Assessing the risk of a clinically significant infection from a Microneedle Array Patch (MAP) product.

Microneedle Array Patches (MAPs) are an emerging dosage form that creates transient micron-sized disruptions in the outermost physical skin barrier, the stratum corneum, to facilitate delivery of active pharmaceutical ingredients to the underlying tissue. Numerous MAP products are proposed and there is significant clinical potential in priority areas such as vaccination. However, since their inception scientists have hypothesized about the risk of a clinically significant MAP-induced infection. Safety data from two major Phase 3 clinical trials involving hundreds of participants, who in total received tens of thousands of MAP applications, does not identify any clinically significant infections. However, the incumbent data set is not extensive enough to make definitive generalizable conclusions. A comprehensive assessment of the infection risk is therefore advised for MAP products, and this should be informed by clinical and pre-clinical data, theoretical analysis and informed opinions. In this article, a group of key stakeholders identify some of the key product- and patient-specific factors that may contribute to the risk of infection from a MAP product and provide expert opinions in the context of guidance from regulatory authorities. Considerations that are particularly pertinent to the MAP dosage form include the specifications of the finished product (e.g. microbial specification), it's design features, the setting for administration, the skill of the administrator, the anatomical application site, the target population and the clinical context. These factors, and others discussed in this article, provide a platform for the development of MAP risk assessments and a stimulus for early and open dialogue between developers, regulatory authorities and other key stakeholders, to expedite and promote development of safe and effective MAP products.

An analysis of the relationship between microneedle spacing, needle force and skin strain during the indentation phase prior to skin penetration.

Microneedle (MN) array patches present a promising new approach for the minimally invasive delivery of therapeutics and vaccines. However, ensuring reproducible insertion of MNs into the skin is challenging. The spacing and arrangement of MNs in an array are critical determinants of skin penetration and the mechanical integrity of the MNs. In this work, the finite element method was used to model the effect of MN spacing on needle reaction force and skin strain during the indentation phase prior to skin penetration. Spacings smaller than 2-3 mm (depending on variables, e.g., skin stretch) were found to significantly increase these parameters.

Models and methods to characterise levonorgestrel release from intradermally administered contraceptives.

Microneedle (MN)-based technologies have been proposed as a means to facilitate minimally invasive sustained delivery of long-acting hormonal contraceptives into the skin. Intradermal administration is a new route of delivery for these contraceptives and therefore no established laboratory methods or experimental models are available to predict dermal drug release and pharmacokinetics from candidate MN formulations. This study evaluates an in vitro release (IVR) medium and a medium supplemented with ex vivo human skin homogenate (SH) as potential laboratory models to investigate the dermal release characteristics of one such hormonal contraceptive that is being tested for MN delivery, levonorgestrel (LNG), and provides details of an accompanying novel two-step liquid-liquid drug extraction procedure and sensitive reversed-phase HPLC-UV assay. The extraction efficiency of LNG was 91.7 ± 3.06% from IVR medium and 84.6 ± 1.6% from the medium supplemented with SH. The HPLC-UV methodology had a limit of quantification of 0.005 µg/mL and linearity between 0.005 and 25 µg/mL. Extraction and detection methods for LNG were exemplified in both models using the well-characterised, commercially available sustained-release implant (Jadelle®). Sustained LNG release from the implant was detected in both media over 28 days. This study reports for the first time the use of biologically relevant release models and a rapid, reliable and sensitive methodology to determine release characteristics of LNG from intradermally administered long-acting drug delivery systems.

Human-centred design of a new microneedle-based hormonal contraceptive delivery system.

