Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles

The stratum corneum (SC) represents a significant barrier to the delivery of gene therapy formulations. In order to realise the potential of therapeutic cutaneous gene transfer, delivery strategies are required to overcome this exclusion effect. This study investigates the ability of microfabricated silicon microneedle arrays to create micron-sized channels through the SC of ex vivo human skin and the resulting ability of the conduits to facilitate localised delivery of charged macromolecules and plasmid DNA (pDNA). Microscopic studies of microneedle-treated human epidermal membrane revealed the presence of microconduits (10-20 microm diameter). The delivery of a macromolecule, beta-galactosidase, and of a 'non-viral gene vector mimicking' charged fluorescent nanoparticle to the viable epidermis of microneedle-treated tissue was demonstrated using light and fluorescent microscopy. Track etched permeation profiles, generated using 'Franz-type' diffusion cell methodology and a model synthetic membrane showed that >50% of a colloidal particle suspension permeated through membrane pores in approximately 2 hours. On the basis of these results, it is probable that microneedle treatment of the skin surface would facilitate the cutaneous delivery of lipid:polycation:pDNA (LPD) gene vectors, and other related vectors, to the viable epidermis. Preliminary gene expression studies confirmed that naked pDNA can be expressed in excised human skin following microneedle disruption of the SC barrier. The presence of a limited number of microchannels, positive for gene expression, indicates that further studies to optimise the microneedle device morphology, its method of application and the pDNA formulation are warranted to facilitate more reproducible cutaneous gene delivery.     

Cutaneous DNA delivery and gene expression in ex vivo human skin explants via wet-etch micro-fabricated micro-needles

Micro-needle arrays increase skin permeability by forming channels through the outer physical barrier, without stimulating pain receptors populating the underlying dermis. It was postulated that micro-needle arrays could facilitate transfer of DNA to human skin epidermis for cutaneous gene therapy applications. Platinum-coated "wet-etch" silicon micro-needles were shown to be of appropriate dimensions to create micro-conduits, approximately 50 microm in diameter, extending through the stratum corneum (SC) and viable epidermis. Following optimisation of skin explant culturing techniques and confirmation of tissue viability, the ability of the micro-needles to mediate gene expression was demonstrated using the beta-galactosidase reporter gene. Preliminary studies confirmed localised delivery, cellular internalisation and subsequent gene expression of pDNA following micro-needle disruption of skin. A combination of this innovative gene delivery platform and the ex vivo skin culture model will be further exploited to optimise cutaneous DNA delivery and address fundamental questions regarding gene expression in skin.     

Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation

The skin is a valuable organ for the development and exploitation of gene medicines. Delivering genes to skin is restricted however by the physico-chemical properties of DNA and the stratum corneum (SC) barrier. In this study, we demonstrate the utility of an innovative technology that creates transient microconduits in human skin, allowing DNA delivery and resultant gene expression within the epidermis and dermis layers. The radio frequency (RF)-generated microchannels were of sufficient morphology and depth to permit the epidermal delivery of 100 nm diameter nanoparticles. Model fluorescent nanoparticles were used to confirm the capacity of the channels for augmenting diffusion of macromolecules through the SC. An ex vivo human organ culture model was used to establish the gene expression efficiency of a beta-galactosidase reporter plasmid DNA applied to ViaDerm treated skin. Skin treated with ViaDerm using 50 microm electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDerm was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDerm) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.     

Characterization of Solid Maltose Microneedles and their Use for Transdermal Delivery

Purpose To characterize solid maltose microneedles and assess their ability to increase transdermal drug delivery. Materials and Methods Microneedles and microchannels were characterized using methylene blue staining and scanning electron microscopy. Diffusion pattern of calcein was observed using confocal scanning laser microscopy. Transepidermal water loss (TEWL) measurements were made to study the skin barrier recovery after treatment. Uniformity in calcein uptake by the pores was characterized and percutaneous penetration of nicardipine hydrochloride (NH) was studied in vitro and in vivo across hairless rat skin. Results Microneedles were measured to be 508.46 ± 9.32 μm long with a radius of curvature of 3 μm at the tip. They penetrated the skin while creating microchannels measuring about 55.42 ± 8.66 μm in diameter. Microchannels were visualized by methylene blue staining. Pretreatment with microneedles resulted in the migration of calcein into the microchannels. TEWL increased after pretreatment and uptake of calcein by the pores was uniform as measured by the pore permeability index values. NH in vitro transport across skin increased significantly after pretreatment (flux 7.05 μg/cm²/h) as compared to the untreated skin (flux 1.72 μg/cm²/h) and the enhanced delivery was also demonstrated in vivo in hairless rats. Conclusion Maltose microneedles were characterized and shown to create microchannels in the skin, which were also characterized and shown to improve the transdermal delivery of NH.     

