Gene delivery to human skin using microneedle arrays

Cutaneous delivery of macromolecules is significantly impeded by the inherent barrier properties of the stratum corneum (SC). Within the last decade sophisticated engineering techniques have enabled the manufacture of microneedle arrays. These are innovative devices consisting of micron-sized needles which when inserted into the skin create physical conduits across the SC but do not impinge upon underlying nerve fibres or blood vessels. This study assessed the ability of microfabricated silicon microneedle arrays to penetrate the SC of ex vivo human skin for the localised delivery and subsequent expression of non-viral gene therapy formulations. Cutaneous gene therapy may represent a new method for the treatment of, or vaccination against, a range of candidate diseases. Microneedle arrays of variant geometries and morphologies, created using dry- and wet-etch microfabrication methods, were characterised by scanning electron microscopy. The potential of these devices for the cutaneous delivery of gene therapy formulations was initially demonstrated by permeation of a size and surface representative fluorescent nanoparticle across microneedle treated human epidermal membrane and observation of these nanoparticles in micron-sized conduits created in excised human skin. The ability to express exogenous genes within ex vivo human skin was subsequently proven by intradermal injection of the pCMVp reporter plasmid. However, a non-viral gene therapy vector failed to enhance cutaneous transfection. Cutaneous plasmid DNA delivery using the microneedle device facilitated effective, if somewhat limited and irreproducible, transfection of epidermal cells proximal to microchannels created in the skin. These investigations confirmed the ability of a silicon microneedle device to deliver macromolecular formulations, including plasmid DNA, to the viable epidermis and have demonstrated exogenous gene expression within human skin. However, limited and unpredictable gene expression following microneedle mediated delivery indicate that further studies to optimise the microneedle array morphology, its method of application and the plasmid DNA formulation are warranted

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 permeation studies the epidermal membranes were visually characterised. Results: At 30 minutes 5.43 μg/cm2 of tetracaine had permeated through untreated membrane compared to 12.13 μg/cm2 in microneedle treated membrane. Insertion of a hypodermic needle created a large single channel in the epidermis (approximately 4250μm2) whilst the punctured surface area following microneedle treatments was estimated to be 75,000μm2. 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

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

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 (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

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

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

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

Objectives: To 1) identify pharmacy students’ preferred achievement goals in a multi-national undergraduate population; 2) investigate achievement goal preferences across comparable degree programs; 3) identify the relationships between achievement goals, academic performance and assessment type. Methods: The Achievement Goal Questionnaire was administered to second year students in four universities in Australia, New Zealand, England and Wales. Academic performance was measured using total scores, multiple choice questions (MCQ) and written answers (short essay). Results: A total of 486 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 MCQ, and written answers scores, were statistically 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

TGFβ 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-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