A calcium phosphate-based gene delivery system

Although nonviral vectors have lower transfection efficiency than viral vectors, the excellent safety profile of nonviral vectors is appealing for gene therapy. An efficient, simple nonviral vector gene delivery system has been designed that includes plasmid DNA-calcium phosphate precipitates (pDNA-CaP) and porous collagen spheres (Cultispherestrade mark). The hypothesis for this study was the pDNA-CaP would achieve efficient plasmid DNA transfection and the porous collagen spheres would provide a suitable delivery carrier system for three-dimensional (3D) administration. To test the hypothesis, plasmid DNA including the LacZ reporter gene encoding beta-galactosidase was precipitated with CaP to form particles of compacted LacZ-CaP and delivered directly or by Cultispherestrade mark to cells in vitro. The transfection efficiency was determined by beta-galactosidase gene expression. Results indicated that pLacZ-CaP promoted 25-84% of transfection efficiency in a broad cell line spectrum and in flexible experimental conditions. Maximum transfection efficiency was achieved by having mostly nano-sized partles (50-200 nm in diameter) of pDNA-CaP precipitates. Seeding density of 0.7-4 x 10(4) cells/cm2 provided sufficient transfection efficiency, and storage of pDNA-CaP at 4 degrees C was most efficient to preserve transfection efficacy for up to 3 days. The pDNA-CaP worked well in the presence of serum and serum-free conditions and was less cytotoxic than the liposomes. Cultispherestrade mark carrying plasmid LacZ-CaP was an effective 3D system for gene delivery. The technique described here is a simple and safe procedure to deliver genes, and may have application to regenerate bone and other tissues.     

Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons

Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25 kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons.     

Improvement of nonviral gene therapy by Epstein-Barr virus (EBV)-based plasmid vectors

The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the beta-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.     

Dual-role self-assembling nanoplexes for efficient gene transfection and sustained gene delivery

Novel cationic pentablock copolymers with poly(diethylamino ethyl methacrylate) blocks covalently attached to parent triblock Pluronic copolymers have been designed and developed as sustained release non-viral gene delivery vectors. These copolymers electrostatically condense plasmid DNA into nanostructures (nanoplexes) and further self-assemble above critical concentration to form thermoreversible hydrogels at physiological temperatures. Unlike other sustained gene delivery systems of non-ionic copolymers that release naked DNA, hydrogels of pentablock copolymer/DNA nanoplexes dissolve in excess buffers to release DNA compacted inside the nanoplexes. These hydrogels permit aqueous pharmaceutical formulations that do not involve organic solvents and are non-invasively injectable with syringes into localized tissues where they instantly form hydrogels in situ. The hydrogels were found to have better mechanical strength than Pluronic gels. Hydrogels of nanoplexes containing 15wt% copolymer dissolved to release nanoplexes up to 5 days in vitro, compared to rapid release of up to 90% entrapped naked DNA from only Pluronic gels by day 1. The release profile of the nanoplexes from the hydrogels could be modulated by changing the concentration of copolymer or plasmid DNA in the hydrogel formulation. Since DNA is electrostatically bound to copolymer molecules, it does not freely diffuse out of the polymeric network, preventing initial release bursts observed with other such controlled release gels/matrices/microspheres. The released nanoplexes were colloidally stable, preserved the integrity of supercoiled plasmid DNA, and gave good transfection efficiencies in vitro upon dissolution. These novel copolymers, thus, act as both nanoscale gene delivery vectors and macroscale sustained delivery agents, and make a clinically viable long-term sustained gene delivery system.     

Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea

Summary We report here on the development of a transient expression system for Chlorella ellipsoidea using a heterologous gene, firefly luciferase. Cells of this unicellular green alga were converted to protoplasts and treated with plasmid pDO432, which bears luciferase under the control of the CaMV 35S promoter. This treatment resulted in detectable luciferase activity in cell extracts. Expression required Cellulysin treatment, active cell metabolism, and the addition of carrier DNA and polyethylene glycol. Linearization of the luciferase plasmid did not significantly alter the activity. A time course of expression showed that luciferase is made rapidly, within about 7 h after addition of DNA, but that the activity disappears over the course of a few days. These experiments represent an important first step in the development of a Chlorella transformation system.     