Microneedles: an emerging transdermal drug delivery system

Objectives One of the thrust areas in drug delivery research is transdermal drug delivery systems (TDDS) due to their characteristic advantages over oral and parenteral drug delivery systems. Researchers have focused their attention on the use of microneedles to overcome the barrier of the stratum corneum. Microneedles deliver the drug into the epidermis without disruption of nerve endings. Recent advances in the development of microneedles are discussed in this review for the benefit of young scientists and to promote research in the area. Key findings Microneedles are fabricated using a microelectromechanical system employing silicon, metals, polymers or polysaccharides. Solid coated microneedles can be used to pierce the superficial skin layer followed by delivery of the drug. Advances in microneedle research led to development of dissolvable/degradable and hollow microneedles to deliver drugs at a higher dose and to engineer drug release. Iontophoresis, sonophoresis and electrophoresis can be used to modify drug delivery when used in concern with hollow microneedles. Microneedles can be used to deliver macromolecules such as insulin, growth hormones, immunobiologicals, proteins and peptides. Microneedles containing ‘cosmeceuticals’ are currently available to treat acne, pigmentation, scars and wrinkles, as well as for skin tone improvement. Summary Literature survey and patents filled revealed that microneedle‐based drug delivery system can be explored as a potential tool for the delivery of a variety of macromolecules that are not effectively delivered by conventional transdermal techniques.     

Gene expression in an intact ex-vivo skin tissue model following percutaneous delivery of cationic liposome-plasmid DNA complexes

The skin represents an attractive site for the localised gene therapy of dermatological pathologies and as a potential antigen bioreactor following transdermal delivery. Potential also exists for the gene therapy of skin as a cosmetic intervention. The most exploited non-viral gene delivery system involves the complexation of cationic liposomes with plasmid DNA (pDNA) to form lipid:pDNA vectors that protect the DNA from nuclease-mediated degradation and improve transgene-cell interactions. Despite numerous studies examining the potential for these vectors in delivering genes to a variety of keratinocyte models, investigations into the topical application of such complexes to intact skin tissue is limited. This ex-vivo study, conducted with intact skin tissue derived from hairless mice, provides quantitative confirmation that topical administration of cationic lipid:pDNA complexes can mediate uptake and expression of reporter pDNA (33-fold higher compared with control) in viable epidermal tissue. The ex-vivo study design provides for intact skin tissue that has not been subjected to depilatory procedures of potential detriment to stratum corneum barrier function, and can be utilised for the quantitative and efficient examination of a potentially wide range of non-viral gene vectors designed for epidermal expression.     

Design of Biodegradable Hydrogel for the Local and Sustained Delivery of Angiogenic Plasmid DNA

Purpose To attain the effective local and sustained delivery of plasmid DNA (pDNA) encoding for a growth factor. Methods We hypothesized that controlling the degradation rate of biomaterials encapsulating pDNA via concurrent physical dissociation of the cross-linked structure and hydrolytic chain breakage of polymers would allow one to significantly broaden the range of pDNA release rate. This hypothesis was examined using ionically cross-linked polysaccharide hydrogels which were previously designed to rapidly degrade via engineering of ionic cross-linking junction and partial oxidation of polysaccharide chains. Results The hydrogel degradation rates were varied over the broad range, and pDNA release correlated with the gel degradation rate. Degradable hydrogels were used for the local and sustained delivery of a pDNA encoding for vascular endothelial growth factor (VEGF) in the ischemic hindlimbs of mice, and local pDNA release significantly improved the recovery of blood perfusion as compared with a bolus injection of VEGFencoding pDNA. Conclusion This strategy to control the hydrogel degradation rate may be useful in regulating the delivery of a broad array of macromolecular drugs, and subsequently improve their therapeutic efficacy.     

