A. Morrissey

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.

Cutaneous DNA delivery and gene expression in ex vivo human skin explants via wet-etch microfabricated microneedles

Microneedle arrays increase skin permeability by forming channels through the outer physical barrier, without stimulating pain receptors populating the underlying dermis. It was postulated that microneedle arrays could facilitate transfer of DNA to human skin epidermis for cutaneous gene therapy applications. Platinum-coated “wet-etch” silicon microneedles were shown to be of appropriate dimensions to create microconduits, approximately 50 μm 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 microneedles to mediate gene expression was demonstrated using the β-galactosidase reporter gene. Preliminary studies confirmed localised delivery, cellular internalisation and subsequent gene expression of pDNA following microneedle 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.

Low temperature fabrication of biodegradable sugar glass microneedles for transdermal drug delivery applications

Transdermal drug delivery is limited by the barrier properties of the outer skin layer. Microneedles (MNs) effectively circumvent the skin barrier to offer this route as a potential alternative to oral and parenteral delivery of therapeutics. Biodegradable microneedles offer particular advantages however processing commonly requires elevated temperatures that may adversely affect heat-labile molecules and macromolecules. In this study, solid amorphous sugar glasses containing low residual quantities of water were created by dehydration of trehalose and sucrose sugar combination solutions. Biodegradable sugar glass MNs were fabricated following optimisation of a simple and novel low temperature vacuum deposition micromoulding methodology. These had absolute morphological fidelity to silicon master structures and demonstrated sufficient structural rigidity to efficiently penetrate excised human breast skin. Sugar glass MNs incorporating a marker compound dissolved rapidly and completely in situ releasing dye into deeper skin layers. The biological activity of a model macromolecule was partially retained over extended storage following incorporation into sugar glass. This is the first demonstration that MNs created from amorphous sugar glasses can be used for incorporating and delivering molecules, and potentially biologically active macromolecules, via the transdermal route.

Development of an ex vivo human skin model for intradermal vaccination: tissue viability and Langerhans cell behaviour.

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

The evolution from convex corner undercut towards microneedle formation: theory and experimental verification

In this paper, we present the mechanism of silicon convex corner undercutting of etch masks in pure aqueous KOH solution (29% KOH, 79 ?C) during microneedle fabrication. The process was documented by images and measurements in an optical microscope. These measurements have been used to determine the Miller indices of the crystal planes comprising the microneedle as well as for mathematical interpretations of etch rates, etch times and mask design. Under these etch conditions, the final structures have convex corner undercutting planes with Miller indices in the {h 1?2} family, in agreement with literature results. However, we show that the index h decreases during the etch process, from more than 14 (nearly vertical walls) to a stable value of 3. The final microneedle is formed by {3?1?2} planes and a small base of {1?2?1} planes. Stable crystal plane formation permits the fabrication of microneedles with different heights with one etch depth using different mask sizes. We demonstrate that the etch bath conditions play an important role in the formation of the needle shape.

A comparison of the puncturing properties of gelatin and hypromellose capsules for use in dry powder inhalers.

This study investigates capsule puncture in dry powder inhalers. Gelatin and hydroxypropylmethyl cellulose (HPMC) capsules (stored at 11 and 33% relative humidities) were punctured using a pin from a Foradil® inhaler, with insertion force measurement via an Instron tester. In HPMC capsules, the force after capsule puncture reduced by half and then increased to a second maximum as the pin shaft entered the hole. In gelatin capsules, the postpuncture force reduced to zero, indicating shell flaps losing contact with the pin. At lower moisture contents, both capsules were less flexible. This provides a tool to measure the shell properties of inhalation capsules.

Biodegradable microneedles for intra-epidermal vaccination [Abstract]

The concept that the time to onset of erythema after the application of the rubefacient and urticant substance methyl nicotinate (MN) indicates skin barrier competence was introduced 30 years ago. MN produces a dose-dependent erythema on topical application to intact skin, the nature of which is known to be fast moving (in the order of minutes) and variable. Using tissue viability imaging (TiVi) the time course and degree of the reaction can be conveniently followed and analysed. Inter-individual variability can be quite marked but intra-individual variability is less pronounced. At the upper end of provocation (higher doses, more sensitive individuals) urtication can occur, which decreases blood flow by increasing pressure on and thus emptying capillaries. The TiVi system can quantitate urtication and inherent (blanched) skin colour. The utility of MN application in the study of individual barrier function and microvascular reactivity is increased by the use of the TiVi system for collection and analysis of data.

An assessment of pain, penetration efficiency and trans-epidermal water loss in human subjects following application of microfabricated microneedle arrays [Abstract]

Objectives The combination of dimethyldioctadecyl ammonium bromide (DDA) and synthetic cord factor, trehalose dibehenate (TDB), can engender protective immune responses against M. tuberculosis infection (Holten-Andersen et al 2004). However, problems such as lack of stability and production of an economical sterile product have motivated investigation into formulation aspects such as freeze drying and g-irradiation sterilisation (Mohammed et al 2006). This study evaluates the immunogenicity of these optimised formulations. Methods DDA/TDB formulations were prepared by lipid hydration. BALB/c mice (n = 5) were immunised with vaccines incorporating a range of cryoprotectants, or vaccines exposed to gamma radiation (25 KGy) as approved by the local ethical review committee. Immunological analysis was as previously outlined (Vangala et al 2007). Briefly; antibody responses were assessed using ELISA; spleen cell proliferation was quantified using uptake of H thymidine following restimulation; and cytokine production was evaluated using DuoSet capture ELISA (R & D Systems). Results Almost all of the antibody mediated immune responses were comparable with no adverse effect on immunogenicity. However, antigen specific cell proliferation (Figure 1) showed that the inclusion of lysine plus sterilisation facilitated superior cell proliferation to any of the other groups except lysine without sterilisation. The inclusion of lysine has not previously been evaluated for immunogenicity and clearly warrants further investigation. IL-2 levels indicated that the increased cell proliferation was probably associated with enhanced T cell expansion. IL-2 and IFN-g production mirrored the cell proliferation results. Conclusions In all tests, the various modifications facilitating production of a sterile and more stable product, do not adversely affect immunogenicity. This study supports the use of these optimisations in the formulation of this vaccine.

Microneedle facilitated delivery of pDNA encoding HBsAg to human skin: potential for genetic vaccination [Abstract]

The calculations necessary to allow infusion of a known drug dosage at micrograms per kilogram of body weight per minute are time-consuming and error prone. A simpler method entails multiplication of the patients weight in kilograms by the factor 15. The resultant figure represents the number of milligrams of drug to be placed in 250 ml of infusate vehicle. The solution, which is delivered through a microdrip chamber (60 gtt per milliliter), will contain 1 microgram per kilogram in each drop. One is thus permitted to define dosage by setting up the solution to have 1 gtt = 1 microgram/kg.