M. A. F. Kendall

A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids.

Synthetic vectors based on reducible polycations consisting of histidine and polylysine residues (HIS RPCs) were evaluated for their ability to deliver nucleic acids. Initial experiments showed that RPC-based vectors with at least 70% histidine content mediated efficient levels of gene transfer without requirement for the endosomolytic agent chloroquine. Significant gene transfer was observed in a range of cell types achieving up to a 5-fold increase in the percentage of transfected cells compared to 25 kDa PEI, a gold standard synthetic vector. In contrast to 25 kDa PEI, HIS RPCs also mediated efficient transfer of other nucleic acids, including mRNA encoding green fluorescent protein in PC-3 cells and siRNA directed against the neurotrophin receptor p75NTR in post-mitotic cultures of rat dorsal root ganglion cell neurons. Experiments to elevate intracellular glutathione and linear profiling of cell images captured by multiphoton fluorescent microscopy highlighted that parameters such as the molecular weight and rate of cleavage of HIS RPCs were important factors in determining transfection activity. Altogether, these results demonstrate that HIS RPCs represent a novel and versatile type of vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids. This should enable different or a combination of therapeutic strategies to be evaluated using a single type of polycation-based vector.

Dry-coated microprojection array patches for targeted delivery of immunotherapeutics to the skin

Dry-coated microprojections (MPs) deliver vaccine to abundant immunogenic cells within the skin to induce immune responses. Success in this targeted vaccine delivery relies on overcoming the challenges of dry-coating the vaccine onto the very small (<or=90 microm length) and densely packed (approximately 20,000 cm(-2)) MPs. In this paper, we show that a gas-jet drying coating method achieves the desired uniform coating. The coating approach is robustly demonstrated on compounds representative of a range of immunotherapeutics (e.g. DNA, proteins), with each uniformly coated on thousands of MPs. Furthermore, the dry-coating remains intact during skin insertion, and then releases within the wet skin cellular environment within 3 min. Finally, we applied ovalbumin protein coated MP patches to immunise mice, achieving comparable antibody levels (p=0.08) with needle and syringe intramuscular injection. In summary, this paper presents a simple, practical and versatile method to achieve uniform coating on very small and densely packed MPs for a needle-free and targeted vaccine delivery technology.

Potent Immunity to Low Doses of Influenza Vaccine by Probabilistic Guided Micro-Targeted Skin Delivery in a Mouse Model

Background Over 14 million people die each year from infectious diseases despite extensive vaccine use [1]. The needle and syringe—first invented in 1853—is still the primary delivery device, injecting liquid vaccine into muscle. Vaccines could be far more effective if they were precisely delivered into the narrow layer just beneath the skin surface that contains a much higher density of potent antigen-presenting cells (APCs) essential to generate a protective immune response. We hypothesized that successful vaccination could be achieved this way with far lower antigen doses than required by the needle and syringe. Methodology/Principal Findings To meet this objective, using a probability-based theoretical analysis for targeting skin APCs, we designed the Nanopatch™, which contains an array of densely packed projections (21025/cm2) invisible to the human eye (110 µm in length, tapering to tips with a sharpness of <1000 nm), that are dry-coated with vaccine and applied to the skin for two minutes. Here we show that the Nanopatches deliver a seasonal influenza vaccine (Fluvax® 2008) to directly contact thousands of APCs, in excellent agreement with theoretical prediction. By physically targeting vaccine directly to these cells we induced protective levels of functional antibody responses in mice and also protection against an influenza virus challenge that are comparable to the vaccine delivered intramuscularly with the needle and syringe—but with less than 1/100th of the delivered antigen. Conclusions/Significance Our results represent a marked improvement—an order of magnitude greater than reported by others—for injected doses administered by other delivery methods, without reliance on an added adjuvant, and with only a single vaccination. This study provides a proven mathematical/engineering delivery device template for extension into human studies—and we speculate that successful translation of these findings into humans could uniquely assist with problems of vaccine shortages and distribution—together with alleviating fear of the needle and the need for trained practitioners to administer vaccine, e.g., during an influenza pandemic.

The effect of strain rate on the precision of penetration of short densely-packed microprojection array patches coated with vaccine

If skins non-linear viscoelastic properties are mechanically exploited for precise antigen placement, there is tremendous promise for improved vaccines. To achieve this, we designed a Nanopatch-densely packed micro-nanoprojections (>20,000/cm(2)) to directly deposit antigen to large numbers of epidermal Langerhans cells and dermal dendritic cells. Here, we controllably applied our Nanopatches with discrete conditions between peak strain rates of approximately 100 s(-1)-7000 s(-1) and quantified resulting penetration depths, delivery payloads and skin mechanics. Increasing the strain rate of application, we overcame key skin variability, achieving increases in both projection penetration depth (by over 50% length) and area coverage of a full array (from 50% to 100%). This delivery depth precision opens the way for more fully utilizing the skins immune function. Furthermore, we yielded new insights on mechanical behaviour of skin, including: 1) internal skin property changes that could affect/facilitate penetration; 2) projection design to dictate penetration depth; 3) puncture mechanics of skin in this strain rate range. Indeed, we show delivery of a model vaccine using our tested range of strain rates achieved functionally relevant tunable systemic antibody generation in mice. These findings could be of great utility in extending skin strata vaccine targeting approaches to human use.

