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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.

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 I²-galactosidase reporter plasmid DNA applied to ViaDermâ„¢ treated skin. Skin treated with ViaDermâ„¢ using 50 I¼m electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDermâ„¢ was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDermâ„¢) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.

Background: Pulmonary gene therapy requires aerosolisation of the gene vectors to the target region of the lower respiratory tract. Pulmonary absorption enhancers have been shown to improve the penetration of pharmaceutically active ingredients in the airway. In this study, we investigate whether certain absorption enhancers may also enhance the aerosolisation properties of spray-dried powders containing non-viral gene vectors. Methods: Spray-drying was used to prepare potentially respirable trehalose-based dry powders containing lipid-polycation-pDNA (LPD) vectors and absorption enhancers. Powder morphology and particle size were characterised using scanning electron microscopy and laser diffraction, respectively, with gel electrophoresis used to assess the structural integrity of the pDNA. The biological functionality of the powders was quantified using in vitro cell (A549) transfection. Aerosolisation from a Spinhaler® dry powder inhaler into a multistage liquid impinger (MSLI) was used to assess the in vitro dispersibility and deposition of the powders. Results: Spray-dried powder containing dimethyl-β-cyclodextrin (DMC) demonstrated substantially altered particle morphology and an optimal particle size distribution for pulmonary delivery. The inclusion of DMC did not adversely affect the structural integrity of the LPD complex and the powder displayed significantly greater transfection efficiency as compared to unmodified powder. All absorption enhancers proffered enhanced powder deposition characteristics, with the DMC-modified powder facilitating high deposition in the lower stages of the MSLI. Conclusions: Incorporation of absorption enhancers into non-viral gene therapy formulations prior to spray-drying can significantly enhance the aerosolisation properties of the resultant powder and increase biological functionality at the site of deposition in an in vitro model. Copyright © 2005 John Wiley & Sons, Ltd.

Spray-drying represents a viable alternative to freeze-drying for preparing dry powder dispersions for delivering macromolecules to the lung. The dispersibility of spray-dried powders is limited however, and needs to be enhanced to improve lung deposition and subsequent biological activity. In this study, we investigate the utility of leucine as a dry powder dispersibility enhancer when added prior to spray-drying a model non-viral gene therapy formulation (lipid:polycation:pDNA, LPD). Freeze-dried lactose-LPD, spray-dried lactose-LPD and spray-dried leucine-lactose-LPD powders were prepared. Scanning electron microscopy showed that leucine, increased the surface roughness of spray-dried lactose particles. Particle size analysis revealed that leucine-containing spray-dried powders were unimodally dispersed with a mean particle diameter of 3.12 μm. Both gel electrophoresis and in vitro cell (A549) transfection showed that leucine may compromise the integrity and biological functionality of the gene therapy vector. The deposition of the leucine containing powder was however significantly enhanced as evidenced by an increase in gene expression mediated by dry powder collected at lower stages of a multistage liquid impinger (MSLI). Further studies are required to determine the potential of leucine as a ubiquitous dispersibility enhancer for a variety of pulmonary formulations. © 2003 Taylor & Francis Ltd.

Summary Background The skin represents an accessible somatic tissue for therapeutic gene transfer. The superficial lipophilic layer of the skin, the stratum corneum, however, constitutes a major obstacle to the cutaneous delivery of charged macromolecules such as DNA. Objectives To determine whether silicon-based microneedles, microfabricated via a novel isotropic etching/BOSCH reaction process, could generate microchannels in the skin of sufficient dimensions to facilitate access of lipid : polycation : pDNA (LPD) nonviral gene therapy vectors. Methods Scanning electron microscopy was used to visualize the microconduits created in heat-separated human epidermal sheets after application of the microneedles. Following confirmation of particle size and particle surface charge by photon correlation spectrocopy and microelectrophoresis, respectively, the diffusion of fluorescent polystyrene nanospheres and LPD complexes through heat-separated human epidermal sheets was determined in vitro using a Franz-type diffusion cell. In-vitro cell culture with quantification by flow cytometry was used to determine gene expression in human keratinocytes (HaCaT cells). Results The diffusion of 100 nm diameter fluorescent polystyrene nanospheres, used as a readily quantifiable predictive model for LPD complexes, through epidermal sheets was significantly enhanced following membrane treatment with microneedles. The delivery of LPD complexes either into or through the membrane microchannels was also demonstrated. In both cases considerable interaction between the particles and the epidermal sheet was observed. In-vitro cell culture was used to confirm that LPD complexes mediated efficient reporter gene expression in human keratinocytes in culture when formulated at the appropriate surface charge. Conclusions These studies demonstrate the utility of silicon microneedles in cutaneous gene delivery. Further studies are required to elucidate fully the influence of the physicochemical characteristics of gene therapy vectors, e.g. particle diameter and surface charge, on their diffusion through microchannels and to quantify gene expression in vivo.