Background: It is estimated that 225 million women worldwide have an unmet need for family planning, and more than half live in low- and middle-income countries. Increasing the choice of contraceptive methods available can reduce this unmet need. Microneedle drug delivery systems represent a new technology for minimally invasive self-administration of contraceptives. We explored stakeholders' views on different aspects of a proposed microneedle-based hormonal contraceptive delivery system. The feedback was used to iteratively develop this delivery system. Methods: Focus group discussions and semi-structured interviews were conducted with potential stakeholders (women and trans males of childbearing age, their partners, and health professionals and organisations that provide family planning advice and contraception services) in Uganda, The Gambia, Malawi, and the UK, exploring concept acceptability and gathering feedback on different aspects of design and usability of the proposed delivery system. Results: Participants viewed the concept of a new, microneedle-based contraceptive favourably. In Uganda, participants were presented with 7 different prototype applicators and identified desirable features of a preferred delivery device; their input reducing the number of prototypes that were subsequently evaluated by stakeholders in The Gambia and the UK. Participants in these countries helped to identify and/or confirm the most desirable characteristics of the applicator, resulting in design consolidation into a refined concept applicator. The final, optimised applicator prototype was validated during user research in Malawi. This human-centred design approach was also used to iteratively develop an information leaflet for the device. During these user studies, other preferred aspects of a contraceptive delivery system were also evaluated, such as anatomical site of application, duration of action, and return to fertility. Conclusions: A new microneedle-based contraceptive delivery system was iteratively developed using a human-centred design approach and was favourably received by potential stakeholders. The product is now being refined for testing in pre-clinical studies.

Using gold nanoparticles for enhanced intradermal delivery of poorly soluble auto-antigenic peptides.

Ultra-small 1-2 nm gold nanoparticles (NP) were conjugated with a poorly-soluble peptide auto-antigen, associated with type 1 diabetes, to modify the peptide pharmacokinetics, following its intradermal delivery. Peptide distribution was characterized, in vivo, after delivery using either conventional intradermal injection or a hollow microneedle device. The poorly-soluble peptide was effectively presented in distant lymph nodes (LN), spleen and draining LN when conjugated to the nanoparticles, whereas peptide alone was only presented in the draining LN. By contrast, nanoparticle conjugation to a highly-soluble peptide did not enhance in vivo distribution. Transfer of both free peptide and peptide-NPs from the skin to LN was reduced in mice lacking lymphoid homing receptor CCR7, suggesting that both are actively transported by migrating dendritic cells to LN. Collectively, these data demonstrate that intradermally administered ultra-small gold nanoparticles can widen the distribution of poorly-soluble auto-antigenic peptides to multiple lymphoid organs, thus enhancing their use as potential therapeutics.

Targeting proinsulin to local immune cells using an intradermal microneedle delivery system; a potential antigen-specific immunotherapy for type 1 diabetes.

Antigen-specific immunotherapy (ASI) has been proposed as an alternative treatment strategy for type 1 diabetes (T1D). ASI aims to induce a regulatory, rather than stimulatory, immune response in order to reduce, or prevent, autoimmune mediated ß-cell destruction, thus preserving endogenous insulin production. The abundance of immunocompetent antigen presenting cells (APCs) within the skin makes this organ an attractive target for immunotherapies. Microneedles (MNs) have been proposed as a suitable drug delivery system to facilitate intradermal delivery of autoantigens in a minimally invasive manner. However, studies to date have employed single peptide autoantigens, which would restrict ASI to patients expressing specific Human Leukocyte Antigen (HLA) molecules, thus stratifying the patient population. This study aims to develop, for the first time, an intradermal MN delivery system to target proinsulin, a large multi-epitope protein capable of inducing tolerance in a heterogeneous (in terms of HLA status) population of T1D patients, to the immunocompetent cells of the skin. An optimized three component coating formulation containing proinsulin, a diluent and a surfactant, facilitated uniform and reproducible coating of >30 µg of the active pharmaceutical ingredient on a stainless steel MN array consisting of thirty 500 µm projections. When applied to a murine model these proinsulin-coated MNs efficiently punctured the skin and after a limited insertion time (150 s) a significant proportion of the therapeutic payload (86%) was reproducibly delivered into the local tissue. Localized delivery of proinsulin in non-obese diabetic (NOD) mice using the coated MN system stimulated significantly greater proliferation of adoptively transferred antigen-specific CD8+ T cells in the skin draining lymph nodes compared to a conventional intradermal injection. This provides evidence of targeted delivery of the multi-epitope proinsulin antigen to skin-resident APCs, in vivo, in a form that enables antigen presentation to antigen-specific T cells in the local lymph nodes. The development of an innovative coated MN system for highly targeted and reproducible delivery of proinsulin to local immune cells warrants further evaluation to determine translation to a tolerogenic clinical outcome.