Skin Vaccination against Cervical Cancer Associated Human Papillomavirus with a Novel Micro-Projection Array in a Mouse Model

Background Better delivery systems are needed for routinely used vaccines, to improve vaccine uptake. Many vaccines contain alum or alum based adjuvants. Here we investigate a novel dry-coated densely-packed micro-projection array skin patch (Nanopatch™) as an alternate delivery system to intramuscular injection for delivering an alum adjuvanted human papillomavirus (HPV) vaccine (Gardasil®) commonly used as a prophylactic vaccine against cervical cancer. Methodology/Principal Findings Micro-projection arrays dry-coated with vaccine material (Gardasil®) delivered to C57BL/6 mouse ear skin released vaccine within 5 minutes. To assess vaccine immunogenicity, doses of corresponding to HPV-16 component of the vaccine between 0.43±0.084 ng and 300±120 ng (mean ± SD) were administered to mice at day 0 and day 14. A dose of 55±6.0 ng delivered intracutaneously by micro-projection array was sufficient to produce a maximal virus neutralizing serum antibody response at day 28 post vaccination. Neutralizing antibody titres were sustained out to 16 weeks post vaccination, and, for comparable doses of vaccine, somewhat higher titres were observed with intracutaneous patch delivery than with intramuscular delivery with the needle and syringe at this time point. Conclusions/Significance Use of dry micro-projection arrays (Nanopatch™) has the potential to overcome the need for a vaccine cold chain for common vaccines currently delivered by needle and syringe, and to reduce risk of needle-stick injury and vaccine avoidance due to the fear of the needle especially among children.

The viscoelastic, hyperelastic and scale dependent behaviour of freshly excised individual skin layers

Micro-devices using mechanical means to target skin for improved drug and vaccine delivery have great promise for improved clinical healthcare. Fully realizing this promise requires a greater understanding of key micro-biomechanical properties for each of the different skin layers - that are both the mechanical barriers and biological targets of these devices. Here, we performed atomic force microscopy indentation on a micro-nano scale to quantify separately, in fresh mouse skin, the viscous and elastic behaviour of the stratum corneum, viable epidermis and dermis. By accessing each layer directly, we examined the response to nanoindentation at sub-cellular and bulk-cellular scale. We found that the dermis showed greatest mechanical stiffness (elastic moduli of 7.33-13.48 MPa for 6.62 μm and 1.90 μm diameter spherical probes respectively). In comparison, the stratum corneum and viable epidermis were weaker at 0.75-1.62 MPa and 0.49-1.51 MPa respectively (again with the lower values resulting from indentations with the large probe 6.62 μm). The living cell layer of the epidermis (viable epidermis) showed greatest viscoelasticity - almost fully relaxing from shallow indentation - whilst the other layers reached a plateau after relaxing by around 40%. With small scale (sub-micron) AFM indentation, we directly determined the effects of different layer constituents - in particular, the dermis showed that some indents contacted collagen fibrils and others contacted ground substance/cellular areas. This work has far reaching implications for the design of micro-devices using mechanical means to deliver drugs or vaccines into the skin; providing key characterized mechanical property values for each constituent of the target delivery material.

Quantum dot penetration into viable human skin.

Abstract Systematic studies probing the effects of nanoparticle surface modification and formulation pH are important in nanotoxicology and nanomedicine. In this study, we use laser-scanning fluorescence confocal microscopy to evaluate nanoparticle penetration in viable excised human skin that was intact or tape-stripped. Quantum dot (QD) fluorescent nanoparticles with three surface modifications: Polyethylene glycol (PEG), PEG-amine (PEG-NH2) and PEG-carboxyl (PEG-COOH) were evaluated for human skin penetration from aqueous solutions at pH 7.0 and at pHs of solutions provided by the QD manufacturer: 8.3 (PEG, PEG-NH2) and 9.0 (PEG-COOH). There was some penetration into intact viable epidermis of skin for the PEG-QD at pH 8.3, but not at pH 7.0 nor for any other QD at the pHs used. Upon tape stripping 30 strips of stratum corneum, all QDs penetrated through the viable epidermis and into the upper dermis within 24 h.

Improved DNA vaccination by skin-targeted delivery using dry-coated densely-packed microprojection arrays

HSV-2-gD2 DNA vaccine was precisely delivered to immunologically sensitive regions of the skin epithelia using dry-coated microprojection arrays. These arrays delivered a vaccine payload to the epidermis and the upper dermis of mouse skin. Immunomicroscopy results showed that, in 43 ± 5% of microprojection delivery sites, the DNA vaccine was delivered to contact with professional antigen presenting cells in the epidermal layer. Associated with this efficient delivery of the vaccine into the vicinity of the professional antigen presenting cells, we achieved superior antibody responses and statistically equal protection rate against an HSV-2 virus challenge, when compared with the mice immunized with intramuscular injection using needle and syringe, but with less than 1/10th of the delivered antigen.

Investigations of gas and particle dynamics in first generation needle-free drug delivery devices

Abstract. Transdermal powdered drug delivery involves the propulsion of solid drug particles into the skin by means of high-speed gas-particle flow. The fluid dynamics of this technology have been investigated in devices consisting of a convergent-divergent nozzle located downstream of a bursting membrane, which serves both to initiate gas flow (functioning as the diaphragm of a shock tube) and to retain the drug particles before actuation. Pressure surveys of flow in devices with contoured nozzles of relatively low exit-to-throat area ratio and a conical nozzle of higher area ratio have indicated a starting process of approximately 200

Nanopatch targeted delivery of both antigen and adjuvant to skin synergistically drives enhanced antibody responses

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