Polyplex Delivery Research Articles

Gold nanoparticles electroporation enhanced polyplex delivery to mammalian cells.

Nonviral methods have been explored as the replacement of viral systems for their low toxicity and immunogenicity. However, they have yet to reach levels competitive to their viral counterparts. In this paper, we combined physical and chemical methods to improve the performance of polyplex delivery of DNA and small interfering RNA. Specifically, gold nanoparticles (AuNPs) were used to carry polyplex (a chemical approach) while electroporation (a physical approach) was applied for fast and direct cytosolic delivery. In this hybrid approach, cationic polymer molecules condense and/or protect genetic probes as usual while AuNPs help fix polycations to reduce their cytotoxicity and promote the transfection efficiency of electroporation. AuNPs of various sizes were first coated with polyethylenimine, which were further conjugated with DNA plasmids or small interfering RNA molecules to form AuNPs-polyplex. The hybrid nanoparticles were then mixed with cells and introduced into cell cytosol by electroporation. The delivery efficiency was evaluated with both model anchor cells (i.e., NIH/3T3) and suspension cells (i.e., K562), together with their impact on cell viability. We found that AuNP-polyplex showed 1.5∼2 folds improvement on the transfection efficiency with no significant increase of toxicity when compared to free plasmid delivery by electroporation alone. Such a combination of physical and chemical delivery concept may stimulate further exploration in the delivery of various therapeutic materials for both in vitro and in vivo applications.

Abstract 520: Peptide and SiRNA Modulation of HSP20 Influences Vasoreactivity

Subarachnoid hemorrhages affect 30,000 people each year and accounts for between 1-7% of all strokes. Nearly 85% of the time that the hemorrhage occurs, it is due to a cerebral aneurysm that leads to delayed vasospasm and stroke. Traditional vasodilators cannot be used because they will cause systemic hypotension leaving an unmet clinical need. HSPB1 is a small heat shock related protein that modulates vasorelaxation, is downregulated in vasospastic vessels, and represents a potential therapeutic modality for the treatment of SAH-induced vasospasm. siRNA against HSPB1 that was encapsulated with a diblock copolymer to enhance uptake, or Rat aortic smooth muscle was tested with a HSPB1 phospho-peptide (HSPB1-p) +/- a polyplex delivery system (polyplexHSPB1). Intracellular calcium was measured concurrently with force using a Fluoroplex sysem. Knockdown of HSPB1 inhibited sodium nitroprusside (SNP)-induced relaxation of rat aorta. In conclusion, HSP20 modulates calcium independent relaxation of vascular smooth muscle and HSPB1-p may represent a novel therapeutic for vasospasm. Tissue treated with HSPB1-p or polyplex HSPB1-p had a dose dependent inhibition of contraction. The polyplex inhibited contraction at a lower dose of HSPB1-p than peptide alone with complete inhibition of contraction at 500 μM). Complete inhibition of contraction occurred without inhibition of increases in intracellular calcium concentrations ([Ca 2+ ] i ).

Polyplex-releasing microneedles for enhanced cutaneous delivery of DNA vaccine

Microneedle (MN)-based DNA vaccines have many advantages over conventional vaccines administered by hypodermic needles. However, an efficient strategy for delivering DNA vaccines to intradermal cells has not yet been established. Here, we report a new approach for delivering polyplex-based DNA vaccines using MN arrays coated with a pH-responsive polyelectrolyte multilayer assembly (PMA). This approach enabled rapid release of polyplex upon application to the skin. In addition to the polyplex-releasing MNs, we attempted to further maximize the vaccination by developing a polymeric carrier that targeted resident antigen presenting cells (APCs) rich in the intradermal area, as well as a DNA vaccine encoding a secretable fusion protein containing amyloid beta monomer (Aβ1–42), an antigenic determinant. The resulting vaccination system was able to successfully induce a robust humoral immune response compared to conventional subcutaneous injection with hypodermal needles. In addition, antigen challenge after immunization elicited an immediate and strong recall immune response due to immunogenic memory. These results suggest the potential utility of MN-based polyplex delivery systems for enhanced DNA vaccination.

