that effects of rh-Rlx on the collagen content depended on microenvironment in solid tumors and that rh-Rlx treatment would enhance electric field-mediated gene delivery only if it could effectively reduce the collagen content in collagen-rich tumors.

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

    Directory of Open Access Journals (Sweden)

    Andrew H. Baker


    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

  10. Role of Cell Membrane-Vector Interactions in Successful Gene Delivery. (United States)

    Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M


    Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors. However, determining the relative importance of these processes and the vector properties necessary for optimization remain a challenge to the field. In this Account, we describe over a decade of studies focused on understanding the interaction of cationic polymer and cationic polymer/oligonucleotide (polyplex) interactions with model lipid membranes, cell membranes, and cells in culture. In particular, we have been interested in how the interaction between cationic polymers and the membrane influences the intracellular transport of intact DNA to the nucleus. Recent advances in microfluidic patch clamp techniques enabled us to quantify polyplex cell membrane interactions at the cellular level with precise control over material concentrations and exposure times. In attempting to relate these findings to subsequent intracellular transport of DNA and expression of protein, we needed to develop an approach that could distinguish DNA that was intact and potentially functional for gene expression from the much larger pool of degraded, nonfunctional DNA within the cell. We addressed this need by developing a FRET oligonucleotide molecular beacon (OMB) to monitor intact DNA transport. The research highlighted in this Account builds to the conclusion that polyplex

  11. Kallistatin in blood pressure regulation transgenic and somatic gene delivery studies. (United States)

    Chao, J; Chao, L


    Kallistatin, first discovered as a human kallikrein-binding protein in the circulation, shares high homology with other plasma serine proteinase inhibitors (serpins). It forms a covalently linked complex with tissue kallikrein and inhibits kallikrein's activity. Substantial evidence has accumulated in recent years indicating that kallistatin may play a role in blood pressure regulation independent of its interaction with tissue kallikrein. Intravenous injection of kallistatin into rats and mice results in a rapid and transient reduction of blood pressure in a dose-dependent manner. Functional analysis in transgenic mice over-expressing rat kallikrein-binding protein, an analogue of human kallistatin, revealed that these mice have significantly lower blood pressure compared with control littermates. Adenovirus-mediated delivery of the human kallistatin gene can cause significant blood pressure reductions for 4 weeks in spontaneously hypertensive rats. Finally, kallistatin can induce vasorelaxation in isolated rat aortic rings and reduce renal perfusion pressure in the isolated, perfused kidney. Together, these findings suggest a direct role for kallistatin in regulating blood pressure and raise the possibility for the development of new pharmacological treatments for hypertension. (Trends Cardiovasc Med 1997;7:307-311). © 1997, Elsevier Science Inc.

  12. Ultrasound-mediated gene and drug delivery using a microbubble-liposome particle system. (United States)

    Yoon, Young Il; Kwon, Yong-Su; Cho, Hee-Sang; Heo, Sun-Hee; Park, Kyeong Soon; Park, Sang Gyu; Lee, Soo-Hong; Hwang, Seung Il; Kim, Young Il; Jae, Hwan Jun; Ahn, Gook-Jun; Cho, Young-Seok; Lee, Hakho; Lee, Hak Jong; Yoon, Tae-Jong


    Theranostic agents present a promising clinical approach for cancer detection and treatment. We herein introduce a microbubble and liposome complex (MB-Lipo) developed for ultrasound (US) imaging and activation. The MB-Lipo particles have a hybrid structure consisting of a MB complexed with multiple Lipos. The MB components are used to generate high echo signals in US imaging, while the Lipos serve as a versatile carrier of therapeutic materials. MB-Lipo allows high contrast US imaging of tumor sites. More importantly, the application of high acoustic pressure bursts MBs, which releases therapeutic Lipos and further enhances their intracellular delivery through sonoporation effect. Both imaging and drug release could thus be achieved by a single US modality, enabling in situ treatment guided by real-time imaging. The MB-Lipo system was applied to specifically deliver anti-cancer drug and genes to tumor cells, which showed enhanced therapeutic effect. We also demonstrate the clinical potential of MB-Lipo by imaging and treating tumor in vivo.

  13. Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery. (United States)

    Thibault, Marc; Lavertu, Marc; Astolfi, Mélina; Buschmann, Michael D


    Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.

  14. Bio-reducible polycations from ring-opening polymerization as potential gene delivery vehicles. (United States)

    Yu, Qing-Ying; Liu, Yan-Hong; Huang, Zheng; Zhang, Ji; Luan, Chao-Ran; Zhang, Qin-Fang; Yu, Xiao-Qi


    Synthetic polycations show great potential for the construction of ideal non-viral gene delivery systems. Several cationic polymers were synthesized by the epoxide ring-opening polymerization between diepoxide and various polyamines. Disulfide bonds were introduced to afford the polymers bio-reducibility, while the oxygen-rich structure might enhance the serum tolerance and biocompatibility. The polycations have much lower molecular weights than PEI 25 kDa, but still could well bind and condense DNA into nano-sized particles. DNA could be released from the polyplexes by addition of reductive DTT. Compared to PEI, the polycations have less cytotoxicity possibly due to their lower molecular weights and oxygen-rich structure. More significantly, these materials exhibit excellent serum tolerance than PEI, and up to 6 times higher transfection efficiency than PEI could be obtained in the presence of serum. The transfection mediated by was seldom affected even at a high concentration of serum. Much lower protein adsorption of polycations than PEI was proved by bovine serum albumin adsorption experiments. Flow cytometry also demonstrates their good serum resistance ability.

  15. Synthesis of water-based cationic polyurethane for antibacterial and gene delivery applications. (United States)

    Wu, Geng-Hsi; Hsu, Shan-Hui


    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.

  16. 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


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

    Directory of Open Access Journals (Sweden)

    Jesse B Owens


    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.

  19. Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genesimplicated in human melanoma%Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genes implicated in human melanoma

    Institute of Scientific and Technical Information of China (English)

    Andrea J. McKinney; Sheri L. Holmen


    The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern. Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%. Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies. However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance. Several different genetic alterations identified in human melanoma have been recapitulated in mice. This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease. We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.

  20. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    Energy Technology Data Exchange (ETDEWEB)

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC


    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  1. Brain-targeted co-delivery of therapeutic gene and peptide by multifunctional nanoparticles in Alzheimer's disease mice. (United States)

    Liu, Yang; An, Sai; Li, Jianfeng; Kuang, Yuyang; He, Xi; Guo, Yubo; Ma, Haojun; Zhang, Yu; Ji, Bin; Jiang, Chen


    Multifunctional nanocarriers are increasingly promising for disease treatment aimed to regulate multiple pathological dysfunctions and overcome barriers in drug delivery. Here we develop a multifunctional nanocarrier for Alzheimer's disease (AD) treatment by achieving therapeutic gene and peptide co-delivery to brain based on PEGylated dendrigraft poly-l-lysines (DGLs) via systemic administration. The dendritic amine-rich structure of DGLs provides plenty reaction sites and positive charge for drug loading. Successful co-delivery of drugs overcoming the blood-brain barrier by brain-targeted ligand modification was demonstrated both in vitro and in vivo. The pharmacodynamics study of the system following multiple-dosing treatment was verified in transgenic AD mice. Down-regulation of the key enzyme in amyloid-β formation was achieved by delivering non-coding RNA plasmid. Simultaneous delivery of the therapeutic peptide into brain leads to reduction of neurofibrillary tangles. Meanwhile, memory loss rescue in AD mice was also observed. Taken together, the multifunctional nanocarrier provides an excellent drug co-delivery platform for brain diseases.

  2. Principles of electrostatic interactions and self-assembly in lipid/peptide/DNA systems: applications to gene delivery. (United States)

    Berezhnoy, Nikolay V; Korolev, Nikolay; Nordenskiöld, Lars


    Recently, great progress has been achieved in development of a wide variety of formulations for gene delivery in vitro and in vivo, which include lipids, peptides and DNA (LPD). Additionally, application of natural histone-DNA complexes (chromatin) in combination with transfection lipids has been suggested as a potential route for gene delivery (chromofection). However, the thermodynamic mechanisms responsible for formation of the ternary lipid-peptide-DNA supramolecular structures have rarely been analyzed. Using recent experimental studies on LPD complexes (including mixtures of chromatin with cationic lipids) and general polyelectrolyte theory, we review and analyze the major determinants defining the internal structure, particle composition and size, surface charge and ultimately, transfection properties of the LPD formulations.

  3. Peroxisome proliferator-activated receptor subtype- and cell-type-specific activation of genomic target genes upon adenoviral transgene delivery

    DEFF Research Database (Denmark)

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G


    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral...... delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes....... We demonstrate that PPARgamma2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARgamma2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (alpha, gamma, and beta...

  4. Gene delivery to brain cells with apoprotein E derived peptide conjugated to polylysine (apoEdp-PLL). (United States)

    Mousazadeh, Mohammad; Palizban, Abbasali; Salehi, Rasool; Salehi, Mansoor


    A promising strategy to carry genetic material to brain cells either in vitro or in vivo is using the LDL receptor (LDLr) on blood-brain barrier. LDLr naturally help to low density lipoproteins (LDL(S)) transporting across the BBB by endocytosis. Here we present the idea of using the LDLr-mediated pathway for transporting genetic material to brain cells. A tandem dimer Sequence of apoprotein-E (apoE) (141-150) conjugated to polylysine sequence was used as a novel DNA Delivery vector for transfecting of brain cells either in vitro or in vivo. DNA condensation occurs with this vector because electrostatic interaction between DNA and polylysine. The vector favors to protection of DNA from enzymatic degradation and also helps to DNA carrying in blood stream to reach BBB and transport it to brain cells and eventually help DNA expression in target cells. These results suggest a novel gene delivery vector for gene therapy of brain disease.

  5. Small-Molecule End-Groups of Linear Polymer Determine Cell-type Gene-Delivery Efficacy. (United States)

    Sunshine, Joel; Green, Jordan J; Mahon, Kerry P; Yang, Fan; Eltoukhy, Ahmed A; Nguyen, David N; Langer, Robert; Anderson, Daniel G


    End-modified polymers are promising for the nonviral delivery of genes to cancer cells, immune cells, and human stem cells and point to polymer end-groups as regulators for cell-type specificity. A library of polymers has been synthesized and, although some polymers are strong transfection agents overall, for each cell type, a particular polymer is most effective. Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Herpesvirus-mediated gene delivery into the rat brain: specificity and efficiency of the neuron-specific enolase promoter. (United States)

    Andersen, J K; Frim, D M; Isacson, O; Breakefield, X O


    1. Herpesvirus infection with genetically engineered vectors is a way to deliver foreign gene products to various cell populations in culture and in vivo. Selective neuronal gene expression can be achieved using the neuron-specific enolase (NSE) promoter regulating expression of a transgene placed in and delivered by a herpesvirus vector. 2. We sought to determine the anatomical specificity and efficiency of herpesvirus-mediated gene transfer into the rat brain following placement of virus particles carrying a transgene (lacZ) under control of the NSE promoter. The virus utilized was thymidine kinase (TK) deficient and therefore replication deficient in the brain. 3. Infusion of 10(6) plaque-forming units of virus into the striatum caused a limited number of striatal neurons to express the lacZ transgene mRNA and protein product 7 days postinfection. In addition, small numbers of neurons expressing the transgene mRNA and protein were found ipsilateral to the viral injection in the frontal cortex, substantia nigra pars compacta, and thalamus. Neurons at these anatomic loci project directly to the striatal injection site. No other cells within the brains of injected animals expressed the lacZ gene. 4. While this herpesvirus NSE vector was capable of introducing novel functional genetic information into postmitotic neurons within defined neuroanatomic constraints, the numbers of neurons expressing detectable levels of beta-galactosidase was minimal. The calculated efficiency of delivery and transgene expression at 7 days postinfection was 1 transgenic neuron per 10(4) virus particles infused. 5. We conclude that NSE probably is not an optimal promoter for use in gene delivery to CNS neurons in herpesvirus vectors and that the efficacy of gene delivery using other neuron-specific promoters placed at various sites in the herpes viral genome needs to be explored.

  7. Cytosolic mRNA Target and Bioavailability of Nanoparticulate siRNA delivery systems for gene silencing. (United States)

    Leucuta, Sorin Emilian


    Recent research in medical and pharmaceutical sciences has benefited from advances in molecular biology and genetics, which made possible a diagnosis at the molecular level in more and more diseases. This implies the drug treatment at the molecular level. The interest in Ribonucleic acid interference (RNAi) is based on the mechanism operates by eliminating the messenger RNAs (mRNAs) coding for multiple proteins, which open solutions for treating many types of diseases. Small (short) interfering RNA (siRNA) has quickly been established as an effective gene-silencing strategy in animal models, and more recently in human clinical trials, as a potential therapeutic approach. Various nanoparticulate drug delivery systems for siRNA delivery have been explored extensively. However, there are many more barriers and challenges that need to be addressed and overcome to achieve the ideal formulation in terms of selectivity, efficacy and safety. One of the major causes of the drawback of these treatments is the difficulty to transport the nucleic acids in the cytosol and organelles. These delivery systems will favorably alter the pharmacokinetics and biodistribution of siRNAs, should be biocompatible and genocompatible to avoid immune stimulation and off-target gene effects. These properties are essential for systemic use, as they prolong siRNA half-lives in blood and increase intracellular bioavailability of siRNA. Future research needs drug delivery systems with more effective design, enhanced biological stability, subcellular bioavailability, and efficient targeted delivery in vivo for improved targeting and specificity of siRNA molecules for any given clinical condition. The paper shows how to overcome physiological barriers to achieve the target, and examples in which significant results were obtained in therapeutic in vitro and in vivo research including nanoparticulate systems.To day, only a few nanoparticle-based siRNA delivery systems have been approved by the Food

  8. Optimization of transfection parameters for ultrasound/SonoVue microbubble-mediated hAng-1 gene delivery in vitro. (United States)

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


    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.

