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Abstract: Microneedle (MN) devices comprise of micron-sized structures that circumvent biological barriers in a minimally invasive manner. MN research continues to grow and evolve; the technology was recently identified as one of the top ten overall emerging technologies of 2020. There is a growing interest in using such devices in cosmetology and dermatological conditions where the MNs mechanically disrupt the outer skin barrier layer, creating transient pathways that allow the passage of materials to underlying skin layers. This review aims to appraise the application of microneedle technologies in skin science, provide information on potential clinical benefits, as well as indicate possible dermatological conditions that can benefit from this technology, including autoimmune-mediated inflammatory skin diseases, skin aging, hyperpigmentation, and skin tumors. A literature review was carried out to select studies that evaluated the use of microneedles as drug delivery enhancement for dermatologic purposes. MN patches create temporary pathways that allow the passage of material to deeper layers of the skin. Given their demonstrable promise in therapeutic applications, it will be essential for healthcare professionals to engage with these new delivery systems.

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. Graphical abstract Supplementary information The online version contains supplementary material available at 10.1007/s13346-021-01091-5.

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 β-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 heterogenous (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.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (

Poly (methyl methacrylate) (PMMA) bone cement is widely used for anchoring joint arthroplasties. In cement brands approved for these procedures, micron-sized particles (usually barium sulphate, BaSO4) act as the radiopacifier. It has been postulated that these particles act as sites for crack initiation and subsequently cement fatigue. This study investigated whether alternative radiopacifiers, anatase titanium dioxide (TiO2) and yttria-stabilised zirconium dioxide (ZrO2), could improve the in vitro mechanical, fatigue crack propagation and biological properties of polymethyl methacrylate (PMMA) bone cement and whether their coating with a silane could further enhance cement performance. Cement samples containing 0, 5, 10, 15, 20 and 25%w/w TiO2or ZrO2and 10%w/w silane-treated TiO2or ZrO2were prepared and characterised in vitro in terms of radiopacity, compressive and bending strength, bending modulus, fatigue crack propagation, hydroxyapatite forming ability and MC3T3-E1 cell attachment and viability. Cement samples with greater than 10%w/w TiO2and ZrO2had a similar radiopacity to the control 10%w/w BaSO4cement and commercial products. The addition of TiO2and ZrO2to bone cement reduced the bending strength and fracture toughness and increased fatigue crack propagation due to the formation of agglomerations and voids. Silane treating TiO2reversed this effect, enhancing the dispersion and adhesion of particles to the PMMA matrix and resulted in improved mechanical properties and fatigue crack propagation resistance. Silane-treated TiO2cements had increased nucleation of hydroxyapatite and MC3T3-E1 cell attachment in vitro, without significantly compromising cell viability. This research has demonstrated that 10%w/w silane-treated anatase TiO2is a promising alternative radiopacifier for PMMA bone cement offering additional benefits over conventional BaSO4radiopacifiers.

International audience; 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.

The powerful and intriguing idea that drives the emerging technology of microneedles—shrinking the standard needle to a micron scale—has fostered an entire field of microneedle study and subsequent exponential growth in research and product development. Originally enabled by microfabrication tools derived from the microelectronic industry, microneedles are now produced through a number of methods in a variety of forms including solid, coated, dissolvable, and hollow microneedles. They are used to deliver a broad spectrum of molecules, including small molecules, biomolecules, and vaccines, as well as various forms of energy into the skin, eye, and other tissues. Microneedles are also being exploited for use in diagnostics, as well as additional medical, cosmetic, and other applications. This review elucidates the relative roles of different aspects of microneedle technology development, as shown through scientific papers, patents, clinical studies, and internet/social media activity. Considering >1000 papers, 750 patents, and almost 80 clinical trials, we analyze different attributes of microneedles such as usage of microneedles, types of microneedles, testing environment, types of patent claims, and phases of clinical trials, as well as which institutions and people in academia and industry from different locations and in different journals are publishing, patenting, and otherwise studying the potential of microneedles. We conclude that there is robust and growing activity in the field of microneedles; the technology is rapidly developing and being used for novel applications to benefit human health and well-being.