A Molecular Dynamics Simulation Study on the Effect of Endogenous Molecules on SiRNA Polyplexes

Small interfering RNA (siRNA) based RNA interference (RNAi) is an important therapeutic strategy in treatment of cancer and hereditary diseases. RNAi is composed of several important stages, the first being siRNA delivery into cells. Polymeric carriers are used in delivery to aggregate siRNA molecules into nano-scale polyplexes. Upon delivery of polyplexes into cytosol, siRNA has to be released to trigger RNAi. However, mechanism of siRNA release from polyplexes is not clear; how endogenous molecules may affect the stability of polyplexes in cytosol is yet to be explored. In this study, we performed a series of molecular dynamics (MD) simulations to study the behavior of siRNA polyplexes in the presence of microRNA (miRNA), the most abundant nucleic acid inside cells. Polyethylenimine (PEI), known as a ‘gold-standard’ among polymeric delivery vehicles, was used as the carrier. miRNAs were introduced into pre-formed siRNA-PEI polyplexes. Quantitative analysis of the MD trajectories was carried out to characterize the change in the structure, compactness and charge distribution of the polyplex upon miRNA addition. PEI affinity toward siRNA and miRNA was determined and compared through individual simulations on siRNA-PEI and miRNA-PEI complexation. Data obtained from the MD simulations was compared with gel electrophoresis assays of siRNA-PEI-miRNA mixtures as well as siRNA-PEI-heparin mixtures; the latter served as a control due to the known capability of heparin to cause siRNA release. Our results revealed the ability of miRNAs to bind to pre-formed siRNA polyplexes through electrostatic interactions with PEI nitrogens. Therefore, upon the addition of miRNA, the size of the polyplex increased and its charge became negative. In agreement with the simulations, in gel electrophoresis, at the same concentrations, heparin was able to release siRNA while polyplexes maintained their integrity in the presence of miRNA.

Mucosal application of gp140 encoding DNA polyplexes to different tissues results in altered immunological outcomes in mice.

Increasing evidence suggests that mucosally targeted vaccines will enhance local humoral and cellular responses whilst still eliciting systemic immunity. We therefore investigated the capacity of nasal, sublingual or vaginal delivery of DNA-PEI polyplexes to prime immune responses prior to mucosal protein boost vaccination. Using a plasmid expressing the model antigen HIV CN54gp140 we show that each of these mucosal surfaces were permissive for DNA priming and production of antigen-specific antibody responses. The elicitation of systemic immune responses using nasally delivered polyplexed DNA followed by recombinant protein boost vaccination was equivalent to a systemic prime-boost regimen, but the mucosally applied modality had the advantage in that significant levels of antigen-specific IgA were detected in vaginal mucosal secretions. Moreover, mucosal vaccination elicited both local and systemic antigen-specific IgG+ and IgA+ antibody secreting cells. Finally, using an Influenza challenge model we found that a nasal or sublingual, but not vaginal, DNA prime/protein boost regimen protected against infectious challenge. These data demonstrate that mucosally applied plasmid DNA complexed to PEI followed by a mucosal protein boost generates sufficient antigen-specific humoral antibody production to protect from mucosal viral challenge.