Muscular gene transfer using nonviral vectors

Skeletal muscle is a target tissue of choice for the gene therapy of both muscle and non-muscle disorders. Investigations of gene transfer into muscle have progressed considerably from the expression of plasmid reporter genes to the production of therapeutic proteins such as trophic factors, hormones, antigens, ion channels or cytoskeletal proteins. Viral vectors are intrinsically the most efficient vehicles to deliver genes into skeletal muscles. But, because viruses are associated with a variety of problems (such as immune and inflammatory responses, toxicity, limited large scale production yields, limitations in the size of the carried therapeutic genes), nonviral vectors remain a viable alternative. In addition, as nonviral vectors allow to transfer genetic structures of various sizes (including large plasmid DNA carrying full-length coding sequences of the gene of interest), they can be used in various gene therapy approaches. However, given the lack of efficiency of nonviral vectors in experimental studies and in the clinical settings, the overall outcome clearly indicates that improved synthetic vectors and/or delivery techniques are required for successful clinical gene therapy. Today, most of the potential muscle-targeted clinical applications seem geared toward peripheral ischemia (mainly through local injections) and cancer and infectious vaccines, and one locoregional administration of naked DNA in Duchenne muscular dystrophy. This review updates the developments in clinical applications of the various plasmid-based non-viral methods under investigation for the delivery of genes to muscles.     

Bioengineered silk protein-based gene delivery systems

Silk proteins self-assemble into mechanically robust material structures that are also biodegradable and non-cytotoxic, suggesting utility for gene delivery. Since silk proteins can also be tailored in terms of chemistry, molecular weight and other design features via genetic engineering, further control of this system for gene delivery can be considered. In the present study, silk-based block copolymers were bioengineered with poly(l-lysine) domains for gene delivery. Ionic complexes of these silk-polylysine based block copolymers with plasmid DNA (pDNA) were prepared for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. The polymers self-assembled in solution and complexed plasmid DNA through ionic interactions. The pDNA complexes with 30 lysine residues prepared at a polymer/nucleotide ratio of 10 and with a solution diameter of 380 nm showed the highest efficiency for transfection. The pDNA complexes were also immobilized on silk films and demonstrated direct cell transfection from these surfaces. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailored gene delivery systems.     

Cationic star polymers consisting of alpha-cyclodextrin core and oligoethylenimine arms as nonviral gene delivery vectors

A series of novel cationic star polymers were synthesized by conjugating multiple oligoethylenimine (OEI) arms onto an alpha-cyclodextrin (alpha-CD) core as nonviral gene delivery vectors. The molecular structures of the alpha-CD-OEI star polymers, which contained linear or branched OEI arms with different chain lengths ranging from 1 to 14 ethylenimine units, were characterized by using size exclusion chromatography, 13C and 1H NMR, and elemental analysis. The alpha-CD-OEI star polymers were studied in terms of their DNA binding capability, formation of nanoparticles with plasmid DNA (pDNA), cytotoxicity, and gene transfection in cultured cells. All the alpha-CD-OEI star polymers could inhibit the migration of pDNA on agarose gel through formation of complexes with pDNA, and the complexes formed nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The star polymers displayed much lower in vitro cytotoxicity than that of branched polyethylenimine (PEI) of molecular weight 25K. The alpha-CD-OEI star polymers showed excellent gene transfection efficiency in HEK293 and Cos7 cells. Generally, the transfection efficiency increased with an increase in the OEI arm length. The star polymers with longer and branched OEI arms showed higher transfection efficiency. The best one of the star polymers for gene delivery showed excellent in vitro transfection efficiency that was comparable to or even higher than that of branched PEI (25K). The novel alpha-CD-OEI star polymers with OEI arms of different chain lengths and chain architectures can be promising new nonviral gene delivery vectors with low cytotoxicity and high gene transfection efficiency for future gene therapy applications.     