A complex assembly of lipids including fatty acids, cholesterol, and ceramides is vital to the integrity of the mammalian epidermal barrier. The formation of this barrier requires oxidation of the substrate fatty acid, linoleic acid (LA), which is initiated by the enzyme 12R-lipoxygenase (LOX). In the epidermis, unoxidized LA is primarily found in long-chain acylceramides termed esterified omega-hydroxy sphingosine (EOS)/phytosphingosine/hydroxysphingosine (collectively EOx). The precise structure and localization of LOX-oxidized EOx in the human epidermis is unknown, as is their regulation in diseases such as psoriasis, one of the most common inflammatory diseases affecting the skin. Here, using precursor LC/MS/MS, we characterized multiple intermediates of EOx, including 9-HODE, 9,10-epoxy-13-HOME, and 9,10,13-TriHOME, in healthy human epidermis likely to be formed via the epidermal LOX pathways. The top layers of the skin contained more LA, 9-HODE, and 9,10,13-TriHOME EOSs, whereas 9,10-epoxy-13-HOME EOS was more prevalent deeper in the stratum corneum. In psoriatic lesions, levels of native EOx and free HODEs and HOMEs were significantly elevated, whereas oxidized species were generally reduced. A transcriptional network analysis of human psoriatic lesions identified significantly elevated expression of the entire biosynthetic/metabolic pathway for oxygenated ceramides, suggesting a regulatory function for EOx lipids in reconstituting epidermal integrity. The role of these new lipids in progression or resolution of psoriasis is currently unknown. We also discovered the central coordinated role of the zinc finger protein transcription factor, ZIC1, in driving the phenotype of this disease. In summary, long-chain oxygenated ceramide metabolism is dysregulated at the lipidomic level in psoriasis, likely driven by the transcriptional differences also observed, and we identified ZIC1 as a potential regulatory target for future therapeutic interventions.

A novel production process flow is presented here for the manufacture of hollow silicon microneedles using deep reactive-ion etching (DRIE) technology. The patent-pending three-step process flow has been developed to produce multiple arrays of sharp-tipped, hollow microneedles, which facilitate easy insertion and controlled fluid injection into excised skin samples. A bevelled tip and vertical sidewalls for the microneedle have been achieved with good uniformity, despite >45% open etch area. Processing steps and etch challenges are discussed, and preliminary skin testing results are presented, showing effective needle insertion and delivery of fluorescent dye into ex vivo skin from human breast tissue.

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 60 μg 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-β 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.

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 (25 mg/ml or 12.5 mg/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 0.37 μg to 2 μg 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.

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.

Background/Aims: Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy. Methods: Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab). Results: The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab. Conclusion: Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent.

Infantile haemangiomas are the most common tumour of infancy. Whilst the majority are left untreated to involute spontaneously, residual skin changes commonly occur, particularly in superficial haemangiomas. The current first-line treatment for problematic lesions is oral propranolol; however due to the risk of systemic adverse effects, the use of off-label topical preparations has recently been investigated. Our systematic review was conducted in accordance with PRISMA guidelines. Four databases were searched to identify original articles evaluating the use of topical propranolol as the primary therapy for infantile haemangiomas. Twelve articles with a total of 597 patients and 632 haemangiomas were included. Three topical propranolol preparations were used, creams, ointments and gels and were all prepared by local pharmaceutical laboratories. The concentration of propranolol ranged from 0.5% to 5%. Treatment duration ranged from two weeks to 16.5 months. Overall, 90% of lesions improved following the initiation of topical propranolol. A good or excellent response, defined as a reduction in the size of at least 50%, was seen in 59% of lesions. Earlier initiation of treatment (less than 3 months of age) was associated with improved outcomes. No systemic adverse effects were reported. Minor local reactions were seen in 1.3% of patients. Topical propranolol is safer than oral propranolol, though may be less effective. Topical propranolol may be more suitable for patients with small, superficial haemangiomas at risk of cosmetic sequelae, where the cosmetic or symptomatic impact does not warrant oral propranolol treatment.

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.