Skin vaccination using microneedles coated with a plasmid DNA cocktail encoding nucleosomal histones of Leishmania spp.

Vaccine delivery using microneedles (MNs) represents a safe, easily disposable and painless alternative to traditional needle immunizations. The MN delivery of DNA vaccines to the dermis may result in a superior immune response and/or an equivalent immune response at a lower vaccine dose (dose-sparing). This could be of special interest for immunization programs against neglected tropical diseases such as leishmaniasis. In this work, we loaded a MN device with 60µg of a plasmid DNA cocktail encoding the Leishmania infantum nucleosomal histones H2A, H2B, H3 and H4 and compared its immunogenicity and protective capacity against conventional s.c. or i.d. injection of the plasmid. Mice immunized with MNs showed increased ratios of IFN-γ/IL-10, IFN-γ/IL-13, IFN-γ/IL-4, and IFN-γ/TGF-ß in the spleens and lymph nodes compared with mice immunized by s.c. and i.d. routes. Furthermore, CCXCL9, CXCL10 and CCL2 levels were also higher. These data suggest that the nucleic acid immunization using MNs produced a better bias towards a Th1 response. However, none of the immunizations strategies were able to control Leishmania major infection in BALB/c mice, as illustrated by an increase in lesion size and parasite burden.

Accelerating Topical Anaesthesia Using Microneedles.

BACKGROUND/AIMS: Topical anaesthetics reduce pain during venous access procedures in children. However, clinical use is hindered by a significant anaesthetic onset time. Restricted diffusion of the topical anaesthetic through the stratum corneum barrier is the principal reason for the delayed onset. Microneedles can painlessly pierce the skin. This study evaluated microneedle pre-treatment of ex vivo human skin as a means to increase the rate of tetracaine permeation, in order to accelerate the onset of anaesthesia. METHODS: Franz-type diffusion cells were used to determine permeation of a commercial tetracaine formulation, Ametop gel, through human skin epidermis. Microneedle-assisted permeation was compared to untreated epidermis. Upon completion of the permeation studies, the epidermal membranes were visually characterised. RESULTS: At 30 min, 5.43 µg/cm2 of tetracaine had permeated through the untreated membrane compared to 12.13 µg/cm2 through the microneedle-treated membrane. Insertion of a hypodermic needle created a large single channel in the epidermis (approx. 4,250 µm2) whilst the punctured surface area following microneedle treatments was estimated to be 75,000 µm2. CONCLUSION: Pre-treatment of skin with microneedles significantly enhances the permeation of tetracaine. Microneedles have the potential to more than halve the onset time for anaesthesia when applying Ametop gel.

Formulation of hydrophobic peptides for skin delivery via coated microneedles.

Microneedles (MNs) have been investigated as a minimally-invasive delivery technology for a range of active pharmaceutical ingredients (APIs). Various formulations and methods for coating the surface of MNs with therapeutics have been proposed and exemplified, predominantly for hydrophilic drugs and particulates. The development of effective MN delivery formulations for hydrophobic drugs is more challenging with dosing restrictions and the use of organic solvents impacting on both the bioactivity and the kinetics of drug release. In this study we propose a novel formulation that is suitable for MN coating of hydrophobic auto-antigen peptides currently being investigated for antigen specific immunotherapy (ASI) of type 1 diabetes. The formulation, comprising three co-solvents (water, 2-methyl-2-butanol and acetic acid) and polyvinylalcohol 2000 (PVA2000) can dissolve both hydrophilic and hydrophobic peptide auto-antigens at relatively high, and clinically relevant, concentrations (25mg/ml or 12.5mg/ml). The drug:excipient ratio is restricted to 10:1 w/w to maximise dose whilst ensuring that the dry-coated payload does not significantly impact on MN skin penetration performance. The coating formulation and process does not adversely affect the biological activity of the peptide. The delivery efficiency of the coated peptide into skin is influenced by a number of parameters. Electropolishing the metal MN surface increases delivery efficiency from 2.0±1.0% to 59.9±6.7%. An increased mass of peptide formulation per needle, from 0.37µg to 2µg peptide dose, resulted in a thicker coating and a 20% reduction in the efficiency of skin delivery. Other important performance parameters for coated MNs include the role of excipients in assisting dissolution from the MNs, the intrinsic hydrophobicity of the peptide and the species of skin model used in laboratory studies. This study therefore both exemplifies the potential of a novel formulation for coating hydrophobic and hydrophilic peptides onto MN devices and provides new insight into the factors that influence delivery efficiency from coated MNs. Importantly, the results provide guidance for identifying critical attributes of the formulation, coating process and delivery device, that confer reproducible and effective delivery from coated MNs, and thus contribute to the requirements of the regulators appraising these devices.