Cationized gelatin hydrogels mixed with plasmid DNA induce stronger and more sustained gene expression than atelocollagen at calvarial bone defects in vivo

Gene transduction of exogenous factors at local sites in vivo is a promising approach to promote regeneration of tissue defects owing to its simplicity and capacity for expression of a variety of genes. Gene transduction by viral vectors is highly efficient; however, there are safety concerns associated with viruses. As a method for nonviral gene transduction, plasmid DNA delivery is safer and simpler, but requires an efficient carrier substance. Here, we aimed to develop a simple, efficient method for bone regeneration by gene transduction and to identify optimal conditions for plasmid DNA delivery at bone defect sites. We focused on carrier substances and compared the efficiencies of two collagen derivatives, atelocollagen, and gelatin hydrogel, as substrates for plasmid DNA delivery in vivo. To assess the efficiencies of these substrates, we examined exogenous expression of green fluorescence protein (GFP) by fluorescence microscopy, polymerase chain reaction, and immunohistochemistry. GFP expression at the bone defect site was higher when gelatin hydrogel was used as a substrate to deliver plasmids than when atelocollagen was used. Moreover, the gelatin hydrogel was almost completely absorbed at the defect site, whereas some atelocollagen remained. When a plasmid harboring bone morphogenic protein 2 was delivered with the substrate to bony defect sites, more new bone formation was observed in the gelatin group than in the atelocollagen group. These results suggested that the gelatin hydrogel was more efficient than atelocollagen as a substrate for local gene delivery and may be a superior material for induction of bone regeneration.     

Topical application of plasmid DNA to mouse and human skin

Gene expression following direct injection of naked plasmid DNA into the skin has been demonstrated in the past. Topical application of plasmid DNA represents an attractive route of gene delivery. If successful, it would have great prospects in skin gene therapy since it is painless and easy to apply. In this study, we analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA in various liposomal spray formulations. Therefore, different concentrations of plasmid DNA expressing enhanced green fluorescent protein (pEGFP-N1) were sprayed onto mouse or human skin once daily for three consecutive days and compared with direct injection. Gene expression was assessed 24 h after the final topical application of various liposomal DNA formulations. The results showed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, respectively. However, epicutaneously applied EGFP plasmid DNA did not lead to microscopically detectable EGFP protein, when assessed by confocal laser microscopy or fluorescence-activated cell sorting in contrast to about 4% of fluorescent keratinocytes following intradermal injection. In an in vivo mouse model, the application of pEGFP-N1 DNA led to the generation of GFP-specific antibodies. These results indicate that topical spray application of pEGFP-N1 liposomal DNA formulations is a suitable method for plasmid DNA delivery to the skin, yielding limited gene expression. This spray method may thus be useful for DNA vaccination. To increase its attractiveness for skin gene therapy, the improvement of topical formulations with enhanced DNA absorption is desirable.     

Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates nonviral gene insertion and stable expression in target cells and tissues. Here, we demonstrate use of the nonviral SB transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (a) green fluorescent protein coupled to the zeocin gene or (b) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells cotransfected with transposon- and transposase-encoding sequences exhibited transgene expression after 1 week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to 5 months in human ES cells cotransfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells.     