  9. 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: [Second Affiliated Hospital, Sun Yat-sen (Zhongshan) University, Guangzhou 510102 (China); Zhang, Li-Ming, E-mail: [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)


    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.

  10. Amphiphilic graft copolymer based on poly(styrene-co-maleic anhydride with low molecular weight polyethylenimine for efficient gene delivery

    Directory of Open Access Journals (Sweden)

    Duan XP


    Full Text Available Xiaopin Duan,1,2 Jisheng Xiao,2 Qi Yin,2 Zhiwen Zhang,2 Shirui Mao,1 Yaping Li21School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 2Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, ChinaBackground and methods: A new amphiphilic comb-shaped copolymer (SP was synthesized by conjugating poly(styrene-co-maleic anhydride with low molecular weight polyethyleneimine for gene delivery. Fourier transform infrared spectrum, 1H nuclear magnetic resonance, and gel permeation chromatography were used to characterize the graft copolymer.Results: The buffering capability of SP was similar to that of polyethyleneimine within the endosomal pH range. The copolymer could condense DNA effectively to form complexes with a positive charge (13–30 mV and a small particle size (130–200 nm at N/P ratios between 5 and 20, and protect DNA from degradation by DNase I. In addition, SP showed much lower cytotoxicity than polyethyleneimine 25,000. Importantly, the gene transfection activity and cellular uptake of SP-DNA complexes were all markedly higher than that of complexes of polyethyleneimine 25,000 and DNA in MCF-7 and MCF-7/ADR cell lines.Conclusion: This work highlights the promise of SP as a safe and efficient synthetic vector for DNA delivery.Keywords: poly(styrene-co-maleic anhydride, polyethylenimine, DNA, gene delivery

  11. Herpesvirus saimiri-mediated delivery of the adenomatous polyposis coli tumour suppressor gene reduces proliferation of colorectal cancer cells. (United States)

    Macnab, Stuart A; Turrell, Susan J; Carr, Ian M; Markham, Alex F; Coletta, P Louise; Whitehouse, Adrian


    Colorectal cancer (CRC) is a major cause of cancer-related mortality. A contributing factor to the progression of this disease is sporadic or hereditary mutation of the adenomatous polyposis coli (APC) gene, a negative regulator of the Wnt signalling pathway. Inherited mutations in APC cause the disorder familial adenomatous polyposis (FAP), which leads to CRC development in early adulthood. However, the gene is also disrupted in some 60% of sporadic cancers. Restoration of functional APC may slow the growth of CRC by negatively regulating proliferation-associated genes such as c-myc. Therefore, we have cloned the cDNA of the APC tumour suppressor gene into a replication competent Herpesvirus saimiri (HVS)-based vector to assess APC gene delivery in SW480 and SW620 CRC cell lines. Our results demonstrate that full length APC protein was efficiently expressed from the HVS vector and that transgene expression inhibited proliferation of both the SW480 and the metastatic SW620 cancer cell lines. Moreover, a sustained effect could be observed for at least 8 weeks after initial infection in SW480 cells. In addition, monolayer wounding assays showed a marked reduction in proliferation and migration in HVS-GFP-APC infected cells. We believe that this is the first instance of infectious delivery and APC cDNA expression from a virus-based vector.

  12. In Vivo Bio-distribution and Efficient Tumor Targeting of Gelatin/Silica Nanoparticles for Gene Delivery (United States)

    Zhao, Xueqin; Wang, Jun; Tao, SiJie; Ye, Ting; Kong, Xiangdong; Ren, Lei


    The non-viral gene delivery system is an attractive alternative to cancer therapy. The clinical success of non-viral gene delivery is hampered by transfection efficiency and tumor targeting, which can be individually overcome by addition of functional modules such as cell penetration or targeting. Here, we first engineered the multifunctional gelatin/silica (GS) nanovectors with separately controllable modules, including tumor-targeting aptamer AGRO100, membrane-destabilizing peptide HA2, and polyethylene glycol (PEG), and then studied their bio-distribution and in vivo transfection efficiencies by contrast resonance imaging (CRI). The results suggest that the sizes and zeta potentials of multifunctional gelatin/silica nanovectors were 203-217 nm and 2-8 mV, respectively. Functional GS-PEG nanoparticles mainly accumulated in the liver and tumor, with the lowest uptake by the heart and brain. Moreover, the synergistic effects of tumor-targeting aptamer AGRO100 and fusogenic peptide HA2 promoted the efficient cellular internalization in the tumor site. More importantly, the combined use of AGRO100 and PEG enhanced tumor gene expression specificity and effectively reduced toxicity in reticuloendothelial system (RES) organs after intravenous injection. Additionally, low accumulation of GS-PEG was observed in the heart tissues with high gene expression levels, which could provide opportunities for non-invasive gene therapy.

  13. Gene delivery of TGF-β1 induces arthrofibrosis and chondrometaplasia of synovium in vivo. (United States)

    Watson, Rachael S; Gouze, Elvire; Levings, Padraic P; Bush, Marsha L; Kay, Jesse D; Jorgensen, Marda S; Dacanay, E Anthony; Reith, John W; Wright, Thomas W; Ghivizzani, Steven C


    To understand the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver and overexpress transforming growth factor-beta 1 (TGF-β1) cDNA (Ad.TGF-β1) in the knee joints of immunocompromised rats. Following delivery, animals were killed periodically, and joint tissues were examined macroscopically and histologically. PCR-array was used to assay alterations in expression patterns of extracellular matrix (ECM)-associated genes. By days 5 and 10, TGF-β1 induced an increase in knee diameter and complete encasement of joints in dense scar-like tissue, locking joints at 90° of flexion. Histologically, massive proliferation of synovial fibroblasts was seen, followed by their differentiation into myofibroblasts. The fibrotic tissue displaced the normal architecture of the joint capsule and fused with articular cartilage. RNA expression profiles showed high levels of transcription of numerous MMPs, matricellular and ECM proteins. By day 30, the phenotype of the fibrotic tissue had undergone chondrometaplasia, indicated by cellular morphology, matrix composition and >100-fold increases in expression of collagen type II and cartilage link protein. Pre-labeling of articular cells by injection with recombinant lentivirus containing eGFP cDNA showed fibrotic/cartilaginous tissues appeared to arise almost entirely from local proliferation and differentiation of resident fibroblasts. Altogether, these results indicate that TGF-β1 is a potent inducer of arthrofibrosis, and illustrate the proliferative potential and plasticity of articular fibroblasts. They suggest the mechanisms causing arthrofibrosis share many aspects with tumorigenesis.

  14. Association of Combined Maternal-Fetal TNF-α Gene G308A Genotypes with Preterm Delivery: A Gene-Gene Interaction Study

    Directory of Open Access Journals (Sweden)

    Mingbin Liang


    Full Text Available Preterm delivery (PTD is a complicated perinatal adverse event. We were interested in association of G308A polymorphism in tumor necrosis factor-α (TNF-α gene with PTD; so we conducted a genetic epidemiology study in Anqing City, Anhui Province, China. Case families and control families were all collected between July 1999 and June 2002. To control potential population stratification as we could, all eligible subjects were ethnic Han Chinese. 250 case families and 247 control families were included in data analysis. A hybrid design which combines case-parent triads and control parents was employed, to test maternal-fetal genotype (MFG incompatibility. The method is based on a log-linear modeling approach. In summary, we found that when the mother's or child's genotype was G/A, there was a reduced risk of PTD; however when the mother's or child's genotype was genotype A/A, there was a relatively higher risk of PTD. Combined maternal-fetal genotype GA/GA showed the most reduced risk of PTD. Comparison of the LRTs showed that the model with maternal-fetal genotype effects fits significantly better than the model with only maternal and fetal genotype main effects (log-likelihood = −719.4, P=.023, significant at 0.05 level. That means that the combined maternal-fetal genotype incompatibility was significantly associated with PTD. The model with maternal-fetal genotype effects can be considered a gene-gene interaction model. We claim that both maternal effects and fetal effects should be considered together while investigating genetic factors of certain perinatal diseases.

  15. α, ω-Cholesterol-functionalized low molecular weight polyethylene glycol as a novel modifier of cationic liposomes for gene delivery. (United States)

    Ma, Cui-Cui; He, Zhi-Yao; Xia, Shan; Ren, Ke; Hui, Li-Wei; Qin, Han-Xiao; Tang, Ming-Hai; Zeng, Jun; Song, Xiang-Rong


    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.

  16. Balancing Cell Migration with Matrix Degradation Enhances Gene Delivery to Cells Cultured Three-Dimensionally Within Hydrogels (United States)

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


    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

  17. Targeted delivery of Bcl-2 conversion gene by MPEG-PCL-PEI-FA cationic copolymer to combat therapeutic resistant cancer. (United States)

    Li, Zibiao; Liu, Xuan; Chen, Xiaohong; Chua, Ming Xuan; Wu, Yun-Long


    Deregulation of anti-apoptosis Bcl-2 protein expression was a key feature in human cancers with therapeutic resistance. Nuclear receptor Nur77 could induce the conformation change of Bcl-2 protein and converted it into an apoptosis inducer by "enemy to friend" strategy. However, the safe and effective delivery of this gene to combat therapeutic resistant cancer remained largely unexplored. In this report, we designed an amphiphilic cationic MPEG-PCL-PEI-FA copolymer, comprising biocompatible and hydrophilic methoxy-poly(ethylene glycol) (MPEG), biodegradable and hydrophobic poly(ε-caprolactone) (PCL), cationic poly(ethylene imine) (PEI) segments, and folic acid (FA) as targeting group, as a high efficient Nur77 gene carrier to folate receptor (FR) highly expressed and therapeutic resistant HeLa/Bcl-2 cancer cells. Interestingly, due to the incorporation of PCL and PEG segments, this MPEG-PCL-PEI-FA copolymer showed less toxicity but better gene transfection efficiency than non-viral gene carrier gold standard PEI (25kDa). This might be due to the formation of micelles to stabilize polyplex for enhanced gene transfection ability. More importantly, MPEG-PCL-PEI-FA copolymer exhibited excellent growth inhibition ability on therapeutic resistant HeLa/Bcl-2 cancer cells, which was FR overexpressed HeLa cervical cancer cells with high expression of Bcl-2 protein, thanks to its FA induced targeting ability, high gene transfection efficiency, and low cytotoxicity. This work signifies the first time that cationic amphiphilic MPEG-PCL-PEI-FA copolymers could be utilized for the gene delivery to therapeutic resistant cancer cells with high expression of anti-apoptosis Bcl-2 protein and the positive results are encouraging for the further design of polymeric platforms for combating drug resistant tumors. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems

    Energy Technology Data Exchange (ETDEWEB)

    Pan Bifeng; Cui Daxiang; Xu Ping; Feng Gao; Huang Tuo; Li Qing; He Rong [Department of Bio-Nano-Science and Engineering, National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro-Nano Science and Technology, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240 (China); Ozkan, Cengiz [Mechanical Engineering Department, University of California Riverside, 900 University Avenue-Riverside, CA 92521 (United States); Ozkan, Mihri [Electrical Engineering Department, University of California Riverside, 900 University Avenue, Riverside, CA 92521 (United States); Chu, Bingfeng [Department of Stomatology, General Hospital of PLA, 28 Fuxing Road, Beijing100853 (China); Hu Guohan [Department of Neurosurgery of Changzheng Hospital, 415 Fengyang Road, Second Military Medical University, Shanghai 20003 (China)], E-mail:, E-mail:


    With the aim of improving the amount and delivery efficiency of genes taken by carbon nanotubes into human cancer cells, different generations of polyamidoamine dendrimer modified multi-walled carbon nanotubes (dMNTs) were fabricated, and characterized by high-resolution transmission electron microscopy, atomic force microscopy, x-ray photoelectron spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis, revealing the presence of dendrimer capped on the surface of carbon nanotubes. The dMNTs fully conjugated with FITC-labeled antisense c-myc oligonucleotides (asODN), those resultant asODN-dMNTs composites were incubated with human breast cancer cell line MCF-7 cells and MDA-MB-435 cells, and liver cancer cell line HepG2 cells, and confirmed to enter into tumor cells within 15 min by laser confocal microscopy. These composites inhibited the cell growth in time- and dose-dependent means, and down-regulated the expression of the c-myc gene and C-Myc protein. Compared with the composites of CNT-NH{sub 2}-asODN and dendrimer-asODN, no. 5 generation of dendrimer-modified MNT-asODN composites exhibit maximal transfection efficiencies and inhibition effects on tumor cells. The intracellular gene transport and uptake via dMNTs should be generic for the mammalian cell lines. The dMNTs have potentials in applications such as gene or drug delivery for cancer therapy and molecular imaging.