Background Translation of cell therapies to the clinic is accompanied by numerous challenges, including controlled and targeted delivery of the cells to their site of action, without compromising cell viability and functionality. Objectives To explore the use of hollow microneedle devices (to date only used for the delivery of drugs and vaccines into the skin and for the extraction of biological fluids) to deliver cells into skin in a minimally-invasive, user-friendly and targeted fashion. Methods Melanocyte, keratinocyte and mixed epidermal cell suspensions were passed through various types of microneedles and subsequently delivered into the skin. Results Cell viability and functionality is maintained after injection through hollow microneedles with a bore size ≥75 μm. Healthy cells are delivered into skin at clinically relevant depths. Conclusions Hollow microneedles provide an innovative and minimally-invasive method for delivering functional cells into the skin. Microneedle cell delivery represents a potential new treatment option for cell therapy approaches including skin re-pigmentation, wound repair, scar and burn remodelling, immune therapies, and cancer vaccines.

International audience; Microneedle devices have been proposed as aminimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600 (R) (MicronJet hereafter) device was used to deliver reporter plasmids (pCMV beta and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50 mu L bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMV beta reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery. (C) 2017 Elsevier B.V. All rights reserved.

Topical delivery of gabapentin is desirable to treat peripheral neuropathic pain conditions whilst avoiding systemic side effects. To date, reports of topical gabapentin delivery in vitro have been variable and dependent on the skin model employed, primarily involving rodent and porcine models. In this study a variety of topical gabapentin formulations were investigated, including Carbopol® hydrogels containing various permeation enhancers, and a range of proprietary bases including a compounded Lipoderm® formulation; furthermore microneedle facilitated delivery was used as a positive control. Critically, permeation of gabapentin across a human epidermal membrane in vitro was assessed using Franz-type diffusion cells. Subsequently this data was contextualised within the wider scope of the literature. Although reports of topical gabapentin delivery have been shown to vary, largely dependent upon the skin model used, this study demonstrated that 6% (w/w) gabapentin 0.75% (w/w) Carbopol® hydrogels containing 5% (w/w) DMSO or 70% (w/w) ethanol and a compounded 10% (w/w) gabapentin Lipoderm® formulation were able to facilitate permeation of the molecule across human skin. Further pre-clinical and clinical studies are required to investigate the topical delivery performance and pharmacodynamic actions of prospective formulations.

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.

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.

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-β 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 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 immunization. In this study excised human skin explants were used to investigate and optimize 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. Optimization of a dip-coating method enabled significant increases in the loading capacity, up to 100 micrograms of pDNA per 5-microneedle array. Coated microneedles were able to reproducibly perforate human skin at low (

PURPOSE: To gather sub-surface in situ images of microneedle-treated human skin, in vivo, using optical coherence tomography (OCT). This is the first study to utilise OCT to investigate the architectural changes that are induced in skin following microneedle application. METHODS: Steel, silicon and polymer microneedle devices, with different microneedle arrangements and morphologies, were applied to two anatomical sites in human volunteers following appropriate ethical approval. A state-of-the-art ultrahigh resolution OCT imaging system operating at 800 nm wavelength and

Aerosolised DNA administration could potentially advance the treatment of inheritable lung diseases, lung malignancies and provide genetic immunisation against infection. Jet nebulisation, the current standard for introducing DNA formulations into the lung, is inherently inefficient. Pressurised metered dose inhalers (pMDIs) offer a potentially more efficacious and convenient alternative, especially for repeat administration. We aim to modify a novel low-energy nanotechnology process to prepare surfactant-coated pDNA nanoparticles for pulmonary gene delivery via a pMDI. Water-in-oil microemulsions containing green fluorescent protein reporter plasmid were snap-frozen and lyophilised. Lyophilised pDNA, in some cases following a surfactant wash, was incorporated into pMDIs with hydrofluoroalkane 134a (HFA134a) propellant and ethanol as cosolvent. To assess biological functionality, A549 human lung epithelial cells were exposed to aerosolised pDNA particles in the presence of dioleoyl-trimethylammonium propane (DOTAP). Transfection studies demonstrated that pDNA biological functionality was maintained following aerosolisation. In vitro toxicity assays (MTT) showed no significant cell viability loss following aerosolised pDNA treatment. We have demonstrated that pDNA particles can be incorporated into an HFA134a formulation and aerosolised using a standard valve and actuator. Particles prepared by this novel process have potential for stable and efficient delivery of pDNA to the lower respiratory tract via standard pMDI technology.