Challenges and strategies in developing microneedle patches for transdermal delivery of protein and peptide therapeutics

The birth of microneedles, an array of needles sufficiently long to penetrate epidermis but small enough to do not cause skin injury and pain feeling, has offered a highly promising solution for non-invasive delivery of protein and peptide drugs, a long-cherished desire over eighty years. However, the attempts to develop clinically feasible microneedle transdermal delivery methods encountered series of difficulties, for which a decade research efforts have yet to result in a single product. Microneedles may be incorporated into devices as skin pre-treatment tools, skin microinjectors as well as transdermal patches by their functions in drug delivery. They may also be categorized to insoluble solid microneedles, hollow microneedles, soluble/degradable solid microneedles and phase-transition microneedles by their structure and forming materials. This review article is aimed to update the progress and discuss the technical challenges raised in developing protein/peptide loaded microneedle patches.     

Nanoparticle Coated Submicron Emulsions: Sustained In-vitro Release and Improved Dermal Delivery of All-trans-retinol

Purpose  The aim of this research is to investigate the dermal delivery of all-trans-retinol from nanoparticle-coated submicron oil-in-water emulsions as a function of the initial emulsifier type, the loading phase of nanoparticles, and the interfacial structure of nanoparticle layers. Methods  The interfacial structure of emulsions was characterized using freeze-fracture-SEM. In-vitro release and skin penetration of all-trans-retinol were studied using Franz diffusion cells with cellulose acetate membrane, and excised porcine skin. The distribution profile was obtained by horizontal sectioning of the skin using microtome-cryostat and HPLC assay. Results  The steady-state flux of all-trans-retinol from silica-coated lecithin emulsions was decreased (up to 90%) and was highly dependent on the initial loading phase of nanoparticles; incorporation from the aqueous phase provided more pronounced sustained release. For oleylamine emulsions, sustained release effect was not affected by initial location of nanoparticles. The skin retention significantly (p ≤ 0.05) increased and was higher for positive oleylamine-stabilised droplets. All-trans-retinol was mainly localized in the epidermis with deeper distribution to viable skin layers in the presence of nanoparticles, yet negligible permeation (∼1% of topically applied dose) through full-thickness skin. Conclusions  Sustained release and targeted dermal delivery of all-trans-retinol from oil-in-water emulsions by inclusion of silica nanoparticles is demonstrated.     

Polyvinyl Derivatives as Novel Interactive Polymers for Controlled Gene Delivery to Muscle

Purpose. DNA plasmids (pDNA) can be taken up by and expressed in striated muscle after direct intramuscular injection. We have developed interactive polymeric gene delivery systems that increase pDNA bioavailability to muscle cells by both protecting pDNA from nucleases and controlling the dispersion and retention of pDNA in muscle tissue. Methods. A DNA plasmid, containing a CMV promoter and a -galactosidase reporter gene (CMV--gal), was injected either in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) solutions. Interactions between PVP and pDNA were assessed by dynamic dialysis, Isothermal Titration Calorimetry (ITC), and Fourier-Transformed Infra Red (FT-IR) spectroscopy. Formulations (50 µl) were injected into rat tibialis muscles after surgical exposure. Immuno-histochemistry for -gal was used to visualize the sites of expression in muscle. Results. -gal expression using pDNA in saline reached a plateau while -gal expression using PVP formulations increased linearly in the dose range studied (12.5–150 µg pDNA injected) and resulted in an increase in the number and distribution of cells expressing -gal. The interaction between PVP and pDNA was found to be an endothermic process governed largely by hydrogen-bonding and results in protection of pDNA from extracellular nucleases. Conclusions. Significant enhancement of gene expression using interactive polyvinyl-based delivery systems has been observed. The improved tissue dispersion and cellular uptake of pDNA using polyvinyl-based systems after direct injection into muscle is possibly due to osmotic effects.     

New Cyclodextrin Hydrogels Cross-Linked with Diglycidylethers with a High Drug Loading and Controlled Release Ability