  19. Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems (United States)

    Pan, Bifeng; Cui, Daxiang; Xu, Ping; Ozkan, Cengiz; Feng, Gao; Ozkan, Mihri; Huang, Tuo; Chu, Bingfeng; Li, Qing; He, Rong; Hu, Guohan


    With the aim of improving the amount and delivery efficiency of genes taken by carbon nanotubes into human cancer cells, different generations of polyamidoamine dendrimer modified multi-walled carbon nanotubes (dMNTs) were fabricated, and characterized by high-resolution transmission electron microscopy, atomic force microscopy, x-ray photoelectron spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis, revealing the presence of dendrimer capped on the surface of carbon nanotubes. The dMNTs fully conjugated with FITC-labeled antisense c-myc oligonucleotides (asODN), those resultant asODN-dMNTs composites were incubated with human breast cancer cell line MCF-7 cells and MDA-MB-435 cells, and liver cancer cell line HepG2 cells, and confirmed to enter into tumor cells within 15 min by laser confocal microscopy. These composites inhibited the cell growth in time- and dose-dependent means, and down-regulated the expression of the c-myc gene and C-Myc protein. Compared with the composites of CNT-NH2-asODN and dendrimer-asODN, no. 5 generation of dendrimer-modified MNT-asODN composites exhibit maximal transfection efficiencies and inhibition effects on tumor cells. The intracellular gene transport and uptake via dMNTs should be generic for the mammalian cell lines. The dMNTs have potentials in applications such as gene or drug delivery for cancer therapy and molecular imaging.

  20. Self-assembled ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates for targeted gene delivery. (United States)

    Liang, Kun; Bae, Ki Hyun; Lee, Fan; Xu, Keming; Chung, Joo Eun; Gao, Shu Jun; Kurisawa, Motoichi


    Nanosized polyelectrolyte complexes are attractive delivery vehicles for the transfer of therapeutic genes to diseased cells. Here we report the application of self-assembled ternary complexes constructed with plasmid DNA, branched polyethylenimine and hyaluronic acid-green tea catechin conjugates for targeted gene delivery. These conjugates not only stabilize plasmid DNA/polyethylenimine complexes via the strong DNA-binding affinity of green tea catechin, but also facilitate their transport into CD44-overexpressing cells via receptor-mediated endocytosis. The hydrodynamic size, surface charge and physical stability of the complexes are characterized. We demonstrate that the stabilized ternary complexes display enhanced resistance to nuclease attack and polyanion-induced dissociation. Moreover, the ternary complexes can efficiently transfect the difficult-to-transfect HCT-116 colon cancer cell line even in serum-supplemented media due to their enhanced stability and CD44-targeting ability. Confocal microscopic analysis demonstrates that the stabilized ternary complexes are able to promote the nuclear transport of plasmid DNA more effectively than binary complexes and hyaluronic acid-coated ternary complexes. The present study suggests that the ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates can be widely utilized for CD44-targeted delivery of nucleic acid-based therapeutics.

  1. Broad functional correction of molecular impairments by systemic delivery of scAAVrh74-hSGSH gene delivery in MPS IIIA mice. (United States)

    Duncan, F Jason; Naughton, Bartholomew J; Zaraspe, Kimberly; Murrey, Darren A; Meadows, Aaron S; Clark, Kelly Reed; Newsom, David E; White, Peter; Fu, Haiyan; McCarty, Douglas M


    Mucopolysaccharidosis (MPS) IIIA is a neuropathic lysosomal storage disease caused by deficiency in N-sulfoglucosamine sulfohydrolase (SGSH). Genome-wide gene expression microarrays in MPS IIIA mice detected broad molecular abnormalities (greater than or equal to twofold, false discovery rate ≤10) in numerous transcripts (314) in the brain and blood (397). Importantly, 22 dysregulated blood transcripts are known to be enriched in the brain and linked to broad neuronal functions. To target the root cause, we used a self-complementary AAVrh74 vector to deliver the human SGSH gene into 4-6 weeks old MPS IIIA mice by an intravenous injection. The treatment resulted in global central nervous system (CNS) and widespread somatic restoration of SGSH activity, clearance of CNS and somatic glycosaminoglycan storage, improved behavior performance, and significantly extended survival. The scAAVrh74-hSGSH treatment also led to the correction of the majority of the transcriptional abnormalities in the brain (95.9%) and blood (97.7%), of which 182 and 290 transcripts were normalized in the brain and blood, respectively. These results demonstrate that a single systemic scAAVrh74-hSGSH delivery mediated efficient restoration of SGSH activity and resulted in a near complete correction of MPS IIIA molecular pathology. This study also demonstrates that blood transcriptional profiles reflect the biopathological status of MPS IIIA, and also respond well to effective treatments.

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

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


    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.

  3. Achieving high gene delivery performance with caveolae-mediated endocytosis pathway by (l)-arginine/(l)-histidine co-modified cationic gene carriers. (United States)

    Li, Hui; Luo, Ting; Sheng, Ruilong; Sun, Jingjing; Wang, Zhao; Cao, Amin


    Developing new amphiphilic polymers with natural product moieties has been regarded as a promising way to achieve biocompatibility and certain biological functions. In prior work, we developed some natural (l)-arginine modified cationic polymers (PAHMAA-Rs) as cationic gene carriers. For the sake of continuing optimize the gene delivery performance, herein, a new series of (l)-arginine and (l)-histidine co-modified cationic poly (ω-aminohexyl methacrylamide)s (PAHMAA-R-H) were synthesized and characterized with (1)H NMR, GPC-SLS and FT-IR. Their proton buffering capacities were studied by acid-base titration assay. pDNA binding affinity and self-assembly properties of the polyplexes were analyzed by agarose gel retardation assay, DLS and AFM, respectively. In vitro cytotoxicity of the PAHMAA-R-H was determined by MTT and LDH assays in H1299 cells, the gene transfection efficacy and intracellular uptake capability were evaluated by luciferase assay and FACS, respectively. Moreover, the endocytosis pathways and intracellular distribution of the polyplexes were investigated by using specific endocytic inhibitors and fluorescent co-localization techniques. The results demonstrated that co-modification of (l)-arginine and (l)-histidine onto the PAHMAA polymer could enhance proton buffering capacity, shield surface charge, decrease cytotoxicity, and improve gene transfection efficiency and serum-compatibility. Moreover, the gene transfection and intracellular uptake behaviors were disclosed strongly rely on the (l)-arginine/(l)-histidine modification ratios. The polyplexes tend to be internalized through caveolae-mediated endocytosis gateway and localized with endosomes/lysosomes in H1299 cells. Notably, among the polymers, the PAHMAA-R18-H6 exhibited remarkable gene delivery efficiency and serum compatibility, which made it promising gene transfection agent for practical application.

  4. Magnetic resonance-guided regional gene delivery strategy using a tumor stroma-permeable nanocarrier for pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Wang Q


    Full Text Available Qingbing Wang,1,2 Jianfeng Li,3 Sai An,3 Yi Chen,1 Chen Jiang,3 Xiaolin Wang1,2 1Department of Interventional Radiology, Zhongshan Hospital, Fudan University, 2Shanghai Institute of Medical Imaging, 3Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China Background: Gene therapy is a very promising technology for treatment of pancreatic ductal adenocarcinoma (PDAC. However, its application has been limited by the abundant stromal response in the tumor microenvironment. The aim of this study was to prepare a dendrimer-based gene-free loading vector with high permeability in the tumor stroma and explore an imaging-guided local gene delivery strategy for PDAC to promote the efficiency of targeted gene delivery.Methods: The experimental protocol was approved by the animal ethics committee of Zhongshan Hospital, Fudan University. Third-generation dendrigraft poly-L-lysines was selected as the nanocarrier scaffold, which was modified by cell-penetrating peptides and gadolinium (Gd chelates. DNA plasmids were loaded with these nanocarriers via electrostatic interaction. The cellular uptake and loaded gene expression were examined in MIA PaCa-2 cell lines in vitro. Permeability of the nanoparticles in the tumor stroma and transfected gene distribution in vivo were studied using a magnetic resonance imaging-guided delivery strategy in an orthotopic nude mouse model of PDAC.Results: The nanocarriers were synthesized with a dendrigraft poly-L-lysine to polyethylene glycol to DTPA ratio of 1:3.4:8.3 and a mean diameter of 110.9±7.7 nm. The luciferases were strictly expressed in the tumor, and the luminescence intensity in mice treated by Gd-DPT/plasmid luciferase (1.04×104±9.75×102 p/s/cm2/sr was significantly (P<0.05 higher than in those treated with Gd-DTPA (9.56×102±6.15×10 p/s/cm2/sr and Gd-DP (5.75×103± 7.45×102 p/s/cm2/sr

  5. Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach (United States)

    Das, Debobrato

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

  6. Adeno-Associated Viral Vectors Serotype 8 for Cell-Specific Delivery of Therapeutic Genes in the Central Nervous System (United States)

    Pignataro, Diego; Sucunza, Diego; Vanrell, Lucia; Lopez-Franco, Esperanza; Dopeso-Reyes, Iria G.; Vales, Africa; Hommel, Mirja; Rico, Alberto J.; Lanciego, Jose L.; Gonzalez-Aseguinolaza, Gloria


    Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters. PMID:28239341

  7. Asialoglycoprotein receptor targeted gene delivery using galactosylated polyethylenimine-graft-poly(ethylene glycol): in vitro and in vivo studies. (United States)

    Kim, Eun-Mi; Jeong, Hwan-Jeong; Park, In-Kyu; Cho, Chong-Su; Moon, Hyung-Bae; Yu, Dae-Yeul; Bom, Hee-Seung; Sohn, Myung-Hee; Oh, In-Joon


    The asialoglycoprotein receptor (ASGP-R) on the hepatocyte membrane is a specific targeting marker for gene and drug delivery. Polyethylenimine (PEI) is a polycationic nonviral vector that is used for gene transfer. We have synthesized galactosylated polyethylenimine-graft-poly(ethylene glycol) (GPP) for performing gene delivery to the hepatocytes. The present study reports on the in vitro and in vivo data that was achieved in hepatoma bearing transgenic mice. The cytotoxicity was decreased with the increasing PEG content. The particle size of the complex was increased with the increasing PEG at an N/P ratio of 3.0, while the zeta potentials were decreased. The (99m)Tc labeled complexes were transfected into HepG2 and HeLa cells, while the GFP reporter genes were mainly expressed in the HepG2 cells. The in vivo data was achieved in ALB/c-Ha-ras transgenic mice. (99m)Tc labeled GPP(50)/DNA was injected into the mice via the tail vein, and the gamma images were acquired at 5, 15 and 30 min. The (99m)Tc labeled complexes were mainly localized in the heart and liver, and they were excreted through the kidneys. The GFP gene was mainly expressed in the proliferating cells at the tumor periphery. This result was confirmed by PCNA staining. The GPP(50)/DNA complexes were bound to ASGP-R of the proliferating hepatocytes in vitro and in vivo. The present results demonstrate the feasibility of nonviral gene transfer using galactosylated PEI-PEG in vivo.

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

    Directory of Open Access Journals (Sweden)

    Zhao W


    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

  9. Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear. (United States)

    Yoon, Ji Young; Yang, Keum-Jin; Kim, Da Eun; Lee, Kyu-Yup; Park, Shi-Nae; Kim, Dong-Kee; Kim, Jong-Duk


    A drug delivery system to the inner ear using nanoparticles consisting of oligoarginine peptide (Arg8) conjugated to poly(amino acid) (poly(2-hydroxyethyl L-aspartamide; PHEA) was investigated to determine whether the limitations of low drug transport levels across the round window membrane (RWM) and poor transport into inner ear target cells, including hair cells and spiral ganglion, could be overcome. Three types of carrier materials, PHEA-g-C18, PHEA-g-Arg8, and PHEA-g-C18-Arg8, were synthesized to examine the effects of oligoarginine and morphology of the synthesized carriers. Nile red (NR) was used as a fluorescent indicator as well as to model a hydrophobic drug. Compared with PHEA-g-C18-NR nanoparticles, the oligoarginine-conjugated nanoparticles of PHEA-g-C18-Arg8-NR and PHEA-g-Arg8-NR entered into HEI-OC1 cells at significant levels. Furthermore, the strongest fluorescence intensity was observed in nuclei when PHEA-g-C18-Arg8 nanoparticles were used. The high uptake rates of PHEA-g-C18 and PHEA-g-C18-Arg8 nanoparticles were observed in ex vivo experiments using hair cells. After the delivery of PHEA-g-C18-Arg8 nanoparticles with reporter gene transfer, EGFP (enhanced green fluorescent protein) expression was monitored as an indicator of gene delivery. In the inner ear cells, PHEA-g-C18-Arg8 nanoparticles showed comparable or better transfection capabilities than the commercially available Lipofectamine reagent. PHEA-g-C18-Arg8 penetrated in vivo across the RWM of C57/BL6 mice with Nile red staining and GFP expression in various inner ear tissues. In conclusion, PHEA-g-C18-Arg8 nanoparticles were successfully transported into the inner ear through the intratympanic route and are proposed as promising candidates as delivery carriers to address inner ear diseases.