The lung is an appropriate present and future target for gene therapy approaches designed to treat inherited monogenic diseases, eradicate bronchial tumours, transfer pharmacologically active products to the general circulation, express enzymes to catabolise toxins, manage pulmonary hypertension and lung injury and vaccinate against infection. Despite 35 years of gene therapy research and some significant milestones in molecular biology, the clinical potential of gene therapy has yet to be realised. In pulmonary gene therapy the nucleic acid cargo needs to be delivered to cells in the target region of the lung, and even in cases when these targets are well defined this is severely limited by the pulmonary architecture, clearance mechanisms, immune activation, the presence of respiratory mucus and the availability of a truly representative biological model. The challenge from a drug delivery perspective is to consider the suitability of conventional nebulisers and inhalers for delivering DNA to the lung and design and apply integrated formulation and device solutions specific to nucleic acid delivery.

In this study, the amino acids arginine, aspartic acid, leucine, phenylalanine and threonine were investigated as ‘dispersibility enhancers’ in spray-dried powders for inhalation. Parameters such as spray-dried yield, tapped density, and Carr's Index were not predictive of aerosolisation performance. In addition, whilst the majority of amino acid-modified powders displayed suitable particle size distribution for pulmonary administration and potentially favourable low moisture content, in vitro particle deposition was only enhanced for the leucine-modified powder. In summary, leucine can be used to enhance the dispersibility and aerosolisation properties of spray-dried powders for pulmonary drug delivery.

Background Non-viral gene delivery vectors are multi-component systems reflecting various functionalities required for effective cell transfection, including DNA condensation, promotion of cell membrane interactions and provision for subcellular targeting through endosomal escape and/or nuclear delivery. Elements mediating these functions will clearly display inter-dependency. In this study we sought to explore the relationship within non-viral vectors of condensation and nuclear localisation. Methods Binary, tertiary and quaternary vectors were prepared with combinations of pDNA, DOTAP lipid, the polycation peptide protamine and either SV40 nuclear localisation sequence peptide (‘SV40 NLS’) or a one amino acid substituted mutant of SV40 NLS (‘mutant sequence’). The efficiency of pDNA condensation was determined by gel electrophoresis and quantitative fluorescence spectroscopy. Transfection efficiency was examined in mammalian cells in vitro using standard methods, by electroporation to bypass the plasma membrane barrier and in cells arrested in G0/G1 cell cycle phase to examine the effect of cell division and nuclear membrane disruption. Results Small NLS peptide sequences, despite possessing a significant proportion of basic amino acids, display minimal pDNA-condensing ability when compared to larger polycations such as protamine. In standard in vitro cell adherent transfection studies the predominant elements affording enhanced gene expression were effective pDNA condensation and lipid enhancement of cell membrane interactions. These features conversely hinder efficient gene expression in cells that have undergone electroporation. The benefit of SV40 NLS was only apparent when used in non-dividing cell populations. Conclusions Whilst effective levels of non-viral-mediated gene expression generally rely on efficient condensation of pDNA and enhanced interactions with cellular membranes, non-covalently associated NLS within a multi-component non-viral gene vector appears to contribute benefit in sustaining gene expression in non-dividing cells. Copyright © 2007 John Wiley & Sons, Ltd.

Delivery of vaccine formulations into the dermis using antigen-coated microneedle patches is a promising and safe approach because of efficient antigen delivery and safety. We evaluated an intradermal vaccine using HIV-1 p24 Gag peptide-conjugated polypropylene sulfide nanoparticles to induce immunity against HIV-1. This peptide-conjugated polypropylene sulfide nanoparticle formulation did not accelerate the maturation of blood- or skin-derived subsets of dendritic cells, either generated in vitro or purified ex vivo, despite efficient uptake in the absence of adjuvant. Moreover, dendritic cell-mediated capture of particulate antigen in this form induced potent HIV-1–specific CD4 + T-cell responses, as well as B-cell–mediated antibody production. Nanoparticle-based intradermal antigen delivery may therefore provide a new option in the global effort to develop an effective vaccine against HIV-1.