Purpose The goal of the study is to develop new hydrogels based on cyclodextrins cross-linked with ethyleneglycol diglycidylether (EGDE) under mild conditions, to be used as carriers of amphiphilic drugs. Also, it aims to characterize the cross-linking and the drug loading and release processes. Methods The cross-linking of hydroxypropyl-β-cyclodextrin (HPβCD) with EGDE, in the absence or presence of hydroxypropylmethylcellulose (HPMC) Methocel? K4M, was optimized applying oscillatory rheometry and Fourier transform infrared. Hydrogels were characterized regarding swelling in water, ability to load diclofenac, and release after different drying treatments. Results Solutions of HPβCD (14.28%), without or with HPMC (0.2–1.0%), provided firm and transparent hydrogels after cross-linking with EGDE (14.28%), in which around two thirds of the OH groups were cross-linked. The incorporation of HPMC progressively reduced the gel time and the swelling degree of hydrogels. HPβCD hydrogels efficiently loaded diclofenac and sustained the release for several hours. The presence of HPMC slowed the release from swollen hydrogels, but promoted it from hydrogels dried before the loading and also before the release. Conclusions HPβCD hydrogels with good mechanical properties and tunable loading and release ability can be obtained by direct cross-linking with EGDE. *The work described in this paper is the subject of patent applications filed by the University of Santiago de Compostela.     

Biodegradable Microneedles Array with Dual-Release Behavior and Parameter Optimization by Finite Element Analysis

Microneedles (MNs) are particularly attractive for transdermal administration because of the improved safety, patient compliance and convenience. Dissolving MNs could provide rapid transdermal delivery, but with relatively low mechanical strength and almost no sustainability. On the other hand, hydrogel MNs are complicated to fabricate and have risk concerns. Herein, we developed a biodegradable MNs array composed of biocompatible silk fibroin and poly(vinyl alcohol) to overcome these limitations. Finite element analysis was employed for parameter optimization. The MNs array fabricated by the optimal parameters and material displayed sufficient mechanical strength to disrupt stratum corneum and formed microchannels for transdermal delivery. Dual-release profile was observed in the MNs array, with rapid release in the beginning, and prolonged release afterward. This release behavior fits Weibull release model and is favorable for topical application. The initial immediate release can quickly deliver active compounds to reach the therapeutic effective concentration and facilitate skin penetration, and the sustained release may supply the skin with active compounds over a prolonged period. This biodegradable MNs array is easy to fabricate, mechanically robust, could eliminate safety concerns, and provide the sustainability and advantage for large-scale production.     

Microneedle mediated delivery of nanoparticles into human skin

The development of novel cutaneous delivery technologies that can produce micron-sized channels within the outermost skin layers has stimulated interest in the skin as an interface for localised and systemic delivery of macromolecular and nanoparticulate therapeutics. This investigation assesses the contribution of physicochemical factors to the rate and extent of nanoparticle delivery through microchannels created in a biological tissue, the skin, by novel delivery technologies such as the microneedle array. The hydrodynamic diameter, zeta potential and surface morphology of a representative fluorescent nanoparticle formulation were characterised. Permeation studies using static Franz-type diffusion cells assessed (i) the diffusion of nanoparticle formulations through a model membrane containing uniform cylindrical microchannels of variable diameter and (ii) nanoparticle penetration across microneedle treated human skin. Wet-etch microneedle array devices can be used to significantly enhance the intra/transdermal delivery of nanoparticle formulations. However the physicochemical factors, microchannel size and particle surface charge, have a significant influence on the permeation and subsequent distribution of a nanoparticle formulation within the skin. Further work is required to understand the behaviour of nanoparticle formulations within the biological environment and their interaction with the skin layers following disruption of the skin barrier with novel delivery devices such as the microneedle array.     

Microneedles containing cross-linked hyaluronic acid particulates for control of degradation and swelling behaviour after administration into skin

Microneedles (MN) containing cross-linked hyaluronic acid (X-linked HA) particulates were prepared to control the degradation and swelling behaviour after transdermal drug delivery. The X-linked HA particulates were prepared by cross-linking HA chains and then passing the particulates through a sieve. Then, microneedles were prepared by micromolding method. The rheological properties of X-linked HA were studied. The penetration success rate, mechanical failure and dissolution rate of microneedles containing only hyaluronic acid (HA MN) and microneedles containing X-linked HA were compared. The delivery of fluorescein into the skin with X-linked HA MN was also observed using a confocal microscope. The size of the pulverised particulates in water ranged between 29 and 82?μm in diameter. The HA MN and X-linked HA MN were 270?μm in length. X-linked HA MN with fluorescein was inserted to a depth of 90% of the microneedle length successfully. There was no decrease in the penetration success rate for MN with up to 20% content of X-linked HA particulates. X-linked HA MN with up to 20% of particulate content did not change the dissolution time. Delay in degradation of HA, sustained drug release, and swelling behaviour of the skin layer can be obtained by X-linked HA MN.     