  10. Nanostructured materials in drug and gene delivery: a review of the state of the art. (United States)

    Petkar, Kailash C; Chavhan, Sandip S; Agatonovik-Kustrin, Snezana; Sawant, Krutika K


    A wide variety of drug delivery systems have been developed, each with its own advantages and limitations, but the important goals of all of the systems are to enhance bioavailability, reduce drug toxicity, target to a particular organ, and increase the stability of the drug. The development of nanostructured drug carriers have grasped increased attention from scientific and commercial organizations due to their unique ability to deliver drugs and challenging molecules such as proteins and nucleic acids. These carriers present many technological advantages such as high carrier capacity, high chemical and biological stability, feasibility of incorporating both hydrophilic and hydrophobic substances, and their ability to be administered by a variety of routes (including oral, inhalational, and parenteral) to provide controlled/sustained drug release. Moreover, applications of nanoparticulate formulations in enhancing drug solubility, dissolution, bioavailability, safety, and stability have already been proven. In the view of their multifaceted applications, the present review aims to discuss and summarize some of the interesting findings and applications, methods of preparation, and characterization of various nanostructured carriers useful in drug delivery. Included in this discussion are polymeric nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, dendrimers, cyclodextrins, fullerenes, gold and silica nanoparticles, and quantum dots. Because there are likely to be new applications for nanoparticles in drug delivery, they are expected to solve many problems associated with the delivery of drugs and biomolecules through different delivery routes.

  11. Efficient in vivo gene transfer to xenotransplanted human skin by lentivirus-mediated, but not by AAV-directed, gene delivery

    DEFF Research Database (Denmark)

    Jakobsen, Maria Vad; Askou, Anne Louise; Dokkedahl, Karin Stenderup

    in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human......Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for treatment of skin diseases. We compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin...... skin graft, and firefly luciferase expression was observed primarily in neighboring tissue beneath or surrounding the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin...

  12. Non-viral gene delivery carrier of probe type host molecule --Interactions between DNA and β-cyclodextrin derivative complexes (Ⅰ)

    Institute of Scientific and Technical Information of China (English)


    A host type non-virus gene delivery carrier, phenanthroline-β-cyclodextrin derivative host molecule, was produced which can be used as molecular probe. Interactions between DZY-1 and DNA were investigated by electrophoresis assay. Hind III enzyme inhibition assay was carried out using DNA condensates induced by host molecules or host- guest molecule complexes to explore their ability to inhibit enzyme digestion. Micro-structure of DNA condensates induced by host molecules and host-guest molecule complexes was observed by scanning electron microscope (SEM). Our work indicates the delivery mechanism of DZY-1 used as a gene delivery carrier and also provides a method to design and produce non-virus gene delivery carriers.

  13. 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


    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

  14. Aminated β-Cyclodextrin-Modified-Carboxylated Magnetic Cobalt/Nanocellulose Composite for Tumor-Targeted Gene Delivery

    Directory of Open Access Journals (Sweden)

    Thayyath Sreenivasan Anirudhan


    Full Text Available Gene therapy is a new kind of medicine, which uses genes as drugs in order to treat life threatening diseases. In the present work, a nonviral vector, aminated β-cyclodextrin-modified-carboxylated magnetic cobalt/nanocellulose composite (ACDC-Co/NCC, was synthesized for efficient transfection of genes into tumour cells. The synthesized ACDC-Co/NCC was characterized by means of FTIR, XRD, SEM, and ESR techniques. DNA condensing ability of ACDC-Co/NCC was found to be increased with increase in amount of ACDC-Co/NCC and 84.9% of DNA (1.0 μg/mL inclusion was observed with 6.0 μg/mL of ACDC-Co/NCC. The cytotoxicity of ACDC-Co/NCC was observed to be minimal, even at higher concentration, with respect to the model transfecting agent, poly(ethyleneimine (PEI. 88.2% of the gene was transfected at high dose of DNA, as indicated by the highest luciferase expression. These results indicated that ACDC-Co/NCC might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, minimal cytotoxicity, and relatively high gene transfection efficiency.

  15. In vivo delivery of DN:REST improves transcriptional changes of REST-regulated genes in HD mice. (United States)

    Conforti, P; Mas Monteys, A; Zuccato, C; Buckley, N J; Davidson, B; Cattaneo, E


    Current therapeutic strategies for Huntington's disease (HD) are focused on symptom management of disease progression. Transcriptional dysregulation is one of the major characteristics in HD. REST is a transcriptional repressor that silences gene expression through binding to RE1/NRSE sites found in the regulatory regions of numerous neuronal genes. Dysregulation of REST and its targeted genes has been reported in different cell and mouse HD models, as well as in biopsies from human patients. In this work, we characterized transcriptional dysregulation associated with REST in two different HD mouse models and assessed the therapeutic effect of interfering with REST function by overexpressing a dominant-negative form (DN:REST). We show that delivery of DN:REST in the motor cortex restores brain-derived neurotrophic factor (BDNF) mRNA and protein levels by reducing endogenous REST occupancy at the Bdnf locus. Similarly, expression of other REST-regulated genes such as Synapsin I (Syn1), Proenkephalin (Penk1) and Cholinergic receptor muscarinic 4 (Chrm4) were restored to normal levels while non-REST-regulated genes were unaffected. This is the first study conducted to investigate REST's role in vivo in a neurodegenerative disease. Our data show that DN:REST in motor cortex reversed RESTs repressive effects on target genes. However, the lack of therapeutic effect on motor function suggests that a more widespread rescue of REST-regulated sites in the affected brain regions may be necessary.

  16. Optical tracking of organically modified silica nanoparticles as DNA carriers: A nonviral, nanomedicine approach for gene delivery (United States)

    Roy, Indrajit; Ohulchanskyy, Tymish Y.; Bharali, Dhruba J.; Pudavar, Haridas E.; Mistretta, Ruth A.; Kaur, Navjot; Prasad, Paras N.


    This article reports a multidisciplinary approach to produce fluorescently labeled organically modified silica nanoparticles as a nonviral vector for gene delivery and biophotonics methods to optically monitor intracellular trafficking and gene transfection. Highly monodispersed, stable aqueous suspensions of organically modified silica nanoparticles, encapsulating fluorescent dyes and surface functionalized by cationic-amino groups, are produced by micellar nanochemistry. Gel-electrophoresis studies reveal that the particles efficiently complex with DNA and protect it from enzymatic digestion of DNase 1. The electrostatic binding of DNA onto the surface of the nanoparticles, due to positively charged amino groups, is also shown by intercalating an appropriate dye into the DNA and observing the Förster (fluorescence) resonance energy transfer between the dye (energy donor) intercalated in DNA on the surface of nanoparticles and a second dye (energy acceptor) inside the nanoparticles. Imaging by fluorescence confocal microscopy shows that cells efficiently take up the nanoparticles in vitro in the cytoplasm, and the nanoparticles deliver DNA to the nucleus. The use of plasmid encoding enhanced GFP allowed us to demonstrate the process of gene transfection in cultured cells. Our work shows that the nanomedicine approach, with nanoparticles acting as a drug-delivery platform combining multiple optical and other types of probes, provides a promising direction for targeted therapy with enhanced efficacy as well as for real-time monitoring of drug action. nonviral vector | ORMOSIL nanoparticles | confocal microscopy

  17. Transferrin-PEG-PE modified dexamethasone conjugated cationic lipid carrier mediated gene delivery system for tumor-targeted transfection (United States)

    Wang, Wei; Zhou, Fang; Ge, Linfu; Liu, Ximin; Kong, Fansheng


    Background The main barriers to non-viral gene delivery include cellular and nuclear membranes. As such, the aim of this study was to develop a type of vector that can target cells through receptor-mediated pathways and by using nuclear localization signal (NLS) to increase the nuclear uptake of genetic materials. Methods A dexamethasone (Dexa)-conjugated lipid was synthesized as the material of the solid lipid nanoparticles (SLNs), and transferrin (Tf) was linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) to obtain Tf-PEG-PE ligands for the surface modification of the carriers. The in vitro transfection efficiency of the novel modified vectors was evaluated in human hepatoma carcinoma cell lines, and in vivo effects were observed in an animal model. Results Tf-PEG-PE modified SLNs/enhanced green fluorescence protein plasmid (pEGFP) had a particle size of 222 nm and a gene loading quantity of 90%. Tf-PEG-PE-modified SLNs/pEGFP (Tf-SLNs/pEGFP) displayed remarkably higher transfection efficiency than non-modified SLNs/pEGFP and the vectors not containing Dexa, both in vitro and in vivo. Conclusion It can be concluded that Tf and Dexa could function as an excellent active targeting ligand to improve the cell targeting and nuclear targeting ability of the carriers, and the resulting nanomedicine could be a promising active targeting drug/gene delivery system. PMID:22679364

  18. Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders. (United States)

    Bevan, Adam K; Duque, Sandra; Foust, Kevin D; Morales, Pablo R; Braun, Lyndsey; Schmelzer, Leah; Chan, Curtis M; McCrate, Mary; Chicoine, Louis G; Coley, Brian D; Porensky, Paul N; Kolb, Stephen J; Mendell, Jerry R; Burghes, Arthur H M; Kaspar, Brian K


    Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step is to demonstrate efficient CNS targeting in large animals at various ages. In the present study, we tested systemically injected AAV9 in cynomolgus macaques, administered at birth through 3 years of age for targeting CNS and peripheral tissues. We show that AAV9 was efficient at crossing the blood-brain barrier (BBB) at all time points investigated. Transgene expression was detected primarily in glial cells throughout the brain, dorsal root ganglia neurons and motor neurons within the spinal cord, providing confidence for translation to SMA patients. Systemic injection also efficiently targeted skeletal muscle and peripheral organs. To specifically target the CNS, we explored AAV9 delivery to cerebrospinal fluid (CSF). CSF injection efficiently targeted motor neurons, and restricted gene expression to the CNS, providing an alternate delivery route and potentially lower manufacturing requirements for older, larger patients. Our findings support the use of AAV9 for gene transfer to the CNS for disorders in pediatric populations.

  19. Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry. (United States)

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


    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.

  20. Multifunctional triblock Nanocarrier (PAMAM-PEG-PLL) for the efficient intracellular siRNA delivery and gene silencing. (United States)

    Patil, Mahesh L; Zhang, Min; Minko, Tamara


    A novel triblock poly(amido amine)-poly(ethylene glycol)-poly-l-lysine (PAMAM-PEG-PLL) nanocarrier was designed, synthesized, and evaluated for the delivery of siRNA. The design of the nanocarrier is unique and provides a solution to most of the common problems associated with the delivery and therapeutic applications of siRNA. Every component in the triblock nanocarrier plays a significant role and performs multiple functions: (1) tertiary amine groups in the PAMAM dendrimer work as a proton sponge and play a vital role in the endosomal escape and cytoplasmic delivery of siRNA; (2) PEG, a linker connecting PLL and PAMAM dendrimers renders nuclease stability and protects siRNA in human plasma; (3) PLL provides primary amines to form polyplexes with siRNA through electrostatic interaction and also acts as penetration enhancer; and (4) conjugation to PEG and PAMAM reduced toxicity of PLL and the entire triblock nanocarrier PAMAM-PEG-PLL. The data obtained show that the polyplexes resulted from the conjugation of siRNA, and the proposed nanocarriers were effectively taken up by cancer cells and induced the knock down of the target BCL2 gene. In addition, triblock nanocarrier/siRNA polyplexes showed excellent stability in human plasma.

  1. The novel albumin-chitosan core-shell nanoparticles for gene delivery: preparation, optimization and cell uptake investigation (United States)

    Karimi, Mahdi; Avci, Pinar; Mobasseri, Rezvan; Hamblin, Michael R.; Naderi-Manesh, Hossein


    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 ( 85 %) was shown by flow cytometry and fluorescent microscopy.

  2. 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: [Tarbiat Modares University, Department of Nanobiotechnology, Faculty of Biological Sciences (Iran, Islamic Republic of)


    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.