Evaluating an undergraduate interprofessional education session for medical and pharmacy undergraduates on therapeutics and prescribing: the medical student perspective.

BACKGROUND: The current literature on undergraduate interprofessional education (IPE) for pharmacy and medical students highlights a range of positive outcomes, although to date IPE has focused predominantly on student views and experiences of IPE sessions with these opinions being sought at the end of the sessions. This study aimed to evaluate medical students' experiences of therapeutics and prescribing IPE, with pharmacy students, 1 year following the session. METHODS: Following ethics committee approval, 3rd year medical students at Cardiff University were invited to participate using non-probability sampling. Topic guide development was informed by the literature and research team discussions, including a review of the materials used in the IPE session. Semi-structured one-to-one interviews explored experiences, prior to, during, and after the IPE session. Interviews were audio-recorded, transcribed verbatim, and analyzed thematically. RESULTS: Eighteen medical students were interviewed; 11 were females. Seven themes were identified, namely 1) refinement of pre-session preparation, 2) session value, 3) learning with a pharmacy student, 4) learning about a pharmacist, 5) learning from a pharmacy student, 6) importance and application of what was learnt into practice, and 7) suggestions for change. CONCLUSION: This study provides a valuable insight into medical students' experiences of a therapeutics and prescribing IPE session and emphasizes the value they placed on interaction with pharmacy students. Medical students were able to recall clear learning experiences from the IPE session that had taken place 12 months earlier, which itself is an indicator of the impact of the session on the students. Furthermore, they were able to describe how knowledge and skills learnt had been applied to subsequent learning activities. Those developing IPE sessions should consider the following: clarify professional roles in the session content, incorporate IPE as a series of activities, and use small groups of students to optimize student-student interaction and active learning.

TGFß Induces a SAMHD1-Independent Post-Entry Restriction to HIV-1 Infection of Human Epithelial Langerhans Cells.

Sterile alpha motif (SAM) and histidine-aspartic (HD) domains protein 1 (SAMHD1) was previously identified as a critical post-entry restriction factor to HIV-1 infection in myeloid dendritic cells. Here we show that SAMHD1 is also expressed in epidermis-isolated Langerhans cells (LC), but degradation of SAMHD1 does not rescue HIV-1 or vesicular stomatitis virus G-pseudotyped lentivectors infection in LC. Strikingly, using Langerhans cells model systems (mutz-3-derived LC, monocyte-derived LC [MDLC], and freshly isolated epidermal LC), we characterize previously unreported post-entry restriction activity to HIV-1 in these cells, which acts at HIV-1 reverse transcription, but remains independent of restriction factors SAMHD1 and myxovirus resistance 2 (MX2). We demonstrate that transforming growth factor-ß signaling confers this potent HIV-1 restriction in MDLC during their differentiation and blocking of mothers against decapentaplegic homolog 2 (SMAD2) signaling in MDLC restores cells' infectivity. Interestingly, maturation of MDLC with a toll-like receptor 2 agonist or transforming growth factor-α significantly increases cells' susceptibility to HIV-1 infection, which may explain why HIV-1 acquisition is increased during coinfection with sexually transmitted infections. In conclusion, we report a SAMHD1-independent post-entry restriction in MDLC and LC isolated from epidermis, which inhibits HIV-1 replication. A better understanding of HIV-1 restriction and propagation from LC to CD4(+) T cells may help in the development of new microbicides or vaccines to curb HIV-1 infection at its earliest stages during mucosal transmission.

Microneedle delivery of autoantigen for immunotherapy in type 1 diabetes.