Early events during dengue virus (DENV) infection remain poorly understood. In this issue, Schaeffer and colleagues employ ex vivo human skin cells to investigate viral infection. They show that skin-resident immune cells are infected by DENV and that their infectability is increased in the inflammatory skin conditions (especially those in which IL-4 is released) that accompany the mosquito bites transmitting the virus.

Spray-drying is an effective process for preparing micron-dimensioned particles for pulmonary delivery. Previously, we have demonstrated enhanced dispersibility and fine particle fraction of spray-dried nonviral gene delivery formulations using amino acids or absorption enhancers as dispersibility-enhancing excipients. In this study, we investigate the use of the cationic polymer chitosan as a readily available and biocompatible dispersibility enhancer.Lactose-lipid:polycation:pDNA (LPD) powders were prepared by spray-drying and post-mixed with chitosan or spray-dried chitosan. In addition, the water-soluble chitosan derivative, trimethyl chitosan, was added to the lactose-LPD formulation before spray-drying.Spray-dried chitosan particles, displaying an irregular surface morphology and diameter of less than 2 microm, readily adsorbed to lactose-LPD particles following mixing. In contrast with the smooth spherical surface of lactose-LPD particles, spray-dried trimethyl chitosan-lactose-LPD particles demonstrated increased surface roughness and a unimodal particle size distribution (mean diameter 3.4 microm), compared with the multimodal distribution for unmodified lactose-LPD powders (mean diameter 23.7 microm). The emitted dose and in vitro deposition of chitosan-modified powders was significantly greater than that of unmodified powders. Moreover, the inclusion of chitosan mediated an enhanced level of reporter gene expression.In summary, chitosan enhances the dispersibility and in vitro pulmonary deposition performance of spray-dried powders.

James Birchall is a Reader in Pharmaceutics at the School of Pharmacy and Pharmaceutical Sciences, Cardiff University. Dr Birchall graduated from Bath University in 1993 and completed his PhD researching drug and DNA delivery systems in 1998 (Cardiff University). His current research is focussed on the use of microneedles for cutaneous drug, vaccine and gene delivery, and the exploitation of novel dry powder and metered dose inhaler formulations for pulmonary drug and gene delivery. Dr Birchall has acted as Associate Editor of Critical Reviews in Therapeutic Drug Carrier Systems, is a member of the Editorial Board of Expert Review of Medical Devices and is an Honorary Senior Lecturer and Research Pharmacist with the National Health Service. His research is funded by the NIH, Royal Society, British Skin Foundation and 3M Healthcare Ltd. Dr Birchall speaks to Laura Harvey (Commissioning Editor) on some of the challenges and future directions of transdermal delivery research into gene delivery and microneedle technology today.

Delivery of vaccines into the skin provides many advantages over traditional parenteral vaccination and is a promising approach due to the abundance of antigen presenting cells (APC) residing in the skin including Langerhans cells (LC) and dermal dendritic cells (DDC). However, the main obstacle for transcutaneous immunization (TCI) is the effective delivery of the vaccine through the stratum corneum (SC) barrier to the APC in the deeper skin layers. This study therefore utilized microneedles (MN) and a lipid-based colloidal delivery system (cubosomes) as a synergistic approach for the delivery of vaccines to APC in the skin. The process of vaccine uptake and recruitment by specific types of skin APC was investigated in real-time over 4 hours in B6.Cg-Tg (Itgax-EYFP) 1 Mnz/J mice by two-photon microscopy. Incorporation of the vaccine into a particulate delivery system and the use of MN preferentially increased vaccine antigen uptake by a highly motile subpopulation of skin APC known as CD207+ DC. No uptake of antigen or any response to immunisation by LC could be detected.