  3. Intraneural GJB1 gene delivery improves nerve pathology in a model of X-linked Charcot-Marie-Tooth disease. (United States)

    Sargiannidou, Irene; Kagiava, Alexia; Bashiardes, Stavros; Richter, Jan; Christodoulou, Christina; Scherer, Steven S; Kleopa, Kleopas A


    X-linked Charcot-Marie-Tooth disease (CMT1X) is a common inherited neuropathy caused by mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32). Clinical studies and disease models indicate that neuropathy mainly results from Schwann cell autonomous, loss-of-function mechanisms; therefore, CMT1X may be treatable by gene replacement. A lentiviral vector LV.Mpz-GJB1 carrying the GJB1 gene under the Schwann cell-specific myelin protein zero (Mpz) promoter was generated and delivered into the mouse sciatic nerve by a single injection immediately distal to the sciatic notch. Enhanced green fluorescent protein (EGFP) reporter gene expression was quantified and Cx32 expression was examined on a Cx32 knockout (KO) background. A gene therapy trial was performed in a Cx32 KO model of CMT1X. EGFP was expressed throughout the length of the sciatic nerve in up to 50% of Schwann cells starting 2 weeks after injection and remaining stable for up to 16 weeks. Following LV.Mpz-GJB1 injection into Cx32 KO nerves, we detected Cx32 expression and correct localization in non-compact myelin areas where gap junctions are normally formed. Gene therapy trial by intraneural injection in groups of 2-month-old Cx32 KO mice, before demyelination onset, significantly reduced the ratio of abnormally myelinated fibers (p = 0.00148) and secondary inflammation (p = 0.0178) at 6 months of age compared to mock-treated animals. Gene delivery using a lentiviral vector leads to efficient gene expression specifically in Schwann cells. Restoration of Cx32 expression ameliorates nerve pathology in a disease model and provides a promising approach for future treatments of CMT1X and other inherited neuropathies. © 2015 American Neurological Association.

  4. Hexanoic acid and polyethylene glycol double grafted amphiphilic chitosan for enhanced gene delivery: influence of hydrophobic and hydrophilic substitution degree. (United States)

    Layek, Buddhadev; Haldar, Manas K; Sharma, Gitanjali; Lipp, Lindsey; Mallik, Sanku; Singh, Jagdish


    Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. To overcome this difficulty a series of hexanoic acid (HA) and monomethoxy poly(ethylene glycol) (mPEG) double grafted chitosan-based (HPC) nanomicelles were developed as nonviral gene carrier. HPC polymers with various HA and mPEG substitution degrees were synthesized, and their chemical structures were confirmed by (1)H NMR spectroscopy. HPC nanomicelles exhibited excellent blood compatibility and cell viability, as demonstrated by in vitro hemolysis and MTT assay, respectively. The cationic HPC nanomicelles retained the plasmid DNA (pDNA) binding capacity of chitosan and formed stable HPC/pDNA polyplexes with diameters below 200 nm. Both hydrophobic and hydrophilic substitution resulted in suppressed nonspecific protein adsorption on HPC/pDNA polyplexes and increased pDNA dissociation. However, resistance against DNase I degradation was enhanced by HA conjugation while being inhibited by mPEG substitution. Amphiphilic modification resulted in 3-4.5-fold higher cellular uptake in human embryonic kidney 293 cells (HEK 293) mainly through clathrin-mediated pathway. The optimal HPC/pDNA polyplexes displayed 50-fold and 1.2-fold higher gene transfection compared to unmodified chitosan and Fugene, respectively, in HEK 293 cells. Moreover, both the cellular uptake and in vitro transfection study suggested a clear dependence of gene expression on the extent of HA and mPEG substitution. These findings demonstrate that amphiphilic HPC nanomicelles with the proper combination of HA and mPEG substitution could be used as a promising gene carrier for efficient gene therapy.

  5. Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo (United States)

    Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna


    In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy. PMID:28042335

  6. Identification of chromosomal genes in Yersinia pestis that influence type III secretion and delivery of Yops into target cells.

    Directory of Open Access Journals (Sweden)

    Andrew S Houppert

    Full Text Available Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague.

  7. Isolated limb perfusion for local gene delivery: efficient and targeted adenovirus-mediated gene transfer into soft tissue sarcomas

    NARCIS (Netherlands)

    W.K. de Roos; J.H.W. de Wilt (Johannes); M.E. van der Kaaden; E.R. Manusama (Eric); M.W. de Vries; A. Bout; T.L.M. ten Hagen (Timo); D. Valerio (Dinko); A.M.M. Eggermont (Alexander)


    textabstractOBJECTIVE: To evaluate the potential of isolated limb perfusion (ILP) for efficient and tumor-specific adenovirus-mediated gene transfer in sarcoma-bearing rats. SUMMARY BACKGROUND DATA: A major concern in adenovirus-mediated gene therapy in cancer is the transfer of ge

  8. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery. (United States)

    Sebe, Attila; Ivics, Zoltán


    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.

  9. TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery. (United States)

    Wang, Bing; Yi, Wen-Jing; Zhang, Ji; Zhang, Qin-Fang; Xun, Miao-Miao; Yu, Xiao-Qi


    Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000.

  10. Three-layered polyplex as a microRNA targeted delivery system for breast cancer gene therapy (United States)

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


    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.

  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)


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

    Energy Technology Data Exchange (ETDEWEB)

    Brian Curtis Anderson


    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

  13. Peptide-Mediated Tumor Targeting by a Degradable Nano Gene Delivery Vector Based on Pluronic-Modified Polyethylenimine (United States)

    Wu, Zhaoyong; Zhan, Shuyu; Fan, Wei; Ding, Xueying; Wu, Xin; Zhang, Wei; Fu, Yinghua; Huang, Yueyan; Huang, Xuan; Chen, Rubing; Li, Mingjuan; Xu, Ningyin; Zheng, Yongxia; Ding, Baoyue


    Polyethylenimine (PEI) is considered to be a promising non-viral gene delivery vector. To solve the toxicity versus efficacy and tumor-targeting challenges of PEI used as gene delivery vector, we constructed a novel non-viral vector DR5-TAT-modified Pluronic-PEI (Pluronic-PEI-DR5-TAT), which was based on the attachment of low-molecular-weight polyethylenimine (LMW-PEI) to the amphiphilic polymer Pluronic to prepare Pluronic-modified LMW-PEI (Pluronic-PEI). This was then conjugated to a multifunctional peptide containing a cell-penetrating peptide (TAT) and a synthetic peptide that would bind to DR5—a receptor that is overexpressed in cancer cells. The vector showed controlled degradation, favorable DNA condensation and protection performance. The Pluronic-PEI-DR5-TAT/DNA complexes at an N/P ratio of 15:1 were spherical nanoparticles of 122 ± 11.6 nm and a zeta potential of about 22 ± 2.8 mV. In vitro biological characterization results indicated that Pluronic-PEI-DR5-TAT/DNA complexes had a higher specificity for the DR5 receptor and were taken up more efficiently by tumor cells than normal cells, compared to complexes formed with PEI 25 kDa or Pluronic-PEI. Thus, the novel complexes showed much lower cytotoxicity to normal cells and higher gene transfection efficiency in tumor cells than that exhibited by PEI 25 kDa and Pluronic-PEI. In summary, our novel, degradable non-viral tumor-targeting vector is a promising candidate for use in gene therapy.

  14. Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells. (United States)

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


    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.

  15. Enhanced gene delivery in porcine vasculature tissue following incorporation of adeno-associated virus nanoparticles into porous silicon microparticles. (United States)

    McConnell, Kellie I; Rhudy, Jessica; Yokoi, Kenji; Gu, Jianhua; Mack, Aaron; Suh, Junghae; La Francesca, Saverio; Sakamoto, Jason; Serda, Rita E


    There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells.

  16. Preparation of Concentrated Chitosan/DNA Nanoparticle Formulations by Lyophilization for Gene Delivery at Clinically Relevant Dosages. (United States)

    Veilleux, Daniel; Nelea, Monica; Biniecki, Kristof; Lavertu, Marc; Buschmann, Michael D


    Chitosan/DNA polyplexes have been optimized for efficient and safe in vitro and in vivo gene delivery. Clinical application of this technology requires the development of formulations with higher concentrations to reach therapeutic dosages. Polyplexes were prepared using chitosan and EGFPLuc plasmids. Freeze-thawing and freeze-drying studies were performed to identify and optimize lyoprotectant and buffer contents in formulations. Freeze-dried samples were rehydrated in reduced volumes to increase their final DNA dose. Nanoparticle physicochemical properties were analyzed, and their transfection efficiency and cytotoxicity were measured in human embryonic kidney 293 cells. Data showed that 3.5 mM histidine buffer (pH 6.5) combined with one of 0.5% wt/vol sucrose, dextran 5 kDa, or trehalose was required to prevent polyplex aggregation during freeze-drying. Optimal formulations could be concentrated 20-fold, to a clinically desired ∼1 mg of DNA/mL, while maintaining near physiological pH and tonicity. Polyplexes were predominantly spherical, with diameters below 200 nm, polydispersity indexes below 0.32, and zeta potentials above +19 mV. Rehydrated formulations had transfection efficiencies no less than 65% of fresh polyplexes without excipients and had no effect on viability and metabolic activity of human embryonic kidney 293 cells. These concentrated formulations represent an important step toward clinical use of chitosan-based gene delivery systems.

  17. Development of a Multi-functional Nano-device for Safe and Effective Gene Delivery to Target Organs. (United States)

    Kodama, Yukinobu


     Nucleic acids are expected as novel effective medicines, although they require a drug delivery system (DDS). Complexes of nucleic acids with cationic liposomes and cationic polymers have been mainly used as DDS for clinical use. However, most cationic complexes have disadvantages such as strong cytotoxicity and low biocompatibility. We previously found that a plasmid DNA (pDNA) complex coated with biodegradable γ-polyglutamic acid (γ-PGA) provided adequate gene expression without cytotoxicity. Based on these results, we developed a new DDS (multi-functional Nano-device) of pDNA using biodegradable polyamino acids. A typical cationic polyamino acid, poly-L-lysine (PLL), was complexed with pDNA. The binary complexes, however, showed low gene expression and high cytotoxicity. Gene expression was enhanced by addition of poly-L-histidine (PLH) to the binary complexes. PLH can increase endosome escape of the complexes by inducing pH-buffering effects. The quaternary complexes (pDNA-PLL-PLH-γ-PGA complexes) exhibited high gene expression and low cytotoxicity. Furthermore, we used dendrigraft poly-L-lysine (DGL) instead of PLL and PLH to enhance gene expression. DGL had sterically congested cations and was biodegradable. The ternary complexes (pDNA-DGL-γ-PGA complexes) exhibited markedly high gene expression and low cytotoxicity. The pDNA-DGL-γ-PGA complexes also had high gene expression in the marginal zone (rich dendritic cells) of the spleen after intravenous injection into mice. These results indicate that pDNA-DGL-γ-PGA complexes may be useful as vaccine vectors. Therefore we prepared a novel malaria DNA vaccine using Plasmodium yoelii GPI8p-transamidase-related protein pDNA (PyTAM). The PyTAM-DGL-γ-PGA complexes markedly improved survival time of model mice infected with malaria.

  18. Discovery of antibiotics-derived polymers for gene delivery using combinatorial synthesis and cheminformatics modeling. (United States)

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


    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.

  19. Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

    Directory of Open Access Journals (Sweden)

    Liu K


    Full Text Available Kehai Liu1,2,*, Xiaoyu Wang1,*, Wei Fan1, Qing Zhu2, Jingya Yang2, Jing Gao3, Shen Gao1 1Department of Pharmaceutics, Shanghai Hospital, Second Military Medical University, 2Department of Biopharmaceutics, School of Food Science and Technology, Shanghai Ocean University, 3Department of Pharmaceutics, School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of China*The first two authors contributed equally to this workBackground: To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed.Methods: First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC, in conjunction with the cell-penetrating peptide Tat (49–57, to yield a bifunctional peptide RGDC-Tat (49–57 named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13. The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in avß3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro.Results: The vector showed controlled degradation, strong targeting specificity to avß3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/µg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 µg/mL sodium

  20. Plasmid-encapsulated polyethylene glycol-grafted polyethylenimine nanoparticles for gene delivery into rat mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Chen X


    Full Text Available Xiao-Ai Chen1,5*, Li-Jun Zhang2*, Zhi-Jie He3, Wei-Wei Wang4, Bo Xu1, Qian Zhong1, Xin-Tao Shuai4, Li-Qun Yang4, Yu-Bin Deng11Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; 2Futian Affiliated Hospital, Guangdong Medical College, Shenzhen, China; 3Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China; 4Institute of Polymer Science, School of Chemistry and Chemical Engineering, BME Center, State Key Laboratory of Optoelectronic Materials and Technologies, Sun Yat-sen University, Guangzhou, China; 5Yunnan Cancer Hospital, The Third Affiliated Hospital, Kunming Medical College, Kunming, China*Both authors contributed equally to this workBackground: Mesenchymal stem cell transplantation is a promising method in regenerative medicine. Gene-modified mesenchymal stem cells possess superior characteristics of specific tissue differentiation, resistance to apoptosis, and directional migration. Viral vectors have the disadvantages of potential immunogenicity, carcinogenicity, and complicated synthetic procedures. Polyethylene glycol-grafted polyethylenimine (PEG-PEI holds promise in gene delivery because of easy preparation and potentially targeting modification.Methods: A PEG8k-PEI25k graft copolymer was synthesized. Agarose gel retardation assay and dynamic light scattering were used to determine the properties of the nanoparticles. MTT reduction, wound and healing, and differentiation assays were used to test the cytobiological characteristics of rat mesenchymal stem cells, fluorescence microscopy and flow cytometry were used to determine transfection efficiency, and atomic force microscopy was used to evaluate the interaction between PEG-PEI/plasmid nanoparticles and mesenchymal stem cells.Results: After incubation with the copolymer, the bionomics of mesenchymal stem cells showed no significant change. The mesenchymal stem cells still maintained high viability, resettled the

  1. Enhanced adenoviral gene delivery to motor and dorsal root ganglion neurons following injection into demyelinated peripheral nerves. (United States)

    Zhang, Yongjie; Zheng, Yiyan; Zhang, Yi Ping; Shields, Lisa B E; Hu, Xiaoling; Yu, Panpan; Burke, Darlene A; Wang, Heming; Jun, Cai; Byers, Jonathan; Whittemore, Scott R; Shields, Christopher B


    Injection of viral vectors into peripheral nerves may transfer specific genes into their dorsal root ganglion (DRG) neurons and motoneurons. However, myelin sheaths of peripheral axons block the entry of viral particles into nerves. We studied whether mild, transient peripheral nerve demyelination prior to intraneural viral vector injection would enhance gene transfer to target DRG neurons and motoneurons. The right sciatic nerve of C57BL/6 mice was focally demyelinated with 1% lysolecithin, and the left sciatic nerve was similarly injected with saline (control). Five days after demyelination, 0.5 microl of Ad5-GFP was injected into both sciatic nerves at the site of previous injection. The effectiveness of gene transfer was evaluated by counting GFP(+) neurons in the DRGs and ventral horns. After peripheral nerve demyelination, there was a fivefold increase in the number of infected DRG neurons and almost a 15-fold increase in the number of infected motoneurons compared with the control, nondemyelinated side. Focal demyelination reduced the myelin sheath barrier, allowing greater virus-axon contact. Increased CXADR expression on the demyelinated axons facilitated axoplasmic viral entry. No animals sustained any prolonged neurological deficits. Increased gene delivery into DRG neurons and motoneurons may provide effective treatment for amyotrophic lateral sclerosis, pain, and spinal cord injury.