Development of a tetracycline controlled gene expression system in the parasitic protozoan Giardia lamblia

Giardia lamblia is a very common intestinal protozoan pathogen of humans. Recent development of gene transfection systems in G. lamblia has allowed constitutive expression of selected genes in the organism. To extend the uses of DNA transfection in G. lamblia, an inducible gene expression system was developed by integrating the bacterial tet operator-repressor elements into an episomal DNA transfection vector. Tetracycline-responsive promoters with insertions of multiple tet operator sequences in the vicinity of a synthetic ran promoter were tested for their inducibility of a luciferase reporter gene expression. Stable cell lines transfected with individual plasmid constructs were established under drug selection. By assaying luciferase activity in transfected cells in response to tetracycline, an inducible promoter with insertion of two tet operators downstream of the adjacent synthetic ran promoter was found to confer a 10-fold inducibility in gene expression with co-expression of the tet-repressor driven by a gdh promoter. To further improve its inducibility, several other synthetic promoter contexts were also tested to increase expression of the tet-repressor gene. An optimal inducibility of 50-fold was obtained when a synthetic alpha-giardin promoter was used. Fine tuning of luciferase expression was achieved by adjusting the concentration of tetracycline and duration of drug exposure. The inducible gene expression system provides us an easy way to manipulate the level of gene expression in G. lamblia in a controllable manner that could not previously be achieved.     

Tyroserleutide-based gene vector for suppressing VEGF expression in cancer therapy

A small interfering RNA (siRNA) plasmid DNA (pYr-1.1-hU6-EGFP-siVEGF) was constructed and used for suppressing vascular endothelial growth factor (VEGF) expression and inhibiting tumor growth. Then, a (tyrosyl-seryl-leucine)-polyethyleneimine-poly(ethylene glycol) (YSL-PEI-PEG) conjugate was designed and synthesized as a gene carrier for the delivery of pYr-1.1-hU6-EGFP-siVEGF plasmid. The therapeutic peptide YSL was conjugated to PEI to improve the anti-cancer efficiency, and the PEG chain was introduced to reduce the serum protein adsorption and improve the stability of the complex in the systemic circulation. It was found that YSL-PEI-PEG could efficiently condense plasmid DNA when the vector/DNA weight ratio was higher than 2. Compared with PEI 25?kDa, YSL-PEI-PEG exhibited higher transfection efficiency and lower cytotoxicity. More importantly, the results showed that the gene delivery system owned strong ability to inhibit cancer cell proliferation in?vitro and tumor growth in?vivo. YSL-PEI-PEG has great potential as a gene vector for clinical applications.     

FGF receptor-mediated gene delivery using ligands coupled to polyethylenimine

To obtain new nonviral vectors with high gene delivery efficiency and special cell targeting ability, an attractive strategy is to link ligands to polyethylenimine (PEI). Fibroblast growth factor receptors (FGFRs) are highly expressed on a variety of human cancer cells and are potential targets for cancer gene therapy. In this study, the peptides NH2-Met-Gln-Leu-Pro-Leu-Ala-ThrGly-Gly-Gly-Cys-COOH (MC11) which have been proved to combine specially with the FGFR on cell membrane are coupled to PEI using N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) as a linker with different molar ratios (1 : 0.3, 1 : 0.75, 1 : 1.5, and 1 : 3.0) and the new polymer PEI-MC11 is verified by a series of physicochemical methods including 1H-NMR and FTIR. The agarose gel electrophoresis assay, particle size test, zeta potential test, and electron microscope observation show that PEI-MC11 can efficiently condense plasmid DNA into nanoparticles with about 200 nm in diameter and with positive surface charge at the suitable N/P ratio. The MTT assay suggests the decreased toxicity of the polymers. The results of the gene delivery efficiency in vitro show that PEI-MC11/pDNA polyplexes have significantly greater transgene activity than PEI/pDNA in COS-7 and HepG2 cells which express FGFR positively, while no such effect is observed in PC3 cells which have negative FGFR. The enhanced gene delivery efficiency of PEI-MC11 can be blocked by the co-culture of free peptides MC11 before the gene delivery procedure. The synthesized nonviral vector based on PEI with the targeting peptides MC11 for binding FGFR has improved efficiency of gene delivery and targeting specificity in FGFR positive cells. It may have potential application in cancer gene therapy.     