A Protective Extracellular Matrix-Based Gene Delivery Reservoir Fabricated by Electrostatic Charge Manipulation

Gene therapy is a field that offers hope and promise for the treatment of diseases and traumas of a genetic nature and otherwise. However, progress toward the clinic has been delayed because of concerns over the safety of viral vectors and the efficacy of safer nonviral systems, which have low transfection efficacy and short transgene expression. This study describes the fabrication and characterization of a safe gene delivery reservoir system that has the potential to overcome issues associated with nonviral systems. Harnessing the electrostatic charges of collagen and polystyrene, microspheres were fabricated using a template-based method and characterized by microscopy techniques such as scanning electron microscopy, transmission electron microscopy, and atomic force microscopy, with the removal of the polystyrene template confirmed by Fourier transform infrared spectroscopy analysis. Loading and release of polyplexes confirmed the ability of the system to prolong polyplex delivery, with minimal cytotoxicity observed from viability studies on 3T3 fibroblasts. Finally, biological activity of released polyplexes was confirmed by reporter gene expression. Taken together, these properties indicate the potential of this system as a reservoir for gene delivery.

Preparation of Cationic Nanogels for Nucleic Acid Delivery

Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.

Intra-articular controlled release of anti-inflammatory siRNA with biodegradable polymer microparticles ameliorates temporomandibular joint inflammation

We investigated the in vivo therapeutic efficacy of an intra-articular controlled release system consisting of biodegradable poly(dl-lactic-co-glycolic acid) (PLGA) microparticles (MPs) encapsulating anti-inflammatory small interfering RNA (siRNA), together with branched poly(ethylenimine) (PEI) as a transfecting agent, in a rat model of painful temporomandibular joint (TMJ) inflammation. The in vivo effects of PLGA MP dose and siRNA–PEI polyplex delivery were examined via non-invasive meal pattern analysis and by quantifying the protein level of the siRNA target as well as of several downstream inflammatory cytokines. Controlled release of siRNA–PEI from PLGA MPs significantly reduced inflammation-induced changes in meal patterns compared to untreated rats with inflamed TMJs. These changes correlated to decreases in tissue-level protein expression of the siRNA target to 20–50% of the amount present in the corresponding control groups. Similar reductions were also observed in the expression of downstream inflammatory cytokines, e.g. interleukin-6, whose tissue levels in the siRNA–PEI PLGA MP groups were 50% of the values for the corresponding controls. This intra-articular sustained release system has significant implications for the treatment of severe TMJ pain, and also has the potential to be readily adapted and applied to mitigate painful, chronic inflammation in a variety of conditions.

Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.

Localized Cell Uptake of His-Tagged Polyplexes Immobilized on NTA Self-Assembled Monolayers

A new method to study substrate-mediated gene delivery was developed using a hexahistidine-tagged polymeric gene delivery vehicle (His-tagged polyplex) and nickel nitrilotriacetic acid (Ni-NTA) self-assembled monolayers (SAMs) on gold surfaces. The His-tagged polyplexes showed specific interaction with Ni-NTA surfaces compared to control surfaces, with increasing NTA content in SAM formulations corresponding to increasing amounts of immobilized His-tagged polypexes on the surface. Cells seeded on NTA SAMs demonstrated uptake of His-tagged polyplexes in the presence of imidazole and EDTA with low cytotoxicity. Cells seeded on NTA SAMs without imidazole and EDTA showed minimal a amount of His-tagged polyplex uptake. This showed that the release of polyplexes from the surface by imidazole and EDTA was necessary for cell uptake. Thus, this system provides potential spatial specificity for polyplex delivery controlled by location of NTA surfaces and temporal specificity for polyplex delivery controlled by the addition of imidazole and EDTA.

Chitosan/siRNA Nanoparticle–mediated TNF-α Knockdown in Peritoneal Macrophages for Anti-inflammatory Treatment in a Murine Arthritis Model