  2. In Vitro Gene Delivery Mediated by Asialofetuin-Appended Cationic Liposomes Associated with γ-Cyclodextrin into Hepatocytes

    Directory of Open Access Journals (Sweden)

    Keiichi Motoyama


    Full Text Available The purpose of this study is to evaluate in vitro gene delivery mediated by asialofetuin-appended cationic liposomes (AF-liposomes associating cyclodextrins (CyD/AF-liposomes as a hepatocyte-selective nonviral vector. Of various CyDs, AF-liposomes associated with plasmid DNA (pDNA and γ-cyclodextrin (γ-CyD (pDNA/γ-CyD/AF-liposomes showed the highest gene transfer activity in HepG2 cells without any significant cytotoxicity. In addition, γ-CyD enhanced the encapsulation ratio of pDNA with AF-liposomes, and also increased gene transfer activity as the entrapment ratio of pDNA into AF-liposomes was increased. γ-CyD stabilized the liposomal membrane of AF-liposomes and inhibited the release of calcein from AF-liposomes. The stabilizing effect of γ-CyD may be, at least in part, involved in the enhancing gene transfer activity of pDNA/γ-CyD/AF-liposomes. Therefore, these results suggest the potential use of γ-CyD for an enhancer of transfection efficiency of AF-liposomes.

  3. Delivery of glutamine synthetase gene by baculovirus vectors: a proof of concept for the treatment of acute hyperammonemia. (United States)

    Torres-Vega, M A; Vargas-Jerónimo, R Y; Montiel-Martínez, A G; Muñoz-Fuentes, R M; Zamorano-Carrillo, A; Pastor, A R; Palomares, L A


    Hyperammonemia, a condition present in patients with urea cycle disorders (UCDs) or liver diseases, can cause neuropsychiatric complications, which in the worst cases result in brain damage, coma or death. Diverse treatments exist for the treatment of hyperammonemia, but they have limited efficacy, adverse effects and elevated cost. Gene therapy is a promising alternative that is explored here. A baculovirus, termed Bac-GS, containing the glutamine synthetase (GS) gene was constructed for the in vitro and in vivo treatment of hyperammonemia. Transduction of MA104 epithelial or L6 myoblast/myotubes cells with Bac-GS resulted in a high expression of the GS gene, an increase in GS concentration, and a reduction of almost half of exogenously added ammonia. When Bac-GS was tested in an acute hyperammonemia rat model by intramuscularly injecting the rear legs, the concentration of ammonia in blood decreased 351 μM, in comparison with controls. A high GS concentration was detected in gastrocnemius muscles from the rats transduced with Bac-GS. These results show that gene delivery for overexpressing GS in muscle tissue is a promising alternative for the treatment of hyperammonemia in patients with acute or chronic liver diseases and hepatic encephalopathy or UCD.

  4. Receptor-mediated gene delivery using polyethylenimine (PEI)coupled with polypeptides targeting FGF receptors on cells surface

    Institute of Scientific and Technical Information of China (English)

    LI Da; WANG Qing-qing; TANG Gu-ping; HUANG Hong-liang; SHEN Fen-ping; LI Jing-zhong; YU Hai


    Objective: To construct a novel kind ofnonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. Methods:The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. Results: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. Conclusion: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jing, E-mail: [Airport College, Civil Aviation University of China, Tianjin 300300 (China); Zhang, Xinming, E-mail: [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)


    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.

  6. Chemotherapeutic Drugs Interfere with Gene Delivery Mediated by Chitosan-Graft-Poly(ethylenimine.

    Directory of Open Access Journals (Sweden)

    Wing-Fu Lai

    Full Text Available Combined chemo-gene therapy is one of the treatment modalities that have attracted extensive research interests; however, there is little information regarding the influence of drug application on gene transfer. This study bridges this gap by examining how chemotherapeutic drugs (teniposide, cis-diamminedichloroplatinum(II and temozolomide interfere with polyplex formation and transfection of chitosan-graft-poly(ethylenimine. Our results indicate that the degree of drug interference varies with the mechanism of drug action, with the transgene expression being severely suppressed when the plasmid is co-delivered with cis-diamminedichloroplatinum(II or teniposide but not temozolomide. In addition, the interference with transfection by drugs varies with different gene/drug co-formulations. This is the first study to evidence that, though combined chemo-gene therapy has therapeutic potential, some chemotherapeutic drugs may reduce the treatment efficiency of gene therapy.

  7. Molecular recognition of genomic DNA in a condensate with a model surfactant for potential gene-delivery applications. (United States)

    Singh, Priya; Choudhury, Susobhan; Chandra, Goutam Kumar; Lemmens, Peter; Pal, Samir Kumar


    The functionality of a gene carrying nucleic acid in an artificial gene-delivery system is important for the overall efficiency of the vehicle in vivo. Here, we have studied a well-known artificial gene-delivery system, which is a condensate of calf thymus DNA (CT-DNA) with a model cationic surfactant cetyltrimethylammonium bromide (CTAB) to investigate the molecular recognition of the genomic DNA in the condensate. While dynamic light scattering (DLS) and circular dichroism (CD) reveal structural aspects of the condensate and the constituting DNA respectively, picosecond resolved polarization gated spectroscopy and Förster resonance energy transfer (FRET) reveal molecular recognition of the genomic DNA in the condensate. We have considered ethidium bromide (EB) and crystal violet (CV), which are well known DNA-binding agents through intercalative (specific) and electrostatic (non-specific) interactions, respectively, as model ligands for the molecular recognition studies. A fluorescent cationic surfactant, Nonyl Acridine Orange (NAO) is considered to be a mimic of CTAB in the condensate. The polarization gated fluorescence of NAO at various temperatures has been used to investigate the local microviscosity of the condensate. The excellent spectral overlap of NAO emission and the absorption spectra of both EB and CV allow us to investigate FRET-distances of the ligands with respect to NAO in the condensate at various temperatures and thermal stability of ligand-binding of the genomic DNA. The thermodynamic properties of the molecular recognition have also been explored using Van't Hoff equation. We have also extended our studies to molecular recognition of the genomic DNA in the condensate as dried thin films. This has important implications for its application in bioelectronics.

  8. Serum-resistant complex nanoparticles functionalized with imidazole-rich polypeptide for gene delivery to pulmonary metastatic melanoma. (United States)

    Gu, Jijin; Chen, Xinyi; Xin, Hongliang; Fang, Xiaoling; Sha, Xianyi


    To enhance serum-resistance and overcome the lysosomal barrier are effective and feasible strategies to increase the transfection efficiency of non-viral gene delivery system. For the systemic delivery of therapeutic gene, we previously developed self-assemble carboxymethyl poly(l-histidine) (CM-PLH)/poly(β-amino ester) (PbAE)/pDNA ternary complex nanoparticles based on electrostatic coating as an effective pDNA carrier. Recharging cationic PbAE/pDNA polyplexes with CM-PLH was a promising method to reduce the cytotoxicity and enhance the stability in vivo of positive charged polyplexes. In the present study, the transfection activities of ternary complex nanoparticles were further evaluated in vitro and in vivo. The transfection efficiency of ternary complex nanoparticles showed significant serum-resistance (CM-PLH-containing (51.9±4.35)% in 50% FBS>CM-PLH-free (14.7±5.66)% in 50% FBS), cell line dependent (HEK293>MCF-7>COS7>B16F10>A549>Hela>SPC-A1>CHO>SKOV3) and incubation period dependent (24 h, 20 h, 16 h>12 h>8 h>4 h>2 h>1 h>0.5 h). After transfected with ternary complex nanoparticles loading pGV240-MDA-7/IL-24, the B16F10 cells exhibited significant apoptosis and proliferation inhibition due to the expression of IL-24. Moreover, in the pulmonary metastatic melanoma model, ternary complex nanoparticles loading pGV240-MDA-7/IL-24 showed significant antitumor therapeutic efficacy in vivo. These results suggested that CM-PLH/PbAE/pDNA ternary complex nanoparticles were promising and challenging gene vector for practical application.

  9. Efficient delivery of C/EBP beta gene into human mesenchymal stem cells via polyethylenimine-coated gold nanoparticles enhances adipogenic differentiation (United States)

    Joydeep, Das; Choi, Yun-Jung; Yasuda, Hideyo; Han, Jae Woong; Park, Chankyu; Song, Hyuk; Bae, Hojae; Kim, Jin-Hoi


    The controlled differentiation of stem cells via the delivery of specific genes encoding appropriate differentiation factors may provide useful models for regenerative medicine and aid in developing therapies for human patients. However, the majority of non-viral vectors are not efficient enough to manipulate difficult-to-transfect adult human stem cells in vitro. Herein, we report the first use of 25 kDa branched polyethylenimine-entrapped gold nanoparticles (AuPEINPs) and covalently bound polyethylenimine-gold nanoparticles (AuMUAPEINPs) as carriers for efficient gene delivery into human mesenchymal stem cells (hMSCs). We determined a functional application of these nanoparticles by transfecting hMSCs with the C/EBP beta gene, fused to EGFP, to induce adipogenic differentiation. Transfection efficacy with AuPEINPs and AuMUAPEINPs was 52.3% and 40.7%, respectively, which was 2.48 and 1.93 times higher than that by using Lipofectamine 2000. Luciferase assay results also demonstrated improved gene transfection efficiency of AuPEINPs/AuMUAPEINPs over Lipofectamine 2000 and polyethylenimine. Overexpression of exogenous C/EBP beta significantly enhanced adipogenesis in hMSCs as indicated by both of Oil Red O staining and mRNA expression analyses. Nanoparticle/DNA complexes exhibited favorable cytocompatibility in hMSCs. Taken together, AuPEINPs and AuMUAPEINPs potentially represent safe and highly efficient vehicles for gene delivery to control hMSC differentiation and for therapeutic gene delivery applications. PMID:27677463

  10. Gene delivery to mice spermatogenic stem cells by EffecteneTM reagent

    Institute of Scientific and Technical Information of China (English)

    陈晓光; 王宁; 姚纪花; 陈浩明; 沈琦; 薛京伦


    @@ Spermatogenic (stem) cells, or spermatogonial stem cells, are the only cell type in postnatal mammals, which have the capability to self-renew and to contribute geneticinformation to the next generation. The manipulation of spermatogenic cells and the modification of their genomes have great significance for the treatment of male sterility,for gene therapy via germ cells, as well as for building transgenic animal models. 1 In this assay, we analyzed the efficiency of EffecteneTM reagent-mediated gene transfection into spermatogenic cells. The effect of transplants with different time schedules on transfection efficiency and on gene expression was also investigated.

  11. Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats

    National Research Council Canada - National Science Library

    Cindy Wang; Eric Dobrzynski; Julie Chao; Lee Chao


    .... A single tail vein injection of adenovirus harboring the human AM gene significantly blunted a blood pressure increase that lasted for more than 3 wk in two-kidney one-clip (2K1C) hypertensive rats...

  12. Disulfide-functional poly(amido amine)s with tunable degradability for gene delivery

    NARCIS (Netherlands)

    Elzes, M. Rachel; Akeroyd, N.; Engbersen, Johan F.J.; Paulusse, Jos M.J.