Electrohydrodynamic Comminution: A Novel Technique for the Aerosolisation of Plasmid DNA

Purpose Naked plasmid DNA (pDNA) is a potential gene transfer agent for lung gene therapies but cannot be aerosolised without degradation using conventional nebulisation devices. This study investigated the viability of an alternative nebulisation technique, electrohydrodynamic (EHD) comminution for the aerosol delivery of naked DNA in vivo.Methods Naked pDNA was aerosolised using jet and ultrasonic nebulisers, and by EHD comminution. Degradation associated with the aerosolisation process was investigated using gel electrophoresis and by transfection studies in cell culture. Optimised formulations for EHD aerosolisation of pDNA were developed and in vivo deposition and reporter gene expression were investigated in mice.Results Unlike conventional nebulisation devices, EHD comminution of plasmids up to 15 kb in size resulted in no detectable pDNA degradation. EHD formulations containing up to 1 mg/ml pDNA were developed and shown to produce monodisperse aerosols suitable for targeted lung delivery in humans. Aerosolisation studies in vivo demonstrated detectable levels of pDNA deposition and measurable luciferase reporter gene expression in the lungs of exposed mice.Conclusions This study demonstrates for the first time that respirable aerosols of naked pDNA can be generated without plasmid degradation and that EHD comminution is an appropriate technique for the aerosolisation of delicate gene transfer agents.     

Microneedles for transdermal drug delivery

The success of transdermal drug delivery has been severely limited by the inability of most drugs to enter the skin at therapeutically useful rates. Recently, the use of micron-scale needles in increasing skin permeability has been proposed and shown to dramatically increase transdermal delivery, especially for macromolecules. Using the tools of the microelectronics industry, microneedles have been fabricated with a range of sizes, shapes and materials. Most drug delivery studies have emphasized solid microneedles, which have been shown to increase skin permeability to a broad range of molecules and nanoparticles in vitro. In vivo studies have demonstrated delivery of oligonucleotides, reduction of blood glucose level by insulin, and induction of immune responses from protein and DNA vaccines. For these studies, needle arrays have been used to pierce holes into skin to increase transport by diffusion or iontophoresis or as drug carriers that release drug into the skin from a microneedle surface coating. Hollow microneedles have also been developed and shown to microinject insulin to diabetic rats. To address practical applications of microneedles, the ratio of microneedle fracture force to skin insertion force (i.e. margin of safety) was found to be optimal for needles with small tip radius and large wall thickness. Microneedles inserted into the skin of human subjects were reported as painless. Together, these results suggest that microneedles represent a promising technology to deliver therapeutic compounds into the skin for a range of possible applications.     

Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel

Purpose. Currently, most pDNA delivery systems based on synthetic polymers are either nonbiodegradable or not sensitive to the release environment. The primary objective of this study was to develop and evaluate an aqueous-based, thermosensitive, biodegradable and biocompatible triblock copolymer to control pDNA delivery in vitro and in vivo. Methods. The triblock copolymers, poly[ethylene glycol-b-(D, L-lactic acid-co-glycol acid)-b-ethylene glycol] (PEG-PLGA-PEG), were synthesized as previously described. The molecular weight and polydispersity of PEG-PLGA-PEG were monitored by gel permeation chromatography (GPC). The cytotoxicity of PEG-PLGA-PEG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide assay. The release of ³²P-labeled pDNA entrapped in aqueous dispersion of PEG-PLGA-PEG in 0.1 mol/L sodium phosphate buffer solution (pH 7.4) was studied at 37°C under agitation. Gene transfection efficiency was evaluated in a skin wound model in CD-1 mice. Results. The aqueous dispersion of PEG-PLGA-PEG flows freely at room temperature but form a gel at 37°C body temperature. The in vitro degradation of PEG-PLGA-PEG lasted for more than 30 days. The cytotoxicity of PEG-PLGA-PEG evaluated in HEK 293 cells was significantly lower than that of poly-L-lysine hydrochloride. The release profile of supercoiled pDNA from the polymer followed the zero-order kinetics up to 12 days. Maximal gene expression of luciferase was at 24 h in the skin wound of CD-1 mice and by 72 h, the expression dropped by nearly 94%. Conclusions. These results suggest hydrogel formed by PEG-PLGA-PEG could be a promising platform for delivery of pDNA, which represents a novel strategy that may serve as a non-viral vector for gene therapy in wound healing.     