Antigen specific immunotherapy mediated via the sustained generation of regulatory T cells arguably represents the ideal therapeutic approach to preventing beta cell destruction in type 1 diabetes. However, there is a need to enhance the efficacy of this approach to achieve disease modification in man. Previous studies suggest that prolonged expression of self-antigen in skin in a non-inflammatory context is beneficial for tolerance induction. We therefore sought to develop a dry-coated microneedle (MN) delivery system and combine it with topical steroid to minimise local inflammation and promote prolonged antigen presentation in the skin. Here we show that a combination of surface-modified MNs coated with appropriate solvent systems can deliver therapeutically relevant quantities of peptide to mouse and human skin even with hydrophobic peptides. Compared to conventional "wet" intradermal (ID) administration, "dry" peptide delivered via MNs was retained for longer in the skin and whilst topical hydration of the skin with vehicle or steroid accelerated loss of ID-delivered peptide from the skin, MN delivery of peptide was unaffected. Furthermore, MN delivery resulted in enhanced presentation of antigen to T cells in skin draining lymph nodes (LNs) both 3 and 10days after administration. Repeated administration of islet antigen peptide via MN was effective at reducing antigen-specific T cell proliferation in the pancreatic LN, although topical steroid therapy did not enhance this. Taken together, these data show auto-antigenic peptide delivery into skin using coated MNs results in prolonged retention and enhanced antigen presentation compared to conventional ID delivery and this approach may have potential in individuals identified as being at a high risk of developing type 1 diabetes and other autoimmune diseases.

Evaluating the sensitivity, reproducibility and flexibility of a method to test hard shell capsules intended for use in dry powder inhalers.

Pharmaceutical tests for hard shell capsules are designed for orally administered capsules. The use of capsules in dry powder inhalers is widespread and increasing and therefore more appropriate tests are required to ensure quality and determine if these capsules are fit for purpose. This study aims to determine the flexibility, reproducibility and sensitivity of a quantitative method that is designed to evaluate the puncture characteristics of different capsule shell formulations under different climatic conditions. A puncture testing method was used to generate force displacement curves for five capsule formulations that were stored and tested at two different temperatures (5°C and 19°C). Force-displacement puncture profiles were reproducible for individual capsule shell formulations. The methodology was able to discriminate between capsules produced using different primary materials i.e. gelatin versus hypromellose, as well as more minor changes to capsule formulation i.e. different material grades and excipients. Reduced temperature increased the forces required for capsule puncture however further work is required to confirm its significance. Results indicate the method provides a reproducible and sensitive means of evaluating capsule puncture. Future studies should validate the methodology at different test sites, using different operators and with different capsule shell formulations.

Topical steroid therapy induces pro-tolerogenic changes in Langerhans cells in human skin.

We have investigated the efficacy of conditioning skin Langerhans cells (LCs) with agents to promote tolerance and reduce inflammation, with the goal of improving the outcomes of antigen-specific immunotherapy. Topical treatments were assessed ex vivo, using excised human breast skin maintained in organ bath cultures, and in vivo in healthy volunteers by analysing skin biopsies and epidermal blister roof samples. Following topical treatment with a corticosteroid, tumour necrosis factor-α levels were reduced in skin biopsy studies and blister fluid samples. Blister fluid concentrations of monocyte chemoattractant protein-1, macrophage inflammatory proteins -1α and 1ß and interferon-γ inducible protein-10 were also reduced, while preserving levels of interleukin-1α (IL-1α), IL-6, IL-8 and IL-10. Steroid pre-treatment of the skin reduced the ability of LCs to induce proliferation, while supernatants showed an increase in the IL-10/interferon-γ ratio. Phenotypic changes following topical steroid treatment were also observed, including reduced expression of CD83 and CD86 in blister-derived LCs, but preservation of the tolerogenic signalling molecules immunoglobulin-like transcript 3 and programmed death-1. Reduced expression of HLA-DR, CD80 and CD86 were also apparent in LCs derived from excised human skin. Topical therapy with a vitamin D analogue (calcipotriol) and steroid, calcipotriol alone or vitamin A elicited no significant changes in the parameters studied. These experiments suggest that pre-conditioning the skin with topical corticosteroid can modulate LCs by blunting their pro-inflammatory signals and potentially enhancing tolerance. We suggest that such modulation before antigen-specific immunotherapy might provide an inexpensive and safe adjunct to current approaches to treat autoimmune diseases.