The lower respiratory tract provides a number of disease targets for gene therapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a significant challenge to the maintenance of the physical stability and biological activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebulised lipid-DNA complexes. Complexes prepared from the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) and plasmid DNA (pDNA) at mass (w/w) ratios of 12:1, 6:1 and 3:1, and complexes prepared from DOTAP, the polycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebulised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservoir (pre- and post-nebulisation) and from the aerosol mist were collected and investigated for changes, including: particle diameter, retention of in-vitro transfection activity and the relative concentration and nature of the complexed pDNA remaining after the nebulisation procedure. The process of jet nebulisation adversely affected the physical stability of lipid:pDNA complexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145+/-3 nm pre-nebulisation vs. 142+/-2 nm aerosol mist) and preserve significant pDNA integrity in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD complexes were smaller (102+/-1 nm pre-nebulisation vs. 113+/-2 nm aerosol mist) with considerably greater retention of pDNA integrity in the reservoir (90% of pre-nebulisation pDNA band intensity). In contrast the concentration of pDNA in the aerosol mist for both the 12:1 w/w DOTAP:pDNA and LPD complexes were significantly reduced (10 and 12% of pre-nebulised values, respectively). Despite reduced pDNA concentration the transfection (% cells transfected) mediated by aerosol mist for the nebulised complexes was comparatively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulised complex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physical stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as protamine. The incorporation of the peptide precludes the use of potentially toxic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting.

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.

Cationic liposomes spontaneously interact with negatively charged plasmid DNA to form a transfection competent complex capable of promoting the expression of a therapeutic gene. This work aims to improve the understanding of the poorly defined mechanisms and structural rearrangements associated with the lipid–DNA interaction. Specifically, dimethyl dioctadecylammonium bromide (DDAB):dioleoyl phosphatidylethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) liposomes were mixed with a reporter plasmid (pADβ or pCMVβ) to form lipid–DNA complexes. The size and charge characteristics of the complexes as determined by photon correlation spectroscopy and microelectrophoresis were found to be dependent on the lipid:DNA ratio, with both DDAB:DOPE–DNA and DOTAP–DNA complexes aggregating at around neutral zeta potential. Negative stain transmission electron microscopy demonstrated at least three distinct complex structures being formed at the same DOTAP:DNA ratio. We postulate that two of these aggregates are structural moieties involved in the formation of the efficient transfection particle. Gel electrophoresis was used to determine the efficiency and extent of lipid–DNA complex formation. Results showed that only DOTAP liposomes were capable of preventing ethidium bromide intercalation with DNA and protecting the enclosed plasmid from nuclease digestion. When a range of lipid–DNA complexes were transfected into in vitro cell lines, the efficiency of reporter gene (β-galactosidase) expression was found to depend on the type of liposome used in the complex, the ratio of lipid:DNA and the transfected cell line. Our results challenge the requirement for DOPE to be included in the formulation of cationic lipid vectors, especially in the case of DOTAP containing liposomes.

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.

Transcutaneous (TCI) immunization is a novel vaccination approach that provides many advantages over traditional parenteral vaccination. However, a major barrier to TCI is mediating penetration of vaccine antigens through the stratum corneum (SC) to the deeper tissue layers. Many approaches have been investigated for enhancing drug penetration into the skin including microneedles (MNs) to transiently breach the SC barrier and incorporation of vaccines into penetration-enhancing delivery systems. This study utilized MNs and a lipid-based colloidal delivery system (cubosomes) as a synergistic approach for the delivery of vaccines through the skin. The penetration and permeation of the peptide antigen into and through skin were analysed quantitatively and qualitatively using techniques including Optical Coherence Tomography and two-photon microscopy. It was found that while the use of MNs increased the permeation of an aqueous peptide mixture through skin, cubosome-formulated peptide and cubosomes were retained in the skin. Therefore, it is proposed that a combined approach using MNs and cubosomes will be an efficient system for the local delivery of antigen to immunocompetent cells in the skin.