Secretion of tumor necrosis factor-alpha (TNF-alpha) by macrophages plays a predominant role in the development and progression of rheumatoid arthritis. We demonstrate that knockdown of TNF-alpha expression in systemic macrophages by intraperitoneal (i.p.) administration of chitosan/small interfering RNA (siRNA) nanoparticles in mice downregulates systemic and local inflammation. Chitosan nanoparticles containing an unmodified anti-TNF-alpha Dicer-substrate siRNA (DsiRNA) mediated TNF-alpha knockdown (approximately 66%) in primary peritoneal macrophages in vitro. The presence of Cy3-labeled nanoparticles within peritoneal macrophages and specific TNF-alpha knockdown (approximately 44%) with TNF-alpha siRNA after i.p. injection supports our therapeutic approach. Downregulation of TNF-alpha-induced inflammatory responses arrested joint swelling in collagen-induced arthritic (CIA) mice dosed i.p. with anti-TNF-alpha DsiRNA nanoparticles. The use of 2'-O-Me-modified DsiRNA resulted in the lowest arthritic scores and correlated with reduced type I interferon (IFN) activation in macrophages in vivo compared with unmodified DsiRNA. Histological analysis of joints revealed minimal cartilage destruction and inflammatory cell infiltration in anti-TNF-alpha-treated mice. The onset of arthritis could be delayed using a prophylactic dosing regime. This work demonstrates nanoparticle-mediated TNF-alpha knockdown in peritoneal macrophages as a method to reduce both local and systemic inflammation, thereby presenting a novel strategy for arthritis treatment.

VEGF siRNA Delivery System Using Arginine-Grafted Bioreducible Poly(disulfide amine)

Small interfering RNAs (siRNAs) are able to silence their target genes when they are successfully delivered intact into the cytoplasm. Delivery systems that enhance siRNA localization to the cytoplasm can facilitate gene silencing by siRNA therapeutics. We describe an arginine-conjugated poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH-R)), a bioreducible cationic polymer, as an siRNA carrier for therapeutic gene silencing for cancer. After intracellular uptake of the siRNA/poly(CBA-DAH-R) polyplexes, the reductive environment of the cytoplasm cleaves the disulfide linkages in the polymeric backbone, resulting in decomplexing of the siRNA/poly(CBA-DAH-R) polyplexes and release of siRNA molecules throughout the cytoplasm. The siRNA/poly(CBA-DAH-R) polyplexes, which demonstrate increased membrane permeability with arginine modification, have a similar level of cellular uptake as siRNA/bPEI polyplexes. The VEGF siRNA/poly(CBA-DAH-R) polyplexes, however, inhibit VEGF expression to a greater degree than VEGF siRNA/bPEI in various human cancer cell lines. The improved RNAi activity demonstrated by the VEGF siRNA/poly(CBA-DAH-R) polyplexes is due to enhanced intracellular delivery and effective localization to the cytoplasm of the VEGF siRNAs. These results demonstrate that the VEGF siRNA/poly(CBA-DAH-R) polyplex delivery system may useful for siRNA-based approaches for cancer therapy.

Cardiomyocyte-targeted siRNA delivery by prostaglandin E 2-Fas siRNA polyplexes formulated with reducible poly(amido amine) for preventing cardiomyocyte apoptosis

A cardiomyocyte-targeted Fas siRNA delivery system was developed using prostaglandin E 2 (PGE 2)-modified siRNA polyplexes formed by a reducible poly(amido amine) to inhibit cardiomyocyte apoptosis. PGE 2, which was used as a specific ligand for cardiomyocyte targeting, was conjugated to the terminal-end of the sense siRNA (PGE 2-siRNA). The reducible cationic copolymer, synthesized via Michael-type polyaddition of 1,6-diaminohexane and cystamine bis-acrylamide (poly(DAH/CBA)), tightly condensed the PGE 2-siRNA conjugate to form nanosize polyplexes having a diameter of 100–150 nm. The PGE 2-siRNA/poly(DAH/CBA) polyplexes decomplexed to release PGE 2-siRNA in a cytosolic reducing environment due to the degradation of the reducible poly(DAH/CBA). The cellular uptake of the PGE 2-siRNA/poly(DAH/CBA) polyplex was increased in rat cardiomyocytes (H9C2 cells) due to PGE 2 receptor-mediated endocytosis. When H9C2 cells were transfected with siRNA against Fas, a key regulator of ischemia-induced apoptosis, the PGE 2–Fas siRNA/poly(DAH/CBA) polyplex delivery system led to a significant increase in Fas gene silencing, resulting in inhibition of cardiomyocyte apoptosis. The PGE 2–Fas siRNA/poly(DAH/CBA) polyplex did not induce interferon-alpha in peripheral blood mononuclear cells. These results suggest that the PGE 2–Fas siRNA/poly(DAH/CBA) polyplex formulation may be clinically applicable as a cardiomyocyte-targeted Fas siRNA delivery system to inhibit apoptosis in cardiovascular disease.