    Controlled degradability in response to the local environment is one of the most effective strategies to achieve spatiotemporal release of genes from a polymeric carrier. Exploiting the differences in reduction potential between the extracellular and intracellular environment, disulfides are frequen

  13. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene. (United States)

    Melo, Luis G; Agrawal, Reitu; Zhang, Lunan; Rezvani, Mojgan; Mangi, Abeel A; Ehsan, Afshin; Griese, Daniel P; Dell'Acqua, Giorgio; Mann, Michael J; Oyama, Junichi; Yet, Shaw-Fang; Layne, Matthew D; Perrella, Mark A; Dzau, Victor J


    Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

  14. Sequence-defined cMET/HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene. (United States)

    Urnauer, Sarah; Morys, Stephan; Krhac Levacic, Ana; Müller, Andrea M; Schug, Christina; Schmohl, Kathrin A; Schwenk, Nathalie; Zach, Christian; Carlsen, Janette; Bartenstein, Peter; Wagner, Ernst; Spitzweg, Christine


    The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g (123)I, biological half-life 3 hours) by (123)I-scintigraphy. Therapy studies with three cycles of polyplexes and (131)I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

  15. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism. (United States)

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi


    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)-nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications.

  16. 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

  17. Treatment of Hemophilia A in Utero and Postnatally using Sheep as a Model for Cell and Gene Delivery (United States)

    Porada, Christopher D.; Almeida-Porada, Graça


    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

  18. Low Molecular Weight PEI-Based Vectors via Acid-Labile Ortho Ester Linkage for Improved Gene Delivery. (United States)

    Zhang, Lei; Yu, Min; Wang, Jun; Tang, Rupei; Yan, Guoqing; Yao, Weijing; Wang, Xin


    A series of novel pH-sensitive gene delivery vectors (POEI 1, 2, and 3) are synthesized through Michael addition from low molecular weight PEI (LMW PEI) via acid-labile ortho ester linkage with terminal acrylates (OEAc) by various feed molar ratios. The obtained POEI 1 and POEI 2 can efficiently condense plasmid DNA into nanoparticles with size range of 200-300 nm and zeta-potentials of about +15 mV while protecting DNA from enzymatic digestion compared with POEI 3. Significantly, ortho ester groups of POEI main-chains can make an instantaneous degradation-response to acidic endosomal pH (≈5.0), resulting in accelerated disruption of polyplexes and intracellular DNA release. MTT assay reveals that all POEIs exhibit much lower cytotoxicity in different cells than branched PEI (25 KDa). As expected, POEI 1 and POEI 2 perform improved gene transfection in vitro, suggesting that such polycations might be promising gene vectors based on overcoming toxicity-efficiency contradiction.

  19. Effects of pressure characteristics on transfection efficiency in laser-induced stress wave-mediated gene delivery (United States)

    Ando, Takahiro; Sato, Shunichi; Ashida, Hiroshi; Obara, Minoru


    Laser-induced stress waves (LISWs) generated by irradiating a light-absorbing medium with a pulsed laser can transiently increase the permeability of cell membranes for gene delivery. In this study, we investigated the effects of pressure characteristics of LISWs upon gene transfection efficiency using lasers with different pulse durations: a 6-ns pulsed Nd:YAG laser and 20-ns and 200-µs pulsed ruby lasers. LISWs were generated by irradiating a black rubber disk, on which a transparent plastic sheet was adhered for confinement of the laser-produced plasma. Rat dorsal skin was injected with plasmid DNA coding for luciferase, to which LISWs were applied. With nanosecond laser pulses, transfection efficiency increased linearly with increasing positive peak pressure in the range of 35 to 145 MPa, the corresponding impulse ranging from 10 to 40 Paṡs. With 200-µs laser pulses, on the other hand, efficient gene expression was observed by the application of LISWs even with a 10-fold-lower peak pressure (˜5 MPa), the corresponding impulse being as large as 430 Paṡs. These results indicate that even at low peak pressures, efficient transfection can be achieved by extending the pressure duration and hence by increasing the impulse of LISWs, while the averaged expression efficiencies were relatively low.

  20. Enhanced Delivery of Plasmid Encoding Interleukin-12 Gene by Diethylene Triamine Penta-Acetic Acid (DTPA)-Conjugated PEI Nanoparticles. (United States)

    Dehshahri, Ali; Sadeghpour, Hossein; Keykhaee, Maryam; Khalvati, Bahman; Sheikhsaran, Fatemeh


    Recombinant therapeutic proteins have been considered as an efficient category of medications used for the treatment of various diseases. Despite their effectiveness, there are some reports on the systemic adverse effects of recombinant therapeutic proteins limiting their wide clinical applications. Among different cytokines used for cancer immunotherapy, interleukin-12 (IL-12) has shown great ability as a powerful antitumor and antiangiogenic agent. However, significant toxic reactions following the systemic administration of IL-12 have led researchers to seek for alternative approaches such as the delivery and local expression of the IL-12 gene inside the tumor tissues. In order to transfer the plasmid encoding IL-12 gene, the most extensively investigated polycationic polymer, polyethylenimine (PEI), was modified by diethylene triamine penta-acetic acid (DTPA) to modulate the hydrophobic-hydrophilic balance of the polymer as well as its toxicity. DTPA-conjugated PEI derivatives were able to form complexes in the size range around 100-180 nm with great condensation ability and protection of the plasmid against enzymatic degradation. The highest gene transfer ability was achieved by the DTPA-conjugated PEI at the conjugation degree of 0.1 % where the level of IL-12 production increased up to twofold compared with that of the unmodified PEI. Results of the present study demonstrated that modulation of the surface positive charge of PEI along with the improvement of the polymer hydrophobic balance could be considered as a successful strategy to develop safe and powerful nanocarriers.

  1. Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells. (United States)

    Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N


    The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

  2. Nano-polyplex based on oleoyl-carboxymethy-chitosan (OCMCS) and hyaluronic acid for oral gene vaccine delivery. (United States)

    Liu, Ya; Wang, Fang-Qin; Shah, Zeana; Cheng, Xiao-Jie; Kong, Ming; Feng, Chao; Chen, Xi-Guang


    Here we described nano-polyplexes (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) as novel potential carriers for oral gene vaccines delivery. Aerolysin gene (aerA) of Aeromonas hydrophila as microbial antigen was efficiently loaded to form OCMCS-HA/aerA (OHA) NPs. OHA NPs performed the optimal parameters, i.e. smallest (154.5±9.4nm), positive charged (+7.9±0.5mV) and monodispersed system with the N/P ratio of 5 and OCMCS/HA weight ratio of 4. Upon the introduction of HA, OHA NPs was beneficial for the DNA release in intestinal environments in comparison to OA NPs. The mean fluorescence intensity detected in Caco-2 cells incubated with OHA NPs was about 2.5-fold higher than that of OA NPs; however, it decreased significantly in the presence of excess free HA. The OHA NPs and OA NPs decreased the transepithelial electric resistance (TEER) of Caco-2 monolayers obviously and induced increasing the apparent permeability coefficient (Papp) of DNA by 5.45-6.09 folds compared with free DNA. Significantly higher (P<0.05) antigen-specific antibodies were detected in serum after orally immunized with OHA NPs than that immunized with OA NPs and DNA alone in carps. These results enable the OHA NPs might resolve challenges arising from gastrointestinal damage to gene antigens, and offer an approach applicable for oral vaccination.

  3. Quaternary ammonium salt containing soybean oil: an efficient nanosize gene delivery carrier for halophile green microalgal transformation. (United States)

    Akbari, Fariba; Yari Khosroushahi, Ahmad; Yeganeh, Hamid


    Dunaliella salina, a halophile green microalga, is considered a robust photobioreactor and a remarkable cost beneficial system for the production of therapeutic recombinant proteins. In this study, with low overall cost, a proper cationic lipid was synthesized from renewable soybean oil as an efficient gene delivery carrier for D. salina cells to create appropriate protein-producing transformed cell lines. To obtain an effective carrier, quaternary ammonium salt containing soybean oil (QASSO) was synthesized through the ring opening reaction of the epoxy groups of epoxidized soybean oil with diethylamine. QASSO was characterized using nuclear magnetic resonance and Fourier-transform infrared instruments. QASSO was used to prepare nanolipoplex construct using plasmid DNA molecules containing green fluorescent protein (GFP) as reporter gene. These nanolipoplexes (QASSO-pGFP, N/P=3) and QASSO had diameter of 63.62 and 110.63 nm, and zeta potential of -68.89 and 48.25 mV at pH 7.0, respectively. Results indicated the GFP gene expression and cytoplasmic accumulation of GFP protein in the transformants after incubation under desirable conditions for 48 h and 1 week. The transformation efficiency was quantitatively assayed by flow cytometry, which yielded transformations of 58.87% and 48.34% for QASSO and 38.32% and a negligible percentage for Polyfect® after 48 h and 1 week incubation, respectively.

  4. BDNF gene delivery within and beyond templated agarose multi-channel guidance scaffolds enhances peripheral nerve regeneration (United States)

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


    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.

  5. Photo-irradiation paradigm: Mapping a remarkable facile technique used for advanced drug, gene and cell delivery. (United States)

    Shaker, Mohamed A; Younes, Husam M


    Undoubtedly, the progression of photo-irradiation technique has provided a smart engineering tool for the state-of-the-art biomaterials that guide the biomedical and therapeutic domains for promoting the modern pharmaceutical industry. Many investigators had exploited such a potential technique to create/ameliorate numerous pharmaceutical carriers. These carriers show promising applications that vary from small drug to therapeutic protein delivery and from gene to living cell encapsulation design. Harmony between the properties of precisely engineered precursors and the formed network structure broadens the investigator's intellect for both brilliant creations and effective applications. As well, controlling photo-curing at the formulation level, through manipulating the absorption of light stimuli, photoinitiator system and photo-responsive precursor, facilitates the exploration of novel distinctive biomaterials. Discussion of utilizing different photo-curing procedures in designing/formulation of different pharmaceutical carriers is the main emphasis of this review. In addition, recent applications of these intelligent techniques in targeted, controlled, and sustained drug delivery with understanding of photo-irradiation concept and mechanism are illustrated.

  6. Non-covalent functionalization of single-walled carbon nanotubes with modified polyethyleneimines for efficient gene delivery. (United States)

    Behnam, Behzad; Shier, Wayne T; Nia, Azadeh Hashem; Abnous, Khalil; Ramezani, Mohammad


    Functionalized carbon nanotubes (CNTs) have been recently emerged as important class of vectors for delivery of DNA and other biomolecules into various cells. In this study, single-walled carbon nanotubes (SWNTs) were functionalized by non-covalent binding of hydrophobic moieties, which were covalently linked to polyethyleneimines (PEIs). PEIs of three molecular weights (25, 10 and 1.8kDa) were used. CNTs were functionalized with the PEI series either through phospholipid moiety (via a polyethyleneglycol linker) or through directly-attached long (18 carbons) or intermediate (10 carbons) hydrophobic alkyl moieties. All PEI-functionalized CNTs exhibited good stability and dispersibility in biological media. Visualizing of functionalized CNTs and lack of aggregation were confirmed by atomic force microscopy. The PEI derivatives bound to CNTs retained the ability to fully condense plasmid DNA at low N/P ratios and substantial buffering capacity in the endosomal pH range. PEI-functionalized CNTs exhibited increased transfection efficiency compared to underivatized PEIs up to 19-fold increase being observed in the functionalized CNT with the smallest PEI tested, the smallest hydrophobic attachment moiety tested and no linker. Also PEI-functionalized CNTs were effective gene delivery vectors in vivo following tail vein injection in mice with the largest expression occurring with the vector PEI-functionalized through a polyethyleneglycol linker.

  7. Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

    Institute of Scientific and Technical Information of China (English)

    ZHOU Xi-yuan; LIAO Qing; PU Yi-min; TANG Yong-qiang; GONG Xiao; LI Jia; XU Yan; WANG Zhi-gang


    Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization (CNV), and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor (PEDF) could inhibit CNV.Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure (intravitreal injection); infusing liposomes with the naked plasmid DNA of PEDF into the vitreous (lipofectamine + PEDF); infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately (retrobular injection); infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately (vein injection). The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with

  8. iTRAQ-Based Proteomic Analysis of Visual Cycle-Associated Proteins in RPE of rd12 Mice before and after RPE65 Gene Delivery. (United States)

    Zheng, Qinxiang; Ren, Yueping; Tzekov, Radouil; Hua, Shanshan; Li, Minghan; Pang, Jijing; Qu, Jia; Li, Wensheng


    Purpose. To investigate the iTRAQ-based proteomic changes of visual cycle-associated proteins in RPE of rd12 mice before and after RPE65 gene delivery. Mehtods. The right eyes of rd12 mice underwent RPE65 gene delivery by subretinal injection at P14, leaving the left eyes as control. C57BL/6J mice were served as a wide-type control group. ERGs were recorded at P42, and RPE-choroid-sclera complex was collected to evaluate the proteomic changes in visual cycle-associated proteins by iTRAQ-based analysis. Western blot was used to confirm the changes in the differentially expressed proteins of interest. Results. ERG parameters improved dramatically at P42 after RPE65 delivery. The proteomics analysis identified a total 536 proteins with a global false discovery rate of 0.21%, out of which 7 were visual cycle-associated proteins. RALBP-1, RBP-1, and IRBP were reduced in the untreated rd12 eyes and the former two were improved after gene therapy, confirmed by Western blot analysis. Conclusions. RPE65 gene delivery restored retinal function at P42 and modified the expression of other functional proteins implicated in the visual cycle. The level of RALBP-1 was still below the normal level after gene therapy in rd12 mice, which may explain the delayed dark adaption in LCA patients undergoing similar therapy.