Inhaled heparin appears to provide benefit in the management of airway hyper-reactivity and inflammation. The pharmacodynamics of inhaled heparin are however transient. Providing sustained heparin concentrations in the respiratory tract should provide for an extended duration of action. We examined the in-vivo efficacy of a nebulised controlled-release microparticle formulation of heparin in modifying antigen-induced airway hyper-reactivity (AHR) and lung inflammation. Heparin-loaded biodegradable poly (d,l-lactide-co-glycolide) microparticles were prepared by spray-drying. Aerosol properties for both nebulised heparin solution and heparin microparticles displayed characteristics consistent with heparin delivery to the respiratory tract. In vitro release assays showed heparin to be released from the microparticles over 8–12 h and for the heparin to remain functional. Temporal pharmacodynamic responses were studied in an ovalbumin-sensitised in vivo model exhibiting AHR and airway inflammation. Despite a reduced total dose of heparin deposited in the airways following nebulisation with heparin microparticles, this treatment led to a more sustained inhibitory effect upon AHR and airway inflammation than equivalent doses of nebulised heparin solution. The work supports extended-release heparin as an inhalation dosing strategy in experimental therapeutic applications aimed at improving the pharmacodynamics of heparin in the treatment of AHR and lung inflammation.

The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

Microneedle devices are a promising minimally invasive means of delivering drugs/vaccines across or into the skin. However, there is currently a diversity of microneedle designs and application methods that have, primarily, been intuitively developed by the research community. To enable the rational design of optimised microneedle devices, a greater understanding of human skin biomechanics under small deformations is required. This study aims to develop a representative stratified model of human skin, informed by in vivo data. A multilayer finite element model incorporating the epidermis, dermis and hypodermis was established. This was correlated with a series of in-vivo indentation measurements, and the Ogden material coefficients were optimised using a material parameter extraction algorithm. The finite element simulation was subsequently used to model microneedle application to human skin before penetration and was validated by comparing these predictions with the in-vivo measurements. Our model has provided an excellent tool to predict micron-scale human skin deformation in vivo and is currently being used to inform optimised microneedle designs.

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.

There is a significant gap in our fundamental understanding of early morphological and migratory changes in human Langerhans cells (LCs) in response to vaccine stimulation. As the vast majority of LCs studies are conducted in small animal models, substantial interspecies variation in skin architecture and immunity must be considered when extrapolating the results to humans. This study aims to determine whether excised human skin, maintained viable in organ culture, provides a useful human model for measuring and understanding early immune response to intradermally delivered vaccine candidates. Excised human breast skin was maintained viable in air-liquid-interface organ culture. This model was used for the first time to show morphological changes in human LCs stimulated with influenza virus-like particle (VLP) vaccines delivered via intradermal injection. Immunohistochemistry of epidermal sheets and skin sections showed that LCs in VLP treated skin lost their typical dendritic morphology. The cells were more dispersed throughout the epidermis, often in close proximity to the basement membrane, and appeared vertically elongated. Our data provides for increased understanding of the complex morphological, spatial and temporal changes that occur to permit LC migration through the densely packed keratinocytes of the epidermis following exposure to vaccine. Significantly, the data not only supports previous animal data but also provides new and essential evidence of host response to this vaccination strategy in the real human skin environment.

A simple method suitable for self-administration of vaccine would improve mass immunization, particularly during a pandemic outbreak. Influenza virus-like particles (VLPs) have been suggested as promising vaccine candidates against potentially pandemic influenza viruses, as they confer long-lasting immunity but are not infectious. We investigated the immunogenicity and protective efficacy of influenza H5 VLPs containing the hemagglutinin (HA) of A/Vietnam/1203/04 (H5N1) virus delivered into the skin of mice using metal microneedle patches and also studied the response of Langerhans cells in a human skin model. Prime-boost microneedle vaccinations with H5 VLPs elicited higher levels of virus-specific IgG1 and IgG2a antibodies, virus-specific antibody-secreting cells, and cytokine-producing cells up to 8 months after vaccination compared to the same antigen delivered intramuscularly. Both prime-boost microneedle and intramuscular vaccinations with H5 VLPs induced similar hemagglutination inhibition titers and conferred 100% protection against lethal challenge with the wild-type A/Vietnam/1203/04 virus 16 weeks after vaccination. Microneedle delivery of influenza VLPs to viable human skin using microneedles induced the movement of CD207+Langerhans cells toward the basement membrane. Microneedle vaccination in the skin with H5 VLPs represents a promising approach for a self-administered vaccine against viruses with pandemic potential.