Colloidally stable novel copolymeric system for gene delivery in complete growth media

Novel cationic pentablock copolymers based on poly(2-diethylaminoethylmethacrylate) (PDEAEM) and Pluronic F127 were evaluated as non-viral gene delivery vectors from a physiochemical point of view for stability and transfection efficiency in complete growth media. A novel strategy was introduced to sterically stabilize the polyplexes of such Pluronic-based cationic polymers against aggregation with serum proteins. As cationic pentablock copolymers condense plasmid DNA into nanoplexes of 100–150 nm diameter, unmodified Pluronic added to the formulation self-assemble with the pentablock copolymers on the surface of polyplexes and shield the cationic PDEAEM chains of pentablock copolymers sterically with its long poly(ethyleneoxide) chains. These coated polyplexes formed colloidally stable dispersions of 150–250 nm diameter in serum-supplemented buffers. Cryo-TEM micrographs also showed that coating polyplexes with unmodified Pluronic reduced aggregation in serum proteins. Pentablock copolymers preserved the integrity of plasmid DNA condensed inside the polyplexes and provided efficient resistance to its degradation by nucleases. Though the total amount of DNA retained by ExGen 500® polyplexes after nuclease digestion was more than that retained by pentablock copolymers, the amount of plasmid retained in supercoiled form was not significantly different. Polyplexes coated with unmodified Pluronic provided efficient transfection in SKOV3 cells in complete growth media, comparable to that provided by ExGen 500® in terms of number of cells transfected, and one order less in terms of total transgene protein expressed. These sterically shielded polyplexes also exhibited much lower cytotoxicities than uncoated polyplexes of pentablock copolymers, and significantly lower than the cytotoxicity of ExGen 500® at relevant concentrations. This colloidally stable, versatile, multi-component gene delivery system also forms thermo-reversible injectable hydrogels like Pluronics at physiological temperatures that can be used for sustained delivery of polyplexes, and is promising for systemic applications.

172. Spatially-Controlled Delivery of Cyclodextrin-Based Polyplexes from Solid Surfaces

Localized, nucleic acid delivery from solid surfaces is an important technology for biomedical research, with applications ranging from gene therapy to tissue engineering to functional genomics. The spatially-controlled delivery of growth factor is required for creating a well-organized, complex cellular architecture by tissue engineering. Non-viral gene delivery vehicles have been immobilized to solid surfaces by physical adsorption, biotin-streptavidin interaction, and chemical conjugation1. Here, we present a new approach for surface-mediated gene delivery based on inclusion complex formation between the solid surface and delivery vehicles. Through this method, the delivery of transgenes can be spatially controlled by gene delivery through specific interactions between the delivery vehicles and the surface. Cyclodextrins (CD), cyclic oligomers of glucose, form inclusion complexes with small, hydrophobic compounds such as adamantane. In this work, nanoparticle composites of cyclodextrin polycations and nucleic acids were specifically immobilized on adamantane-coated surfaces. The interaction was characterized by surface plasmon resonance (SPR) spectroscopy and by atomic force microscopy (AFM).