Microneedles and other physical methods for overcoming the stratum corneum barrier for cutaneous gene therapy

The outermost layer of skin, the epidermis, has developed formidable physical and immunological barrier properties that prevent infiltration of deleterious chemicals and pathogens. Consequently, transdermal delivery of medicaments is currently restricted to a limited number of low molecular weight drugs. As a corollary, there has been significant recent interest in providing strategies that disrupt or circumvent the principal physical barrier, the stratum corneum, for the efficient cutaneous delivery of macromolecular and nucleic acid based therapeutics. These strategies include: electrical methods, intradermal injection, follicular delivery, particle acceleration, laser ablation, radiofrequency ablation, microscission, and microneedles. The application of microfabricated microneedle arrays to skin creates transient pathways to enable transcutaneous delivery of drugs and macromolecules. Microneedle use is simple, pain-free, and causes no bleeding, with further advantages of convenient manufacture, distribution, and disposal. To date, microneedles have been shown to deliver drug, peptide, antigen, and DNA efficiently through skin. Robust and efficient microneedle designs and compositions can be inserted into the skin without fracture. Further progress in microneedle array design, microneedle application apparatus, and integrated formulation will confirm this methodology as a realistic clinical strategy for delivering a range of medicaments, including DNA, to and through skin.     

Synergistic Effect of Formulated Plasmid and Needle-Free Injection for Genetic Vaccines

Purpose. A plasmid-based gene expression system was complexed with protective, interactive, and non-condensing (PINC) polymer system and administered with Medi-Jector, a needle-free injection device (NFID), to achieve high and sustained levels of antigen-specific antibodies in blood circulation. Methods. Human growth hormone (hGH) or bacterial -galactosidase gene expression plasmids driven by a cytomegalovirus (CMV) promoter were formulated in saline or complexed with a PINC polymer, polyvinylpyrrolidone (PVP), and intramuscularly or subcutaneously administered into dogs and pigs using a 22-gauge needle or a NFID. The hGH-specific IgG titers in serum were measured by an ELISA. -galactosidase expression was measured in injected muscles by an enzymatic assay or immunohistochemistry. The effect of NFID on DNA stability and topology was assessed by gel electrophoresis. Results. Intramuscular (i.m.) or subcutaneous (s.c.) injection of a hGH expression plasmid pCMV-hGH (0.05-0.5 mg/kg) in dogs and pigs elicited antigen-specific IgG antibody titers to expressed hGH. With both routes of injection, pDNA delivery by a NFID was superior to pDNA injection by needle. The magnitude of hGH-specific IgG titers with NFID was 15–20-fold higher than needle injection when pDNA was complexed with PVP, and only 3–4-fold higher with pDNA in saline. The transfection efficiency in the injected muscle, as measured by -galaclosidase expression, following i.m. injection of pCMV--galaclosidase/PVP, was not significantly different between needle and NFID-injected groups. Conclusions. These data demonstrate that the combination of pDNA/ PVP complexes and a NFID act synergistically to achieve high and sustained levels of antigen-specific IgG response to expressed antigen. This gene delivery approach may offer advantage over needle injection of naked DNA for the development of genetic vaccines.     

Poly(L-lactic acid)/polyethylenimine nanoparticles as plasmid DNA carriers

Non-viral vectors such as liposomes, polycations, and nanoparticles have been used as gene delivery systems. In this study, we prepared and characterized biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) nanoparticles as gene carriers. pCMV/β-gal and pEGFP-C1 were utilized as model plasmid DNAs (pDNA). Nanoparticles were prepared using a double emulsion-solvent evaporation technique, and their pDNA binding capacity was assessed by agarose gel electrophoresis. Transfection was studied in HEK 293 and HeLa cell lines, and the transfection efficiencies were determined by β-galactosidase assay or flow cytometry. Three kinds of PLA/PEI systems were studied by varying the molecular weight of PEI. The PLA/PEI 25K system had a higher transfection efficiency than the PLA/PEI 0.8K or PLA/PEI 750K systems. The transfection efficiency was found to be dependent on the ratio of PLA/PEI nanoparticles to pDNA with an optimum ratio of 60:1 (w/w). The cytotoxicity was dependent on the quantity of PLA/PEI nanoparticles used, but it was comparable to that of commercial Lipofectin™. These results demonstrate the potential of PLA/PEI nanoparticles as gene carriers.