Purpose: Microneedles are being developed to administer vaccines and therapeutics to and through skin. To date there has been no qualitative or quantitative research into public and health professionals’ views on this new delivery technique. Methods: Focus groups (n=7) comprising public and healthcare professionals were convened to capture the perceived advantages for, and concerns with, microneedles. Discussions were audio-recorded and transcribed. Transcript analysis identified themes that were explored using a questionnaire identifying consensus or otherwise. Results: Participants identified many potential benefits of the microneedle delivery system, including reduced pain, tissue damage and risk of transmitting infections compared with conventional injections, as well as potential for self-administration (subject to safeguards such as an indicator to confirm dose delivery). Delayed onset, cost, accurate and reliable dosing and the potential for misuse were raised as concerns. A range of potential clinical applications was suggested. The public (100%) and professional (74%) participants were positive overall about microneedle technology. Conclusions: This exploratory research study captured the views of the eventual end-users of microneedle technology. Microneedle researchers should now reflect on their research and development activities in the context of stakeholder engagement in order to facilitate the transfer of this new technology ‘from bench to bedside.’

Injections using hypodermic needles cause pain, discomfort, localised trauma and apprehension. Additionally, careful use and disposal of needles is required to avoid transmission of blood-borne pathogens. As an alternative, microneedles can facilitate drug delivery without significantly impacting on pain receptors or blood vessels that reside beneath the skin outer layers. In this study we aim to determine the pain and sensory response to the application of wet-etch silicon microneedles, when used in such a way as to reliably penetrate skin, and provide a preliminary indication of how skin responds to microneedle injury with time. Twelve subjects received single-blinded insertions of a 25-G hypodermic needle and two microneedle arrays (36 needles of 180 and 280 mum height). The optimal method for microneedle application was determined in a pilot study. Pain intensity was scored using a visual analogue scale (VAS) and sensory perception determined using an adapted McGill Pain Questionnaire Short Form. Skin penetration was determined by external staining and measurement of trans-epidermal water loss (TEWL). Mean VAS scores, verbal descriptions and questionnaire responses showed that the 180 and 280 mum microneedles caused significantly less pain and discomforting sensation in participants than the hypodermic needle. Methylene blue staining and TEWL analysis confirmed that microchannels were formed in the skin following microneedle application. Evidence of microchannel repair and resealing was apparent at 8-24 h post-application. In summary, this study shows that pyramidal wet-etch microneedles can penetrate human skin with minimal pain and sensory discomfort, creating transient pathways for potential drug, vaccine and DNA delivery.

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 disrupt the stratum corneum barrier layer of skin creating transient pathways for the enhanced permeation of therapeutics into viable skin regions without stimulating pain receptors or causing vascular damage. The cutaneous delivery of nucleic acids has a number of therapeutic applications; most notably genetic vaccination. Unfortunately non-viral gene expression in skin is generally inefficient and transient. This study investigated the potential for improved delivery of plasmid DNA (pDNA) in skin by combining the microneedle delivery system with sustained release pDNA hydrogel formulations.Microneedles were fabricated by wet etching silicon in potassium hydroxide. Hydrogels based on Carbopol polymers and thermosensitive PLGA-PEG-PLGA triblock copolymers were prepared. Freshly excised human skin was used to characterise microneedle penetration (microscopy and skin water loss), gel residence in microchannels, pDNA diffusion and reporter gene (beta-galactosidase) expression.Following microneedle treatment, channels of approximately 150-200 microm depth increased trans-epidermal water loss in skin. pDNA hydrogels were shown to harbour and gradually release pDNA. Following microneedle-assisted delivery of pDNA hydrogels to human skin expression of the pCMVbeta reporter gene was demonstrated in the viable epidermis proximal to microchannels.pDNA hydrogels can be successfully targeted to the viable epidermis to potentially provide sustained gene expression therein.

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.