  9. iTRAQ-Based Proteomic Analysis of Visual Cycle-Associated Proteins in RPE of rd12 Mice before and after RPE65 Gene Delivery

    Directory of Open Access Journals (Sweden)

    Qinxiang Zheng


    Full Text Available Purpose. To investigate the iTRAQ-based proteomic changes of visual cycle-associated proteins in RPE of rd12 mice before and after RPE65 gene delivery. Mehtods. The right eyes of rd12 mice underwent RPE65 gene delivery by subretinal injection at P14, leaving the left eyes as control. C57BL/6J mice were served as a wide-type control group. ERGs were recorded at P42, and RPE-choroid-sclera complex was collected to evaluate the proteomic changes in visual cycle-associated proteins by iTRAQ-based analysis. Western blot was used to confirm the changes in the differentially expressed proteins of interest. Results. ERG parameters improved dramatically at P42 after RPE65 delivery. The proteomics analysis identified a total 536 proteins with a global false discovery rate of 0.21%, out of which 7 were visual cycle-associated proteins. RALBP-1, RBP-1, and IRBP were reduced in the untreated rd12 eyes and the former two were improved after gene therapy, confirmed by Western blot analysis. Conclusions. RPE65 gene delivery restored retinal function at P42 and modified the expression of other functional proteins implicated in the visual cycle. The level of RALBP-1 was still below the normal level after gene therapy in rd12 mice, which may explain the delayed dark adaption in LCA patients undergoing similar therapy.

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

    LENUS (Irish Health Repository)

    Tangney, Mark


    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.

  11. Biomaterial constructs for delivery of multiple therapeutic genes: a spatiotemporal evaluation of efficacy using molecular beacons.

    Directory of Open Access Journals (Sweden)

    Jennifer C Alexander

    Full Text Available Gene therapy is emerging as a potential therapeutic approach for cardiovascular pathogenesis. An appropriate therapy may require multiple genes to enhance therapeutic outcome by modulating inflammatory response and angiogenesis in a controlled and time-dependent manner. Thus, the aim of this research was to assess the spatiotemporal efficacy of a dual-gene therapy model based on 3D collagen scaffolds loaded with the therapeutic genes interleukin 10 (IL-10, a potent anti-inflammatory cytokine, and endothelial nitric oxide synthase (eNOS, a promoter of angiogenesis. A collagen-based scaffold loaded with plasmid IL-10 polyplexes and plasmid eNOS polyplexes encapsulated into microspheres was used to transfect HUVECs and HMSCs cells.The therapeutic efficacy of the system was monitored at 2, 7 and 14 days for eNOS and IL-10 mRNA expression using RT-PCR and live cell imaging molecular beacon technology. The dual gene releasing collagen-based scaffold provided both sustained and delayed release of functional polyplexes in vitro over a 14 day period which was corroborated with variation in expression levels seen using RT-PCR and MB imaging. Maximum fold increases in IL-10 mRNA and eNOS mRNA expression levels occurred at day 7 in HMSCs and HUVECs. However, IL-10 mRNA expression levels seemed dependent on frequency of media changes and/or ease of transfection of the cell type. It was demonstrated that molecular beacons are able to monitor changes in mRNA levels at various time points, in the presence of a 3D scaffolding gene carrier system and the results complemented those of RT-PCR.

  12. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts. (United States)

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


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

  13. Neural stem cell-based dual suicide gene delivery for metastatic brain tumors. (United States)

    Wang, C; Natsume, A; Lee, H J; Motomura, K; Nishimira, Y; Ohno, M; Ito, M; Kinjo, S; Momota, H; Iwami, K; Ohka, F; Wakabayashi, T; Kim, S U


    In our previous works, we demonstrated that human neural stem cells (NSCs) transduced with the cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on glioma and medulloblastoma cells after administration of the prodrug 5-fluorocytosine (5-FC). In addition, herpes simplex virus thymidine kinase (TK) is a widely studied enzyme used for suicide gene strategies, for which the prodrug is ganciclovir (GCV). To apply this strategy to brain metastasis treatment, we established here a human NSC line (F3.CD-TK) expressing the dual suicide genes CD and TK. We examined whether F3.CD-TK cells intensified the antitumor effect on lung cancer brain metastases. In vitro studies showed that F3.CD-TK cells exerted a marked bystander effect on human lung cancer cells after treatment with 5-FC and GCV. In a novel experimental brain metastases model, intravenously administered F3 cells migrated near lung cancer metastatic lesions, which were induced by the injection of lung cancer cells via the intracarotid artery. More importantly, F3.CD-TK cells in the presence of prodrugs 5-FC and GCV decreased tumor size and considerably prolonged animal survival. The results of the present study indicate that the dual suicide gene-engineered, NSC-based treatment strategy might offer a new promising therapeutic modality for brain metastases.

  14. Prenyl Ammonium Salts - New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

    Directory of Open Access Journals (Sweden)

    Emilia Grecka

    Full Text Available Prenyl ammonium iodides (Amino-Prenols, APs, semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation-considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2, introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

  15. 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


    in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion into the brain. The non-mitotic BCECs might, however, not be very susceptible to non-viral gene therapy in vivo, since this strategy is believed to be dependent on active cell division. We have...

  16. Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David G. J. [Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology; McKnight, Timothy E. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Engineering Science and Technology Division; Melechko, Anatoli V. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Simpson, Michael L. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Sayler, Gary S. [Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology


    Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. We used polymerase chain reaction (PCR) and in vitro transcription (IVT) to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. In conclusion,this investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

  17. Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier. (United States)

    Peng, Yu-Shiang; Lai, Po-Liang; Peng, Sydney; Wu, His-Chin; Yu, Siang; Tseng, Tsan-Yun; Wang, Li-Fang; Chu, I-Ming


    Parkinson's disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood-brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson's disease.

  18. Efficiency of adenoviral vector mediated CTLA4Ig gene delivery into mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    邓宇斌; 郭小荑; 原清涛; 李树浓


    Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy. Methods We constructed the recombinant adenovirus containing CTLA4Ig gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4Ig expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model. Results Recombinant adenovirus pAd-CTLA4Ig was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4Ig protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P<0.05). A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4Ig expression was observed in the liver, intestine, kidney and spleen 30 days post- transplantation. Conclusions Our results indicate that adenoviral vectors could efficiently transfer CTLA4Ig gene into MSCs and sustain long-term stable expression in vitro and in vivo.

  19. Targeted delivery of human VEGF gene via complexes of magnetic nanoparticle-adenoviral vectors enhanced cardiac regeneration.

    Directory of Open Access Journals (Sweden)

    Yue Zhang

    Full Text Available This study assessed the concept of whether delivery of magnetic nanobeads (MNBs/adenoviral vectors (Ad-encoded hVEGF gene (Ad(hVEGF could regenerate ischaemically damaged hearts in a rat acute myocardial infarction model under the control of an external magnetic field. Adenoviral vectors were conjugated to MNBs with the Sulfo-NHS-LC-Biotin linker. In vitro transduction efficacy of MNBs/Ad-encoded luciferase gene (Ad(luc was compared with Ad(luc alone in human umbilical vein endothelial cells (HUVECs under magnetic field stimulation. In vivo, in a rat acute myocardial infarction (AMI model, MNBs/Ad(hVEGF complexes were injected intravenously and an epicardial magnet was employed to attract the circulating MNBs/Ad(hVEGF complexes. In vitro, compared with Ad(luc alone, MNBs/Ad(luc complexes had a 50-fold higher transduction efficiency under the magnetic field. In vivo, epicardial magnet effectively attracted MNBs/Ad(hVEGF complexes and resulted in strong therapeutic gene expression in the ischemic zone of the infarcted heart. When compared to other MI-treated groups, the MI-M(+/Ad(hVEGF group significantly improved left ventricular function (p<0.05 assessed by pressure-volume loops after 4 weeks. Also the MI-M(+/Ad(hVEGF group exhibited higher capillary and arteriole density and lower collagen deposition than other MI-treated groups (p<0.05. Magnetic targeting enhances transduction efficiency and improves heart function. This novel method to improve gene therapy outcomes in AMI treatment offers the potential into clinical applications.

  20. Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier

    Directory of Open Access Journals (Sweden)

    Peng YS


    Full Text Available Yu-Shiang Peng,1,* Po-Liang Lai,2,* Sydney Peng,1 His-Chin Wu,3 Siang Yu,1 Tsan-Yun Tseng,4 Li-Fang Wang,5 I-Ming Chu1 1Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 2Department of Orthopedic Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, 3Department of Materials Engineering, Tatung University, Taipei, 4Graduate School of Biotechnology and Bioengineering, College of Engineering, Yuan Ze University, Chung-Li, 5Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan *Yu-Shiang Peng and Po-Liang Lai contributed equally to this work Abstract: Parkinson’s disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood–brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810. This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the

  1. A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Chun-Hong Ma; Xiao-Hong Liang; Wen-Sheng Sun; Pei-Kun Tian; Li-Fen Gao; Su-Xia Liu; Xiao-Yan Wang; Li-Ning Zhang; Ying-Lin Cao; Li-Hui Han


    AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo.METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA. BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured.RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1.The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RTPCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4injections of AFP-enhancing 4-element complex containing 0.2 μg DNA, the diameter of the tumor was 0.995 cm±0.35,which was significantly smaller than that of the control groups (2.215 cm±0.25, P<0.05).CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.

  2. A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma (United States)

    Ma, Chun-Hong; Sun, Wen-Sheng; Tian, Pei-Kun; Gao, Li-Fen; Liu, Su-Xia; Wang, Xiao-Yan; Zhang, Li-Ning; Cao, Ying-Lin; Han, Li-Hui; Liang, Xiao-Hong


    AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo. METHODS: GE7,a 16-peptide specific to EGFR, and HA20, a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA. BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured. RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1.The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4% and 58.5% respectively. RT-PCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2 μg DNA, the diameter of the tumor was 0.995 cm ± 0.35, which was significantly smaller than that of the control groups (2.215 cm ± 0.25, P < 0.05). CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo. PMID:12632498

  3. 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

    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......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....... Results: mRNA expression of proteins with neuroprotective potential in RBEC were enabled. Their expression patters were compared with those of RBE4 and HeLa cells using RT-qPCR analyzes. The evidence for protein synthesis and secretion was obtained by detection of FLAG-tagged to the C-terminal of any...

  4. [Synthesis of GnRH analogs and their application in targeted gene delivery systems]. (United States)

    Iablokova, T V; Chelushkin, P S; Dorosh, M Iu; Efremov, A M; Orlov, S V; Burov, S V


    A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.

  5. Electroporation-mediated Delivery of Genes in Rodent Models of Lung Contusion



    Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated g...

  6. Efficient gene therapy-based method for the delivery of therapeutics to primate cortex. (United States)

    Kells, Adrian P; Hadaczek, Piotr; Yin, Dali; Bringas, John; Varenika, Vanja; Forsayeth, John; Bankiewicz, Krystof S


    Transduction of the primate cortex with adeno-associated virus (AAV)-based gene therapy vectors has been challenging, because of the large size of the cortex. We report that a single infusion of AAV2 vector into thalamus results in widespread expression of transgene in the cortex through transduction of widely dispersed thalamocortical projections. This finding has important implications for the treatment of certain genetic and neurodegenerative diseases.

  7. Translational Advancement of Somatostatin Gene Delivery for Disease Modification and Cognitive Sparing in Intractable Epilepsy (United States)


    Anti-epileptic drugs are not effective anti-epileptogenics, so SST gene transfer offers a novel approach to limiting or even reversing the clinical...8:00 AM -12:00 PM Presenter at Poster: Mon, Nov. 17, 2014, 9:00 AM - 10:00 AM Topic: ++C.07.h. Anticonvulsant and antipileptic therapies Authors: *G...of an SST vector could similarly suppress seizures when delivered after a stable seizure state is established. We tested the putative anticonvulsant

  8. Disulfide-Based Poly(amido amine)s for siRNA Delivery: Effects of Structure on siRNA Complexation, Cellular Uptake, Gene Silencing and Toxicity

    NARCIS (Netherlands)

    Vader, Pieter; Aa, van der Leonardus J.; Engbersen, Johan F.J.; Storm, Gert; Schiffelers, Raymond M.


    Purpose RNA interference (RNAi) is a process by which small interfering RNAs (siRNA) induce sequence-specific gene silencing. Therefore, siRNA is an emerging promise as a novel therapeutic. In order to realize the high expectations for therapeutic applications, efficient delivery systems for siRNA

  9. Synergistic effects in gene delivery-a structure-activity approach to the optimisation of hybrid dendritic-lipidic transfection agents. (United States)

    Jones, Simon P; Gabrielson, Nathan P; Pack, Daniel W; Smith, David K


    Novel gene delivery agents based on combining cholesterol units with spermine-functionalised dendrons exhibit enhanced transfection ability-we report significant synergistic effects in mixed (hybrid) systems which combine aspects of both main classes of synthetic vectors, i.e., cationic polymers and lipids.

  10. Adenovector GAD65