An international comparison study of pharmacy students' achievement goals and their relationship to assessment type and scores.

OBJECTIVE: To identify pharmacy students' preferred achievement goals in a multi-national undergraduate population, to investigate achievement goal preferences across comparable degree programs, and to identify relationships between achievement goals, academic performance, and assessment type. METHODS: The Achievement Goal Questionnaire was administered to second year students in 4 universities in Australia, New Zealand, England, and Wales. Academic performance was measured using total scores, multiple-choice questions, and written answers (short essay). RESULTS: Four hundred eighty-six second year students participated. Students showed an overall preference for the mastery-approach goal orientation across all sites. The predicted relationships between goal orientation and multiple-choice questions, and written answers scores, were significant. CONCLUSION: This study is the first of its kind to examine pharmacy students' achievement goals at a multi-national level and to differentiate between assessment type and measures of achievement motivation. Students adopting a mastery-approach goal are more likely to gain high scores in assessments that measure understanding and depth of knowledge.

An international validation study of two achievement goal measures in a pharmacy education context.

BACKGROUND: Achievement goal theory helps us understand what motivates students to participate in educational activities. However, measuring achievement goals in a precise manner is problematic. Elliot and McGregor's Achievement Goal Questionnaire (AGQ) and Elliot and Murayama's revised Achievement Goal Questionnaire (AGQ-R) are widely used to assess students' achievement goals. Both instruments were developed and validated using undergraduate psychology students in the USA. METHODS: In this study, our aims were to first of all, assess the construct validity of both questionnaires using a cohort of Australian pharmacy students and, subsequently, to test the generalizability and replicability of these tools more widely in schools of pharmacy in other English-speaking countries. The AGQ and the AGQ-R were administered during tutorial class time. Confirmatory factor analysis procedures, using AMOS 19 software, were performed to determine model fit. RESULTS: In contrast to the scale developers' findings, confirmatory factor analysis supported a superior model fit for the AGQ compared with the AGQ-R, in all countries under study. CONCLUSION: Validating measures of achievement goal motivation for use in pharmacy education is necessary and has implications for future research. Based on these results, the AGQ will be used to conduct future cross-sectional and longitudinal analyses of the achievement goals of undergraduate pharmacy students from these countries.

The development of a sensitive methodology to characterise hard shell capsule puncture by dry powder inhaler pins.

In order for hard-shell capsules to function effectively as drug reservoirs in dry powder inhalers, the capsule must be punctured with sharpened pins to release the powdered medicament upon inspiration. Capsule performance in this setting is poorly understood. This study aims to develop a methodology to characterise hard shell capsule penetration by needles from commercial dry powder inhalers, to determine whether changes to capsule materials impact on their performance. Two pin types from two commercial dry powder inhalers were mounted in a material-testing machine, equipped with a 500 N load cell. A stainless steel bush was used to secure a capsule directly below the steel pin. Hypromellose (n=10) and gelatin capsules (n=10) were conditioned in 'normal' or low humidity conditions and were subsequently punctured with both types of pin. Each puncture event was recorded on a load-displacement curve. The force required for puncture was 2.82±0.26 N for hypromellose capsules and 4.54±0.26 N for gelatin capsules, stored in normal humidity. Different capsule materials possessed distinguishable signature profiles but repeated force-displacement profiles were highly reproducible i.e. intra-individual variability was minimal. A rapid, robust yet sensitive methodology has therefore been developed that is able to characterise hard shell capsule materials based on the puncture performance.

An anisotropic, hyperelastic model for skin: experimental measurements, finite element modelling and identification of parameters for human and murine skin.