Poly(glycoamidoamine)s for Gene Delivery: Stability of Polyplexes and Efficacy with Cardiomyoblast Cells

Polymeric vectors have potential as nucleic acid delivery vehicles for novel gene therapy and oligonucleotide treatments for cardiovascular disease. In this report, poly(glycoamidoamine)s that contain four secondary amines and either two or four hydroxyl units in the repeat unit with D-glucarate (D4), meso-galactarate (G4), D-mannarate (M4), and l-tartarate (T4) stereochemistry have been investigated for their pDNA-binding affinity, DNase protection effect, and polyplex stability in the presence of salt and serum. Also, the luciferase gene delivery and cellular internalization of polyplexes formed with these polymers have been investigated with rat cardiomyoblast [H9c2(2-1)] cells. The results demonstrate that the number of hydroxyl groups and the stereochemistry affect the biological properties. Polymers T4 and G4 have higher pDNA binding affinity, protect pDNA from nuclease degradation, and do not release pDNA in the presence of serum. Polymers D4 and M4 bind pDNA with lower affinity, which allows for some pDNA degradation and release in the presence of serum. Although T4 forms the most stable polyplexes, vector G4 reveals the highest luciferase gene expression in serum-free media and the greatest cellular internalization of fluorescein-labeled pDNA both in serum-free and serum-supplemented media. The results of these studies indicate that the polymer-DNA binding affinity, nuclease protection capability, and polyplex stability are important parameters to facilitate effective pDNA delivery with poly(glycoamidoamine)s in cultured cardiomyoblast cells. The carbohydrate type also plays an important role to increase cellular uptake and gene expression where the polymer with the galactarate stereochemistry (in G4) is found to be the most effective vector for pDNA delivery to cardiomyoblast cells in vitro.

583. The Transgene Expression in the Lung after Systemic Injection of DNA Formulated into Nanoparticles with Linear PEI Depends of Bolus Effect and the Presence of Free PEI

Top of pageAbstract Linear polyethylenimine (L-PEI) is the most used non-viral delivery reagent for gene delivery in animals. Various delivery routes of small size defined PEI polyplexes formulated in a 5% glucose solution can be used (intravenous, intracerebral, intraperitoneal, intracardiac, instillation |[hellip]|). Intravenous injection is the route of choice in many gene therapy applications. Following systemic delivery of PEI polyplexes, a non-specific targeting of lungs occurs where c.a. 90% of transgene expression are found. We have addressed the bolus effect caused with the injection volume of polyplexes solution using jetPEI|[trade]| after the rapid injection (few seconds) into the tail vein of mice. We have shown that injection volume less than 200|[mu]|l leads to lower transgene expression in lungs than volumes comprised between 200 and 400|[mu]|l. When the injection volume of polyplexes is increased to 2-2.5 ml and the injection performed by perfusion, the transgene level is slightly affected in lungs. The optimal effect of bolus is obtained following a rapid injection (c.a. 10 seconds) of 400|[mu]|l of a 5% glucose solution. It is generally demonstrated that highly positive polyplexes (N/P ratio >4) are efficient in the lungs. We have purified polyplexes formulated with various N/P ratios by ultracentrifugation. We have shown that as expected, the amount of free PEI removed increased with the initial N/P ratio used to prepare polyplexes and reached more than 50% at N/P ratio of 10. When the optimal injection conditions were applied with polyplexes made initially with a N/P ratio of 10 and then purified, a 10-fold decrease in gene expression in the lung was observed. Increasing the dose of DNA injected using purified polyplexes increased the transgene level comparable to the optimal conditions obtained with crude complexes. It appears that the presence of free polymer in the optimal crude complexes has a co-helper effect. The role of this free polymer to reach a high transgene level in vivo is not yet identified and needs further investigations.

Formulation of a microparticle carrier for oral polyplex-based DNA vaccines.

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.

Stabilization of poly- l-lysine/DNA polyplexes for in vivo gene delivery to the liver

We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly- l-lysine and plasmid DNA. We have characterized poly- l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly- l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly- l-lysine/DNA polyplexes appeared as toroids 25–50 nm in diameter or rods 40–80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50–90 nm. In water, poly- l-lysine and asialoorosomucoid–PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly- l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly- l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components.