Factors that influence transgene expression and cell viability on DNA-PEI-seeded collagen films

Gene delivery from tissue-engineering devices has the potential to improve healing, but better regulation of the level and duration of gene expression is needed. We hypothesized that transgene expression could be controlled by varying the fabrication and soaking parameters used in making collagen- based gene delivery scaffolds. Collagen films were made from acid-insoluble type I bovine dermal collagen and seeded with plasmid DNA encoding firefly luciferase, complexed with polyethylenimine. By varying the thickness of the films, the volume of the DNA soak solution, and the pH of the DNA soak solution, and by cross-linking the films, we identified variable combinations that produce significantly different levels of cell number and transgene expression in L-929 cells in vitro. Increasing film thickness or soak volume increased overall reporter gene expression. Decreasing film thickness or soak volume decreased cell number but did not significantly change reporter gene expression per cell. Cross-linking by ultraviolet irradiation (before adding the DNA) significantly decreased transgene expression, probably because of decreased swelling of the collagen film. These results suggest that collagen-based biomaterials may be designed and fabricated to induce, in a controlled fashion, various levels of cellularity and transgene expression.     

The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded.

A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum. A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E. coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E. coli. All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes. Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster. Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid. Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains. A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII. Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E. coli.     

The assessment of local tolerance,acute toxicity,and DNA biodistribution following particle-mediated delivery of a DNA vaccine to minipigs

Particle-mediated DNA delivery was used to administer a DNA vaccine against Hepatitis B to minipigs. The study represented one arm of the safety evaluation program for this product and was designed to assess local tolerance, acute toxicity, and biodistribution of the DNA plasmid. The vaccine was given to 4 groups of minipigs that were sacrificed at 2, 28, 56, or 141 days after treatment. The procedure was well tolerated with mild local skin reactions at 2 days postdosing and no evidence of systemic toxicity. By 28 days the skin lesions had regressed apart from a low grade perivascular mononuclear cell infiltrate in the upper dermis, together with a small number of phagocytosed gold particles. This infiltrate persisted up to 141 days. The expressed HBsAg was detected by immunohistochemistry in keratinocytes (usually in association with an intranuclear gold particle) at 2 days but not at later time points. Polymerase chain reaction (PCR) was used to assay treatment sites and selected internal organs to evaluate biodistribution and persistence of the DNA plasmid. At 2 days the plasmid was detected in the treatment sites and also in the inguinal lymph nodes. At day 57 it was present in the treatment sites only and by day 141 appeared to have cleared. The results from this study demonstrate that particle-mediated gene delivery was well tolerated in the minipig. The biodistribution and persistence of the plasmid was within acceptable limits for this type of vaccine. As the minipig is regarded as a good model for humans these data support the concept that particle-mediated DNA delivery will be safe in human clinical applications.     

Long-term efficient gene delivery using polyethylenimine with modified Tat peptide

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (≈5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.     

Co-electrospun fibrous scaffold-adsorbed DNA for substrate-mediated gene delivery

Incorporation of gene into electrospun nanofibers for localized gene transfection of target cells represents a robust platform for tissue regeneration. In this study, a new two-step approach was explored to immobilize DNA onto electrospun nanofibers for effective gene delivery, that is, nonviral gene vector of polyethylene glycol (PEG)-modified polyethylenimine (PEI) was incorporated into scaffolds by electrospinning and then target DNA was adsorbed onto the electrospun nanofibers via electrostatic interaction between DNA and PEI-PEG. PEI-PEG/DNA particles formed from the released DNA, and PEI-PEG had a uniform particle size of approximately 200 nm. This nanofiber-based gene delivery system exhibited high transfection efficiency, in which >65% of human embryonic kidney 293 cells and >40% of mesenchymal stem cells were transfected with green fluorescent protein gene. Compared with PEI, PEG modification of PEI had improved the biocompatibility and further increased the transfection efficiency. These results suggest that the combination of nonviral gene carrier with electrospun nanofibers could be used for localized gene delivery, which has multifold potential applications in tissue engineering or as an in vivo substrate for tissue regeneration.