Background Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze-drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non-viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze-drying and spray-drying, a one-step process for producing dry and respirable powders, as methods for preparing non-viral respiratory gene delivery systems. Methods Lipid:polycation:pDNA (LPD) vectors comprising 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP-N1 in 3% lactose solution were either snap-frozen and lyophilised or spray-dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence. Results Lyophilisation produced large, irregularly shaped particles prior to (mean diameter ∼21 µm) and following (mean diameter ∼18 µm) coarse grinding. Spray-drying produced uniformly shaped spherical particles (mean diameter ∼4 µm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray-dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage. Conclusions Spray-drying has utility for producing stable, efficient and potentially respirable non-viral dry powder systems for respiratory gene delivery. Copyright © 2002 John Wiley & Sons, Ltd.

Traditionally, optimisation of a gene delivery formulation utilises a study design that involves altering only one formulation variable at any one time whilst keeping the other variables constant. As gene delivery formulations become more complex, e.g. to include multiple cellular and sub-cellular targeting elements, there will be an increasing requirement to generate and analyse data more efficiently and allow examination of the interaction between variables. This study aims to demonstrate the utility of multifactorial design, specifically a Central Composite Design, in modelling the responses size, zeta potential and in vitro transfection efficiency of some prototypic non-viral gene delivery vectors, i.e. cationic liposome-pDNA complexes, and extending the application of the design strategy to more complex vectors, i.e. tri-component lipid:polycation:DNA (LPD). The modelled predictions of how the above responses change as a function of formulation show consistency with an extensive literature base of data obtained using more traditional approaches, and highlight the robustness and utility of the Central Composite Design in examining key formulation variables in non-viral gene delivery systems. The approach should be further developed to maximise the predictive impact of data across the full range of pharmaceutical sciences.

The impetus to develop non-viral gene delivery vectors has led to examination of synthetic polycationic polymers as plasmid DNA (pDNA) condensing agents. Previous reports have highlighted superiority (up to ×10-fold) in the in-vitro transfection of pDNA complexes formed by poly-( l )-ornithine (PLO) compared to those formed with poly-( l )-lysine (PLL). The apparent basis for this consistent superiority of PLO complexes remains to be established. This comparative study investigates whether physico–chemical differences in the supramolecular properties of polycation:pDNA complexes provide a basis for their observed differential gene transfection. Specifically, particle size distribution and zeta potential of the above complexes formulated over a wide range of polycation:pDNA ratios were found to be consistent with a condensed (150–200 nm) cationic (+30–40 mV) system but not influenced by the type of cationic polymer used. A spectrofluorimetric EtBr exclusion assay showed that polycation:pDNA complexes display different pDNA condensation behaviour, with PLO able to condense pDNA at a lower polycation mass compared to both polylysine isomers, and form complexes that were more resistant to disruption following challenge with anionic counter species, i.e. poly-( l )-aspartic acid and the glycosaminoglycan molecule, heparin. We conclude that particle size and surface potential as gross supramolecular properties of these complexes do not represent, at least in a non-biological system, the basis for the differential transfection behaviour observed between these condensing polymers. However, differences in the ability of the polylysine and polyornithine polymers to interact with pDNA and to stabilise the polymer-pDNA assembly could have profound effects upon the cellular and sub-cellular biological processing of pDNA molecules and contribute to the disparity in cell transfection efficiency observed between these complexes.

Objectives A number of respiratory diseases could potentially benefit from gene transfer to the lung, including cystic fibrosis, asthma and emphysema. Jet nebulisation, the current standard for introducing gene therapy formulations into the lung, is highly inefficient. While newer nebuliser technologies are under development, pressurised metered dose inhalers (pMDIs) offer an alternative with potential advantages of more efficacious and rapid administration. As both solution and suspension pMDI formulations have drawbacks relating to stability and dose reproducibility, we aim to explore the potential of modifying a novel low-energy nanotechnology process (Dickinson et al 2001) to prepare surfactant-coated plasmid DNA (pDNA) nanoparticles for pulmonary gene delivery via a dispersion pMDI. Methods Water-in-oil microemulsions containing pEGFP-N1 reporter plasmid were prepared from sucrose solution (as cryoprotectant and aqueous phase), lecithin: propan-2-ol (as stabilising surfactant) and iso-octane (as organic phase). Resultant microemulsions were snap frozen in liquid nitrogen and lyophilised. Excess surfactant was removed by repeated washes with iso-octane and centrifugation. Scanning electron microscopy (SEM) and gel electrophoresis were used to characterise surface morphology and deduce pDNA integrity, respectively. Results A ternary phase diagram was constructed to identify optimised microemulsion compositions (Figure 1). Microemulsions with a surfactant to water ratio of 1.5 and above formed stable water in oil isotropic systems. Unstable biphasic systems were formed when the surfactant to water ratio fell below 1.5. Optimised formulations resulted in effective incorporation of pDNA into the aqueous pool of reverse micelles. Controlled lyophilisation enabled the formation of novel surfactant- coated pDNA nanoparticles. A qualitative analysis performed using gel electrophoresis showed that the freeze-dried particles retained pDNA structural integrity. SEM images conferred aggregates of DNA-cryoprotectant particles. Conclusions Freeze-drying pDNA microemulsions produced surfactant coated pDNA particles whilst successfully maintaining the integrity of the pDNA. The nanotechnology process used offers the potential for the incorporation of pDNA nanoparticles into pMDI systems for pulmonary delivery of gene vectors.

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.