The mechanical characteristics of skin are extremely complex and have not been satisfactorily simulated by conventional engineering models. The ability to predict human skin behaviour and to evaluate changes in the mechanical properties of the tissue would inform engineering design and would prove valuable in a diversity of disciplines, for example the pharmaceutical and cosmetic industries, which currently rely upon experiments performed in animal models. The aim of this study was to develop a predictive anisotropic, hyperelastic constitutive model of human skin and to validate this model using laboratory data. As a corollary, the mechanical characteristics of human and murine skin have been compared. A novel experimental design, using tensile tests on circular skin specimens, and an optimisation procedure were adopted for laboratory experiments to identify the material parameters of the tissue. Uniaxial tensile tests were performed along three load axes on excised murine and human skin samples, using a single set of material parameters for each skin sample. A finite element model was developed using the transversely isotropic, hyperelastic constitutive model of Weiss et al. (1996) and was embedded within a Veronda-Westmann isotropic material matrix, using three fibre families to create anisotropic behaviour. The model was able to represent the nonlinear, anisotropic behaviour of the skin well. Additionally, examination of the optimal material coefficients and the experimental data permitted quantification of the mechanical differences between human and murine skin. Differences between the skin types, most notably the extension of the skin at low load, have highlighted some of the limitations of murine skin as a biomechanical model of the human tissue. The development of accurate, predictive computational models of human tissue, such as skin, to reduce, refine or replace animal models and to inform developments in the medical, engineering and cosmetic fields, is a significant challenge but is highly desirable. Concurrent advances in computer technology and our understanding of human physiology must be utilised to produce more accurate and accessible predictive models, such as the finite element model described in this study.

Gene silencing following siRNA delivery to skin via coated steel microneedles: In vitro and in vivo proof-of-concept.

The development of siRNA-based gene silencing therapies has significant potential for effectively treating debilitating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be efficacious and widely accepted by physicians and patients, therapeutic siRNAs must access the viable skin layers in a stable and functional form, preferably without painful administration. In this study we explore the use of minimally-invasive steel microneedle devices to effectively deliver siRNA into skin. A simple, yet precise microneedle coating method permitted reproducible loading of siRNA onto individual microneedles. Following recovery from the microneedle surface, lamin A/C siRNA retained full activity, as demonstrated by significant reduction in lamin A/C mRNA levels and reduced lamin A/C protein in HaCaT keratinocyte cells. However, lamin A/C siRNA pre-complexed with a commercial lipid-based transfection reagent (siRNA lipoplex) was less functional following microneedle coating. As Accell-modified "self-delivery" siRNA targeted against CD44 also retained functionality after microneedle coating, this form of siRNA was used in subsequent in vivo studies, where gene silencing was determined in a transgenic reporter mouse skin model. Self-delivery siRNA targeting the reporter (luciferase/GFP) gene was coated onto microneedles and delivered to mouse footpad. Quantification of reporter mRNA and intravital imaging of reporter expression in the outer skin layers confirmed functional in vivo gene silencing following microneedle delivery of siRNA. The use of coated metal microneedles represents a new, simple, minimally-invasive, patient-friendly and potentially self-administrable method for the delivery of therapeutic nucleic acids to the skin.

Microneedle delivery of plasmid DNA to living human skin: Formulation coating, skin insertion and gene expression.

Microneedle delivery of nucleic acids, in particular plasmid DNA (pDNA), to the skin represents a potential new approach for the clinical management of genetic skin diseases and cutaneous cancers, and for intracutaneous genetic immunisation. In this study excised human skin explants were used to investigate and optimise key parameters that will determine stable and effective microneedle-facilitated pDNA delivery. These include (i) high dose-loading of pDNA onto microneedle surfaces, (ii) stability and functionality of the coated pDNA, (iii) skin penetration capability of pDNA-coated microneedles, and (iv) efficient gene expression in human skin. Optimisation of a dip-coating method enabled significant increases in the loading capacity, up to 100µg of pDNA per 5-microneedle array. Coated microneedles were able to reproducibly perforate human skin at low (<1N) insertion forces. The physical stability of the coated pDNA was partially compromised on storage, although this was improved through the addition of saccharide excipients without detriment to the biological functionality of pDNA. The pDNA-coated microneedles facilitated reporter gene expression in viable human skin. The efficiency of gene expression from coated microneedles will depend upon suitable DNA loading, efficient and reproducible skin puncture and rapid in situ dissolution of the plasmid at the site of delivery.