Microprojection arrays applied to skin generate mechanical stress, induce an inflammatory transcriptome and cell death, and improve vaccine-induced immune responses

Abstract: Chemical adjuvants are typically used to improve immune responses induced by immunisation with protein antigens. Here we demonstrate an approach to enhance immune responses that does not require chemical adjuvants. We applied microprojection arrays to the skin, producing a range of controlled mechanical energy to invoke localised inflammation, while administering influenza split virus protein antigen. We used validated computational modelling methods to identify links between mechanical stress and energy generated within the skin strata and resultant cell death. We compared induced immune responses to those induced by needle-based intradermal antigen delivery and used a systems biology approach to examine the nature of the induced inflammatory response, and correlated this with markers of cell stress and death. Increasing the microprojection array application energy and the addition of QS-21 adjuvant were each associated with enhanced antibody response to delivered antigen and with induction of gene transcriptions associated with TNF and NF-κB signalling pathways. We concluded that microprojection intradermal antigen delivery inducing controlled local cell death could potentially replace chemical adjuvants to enhance the immune response to protein antigen

Determination of stress field caused by microprojection arrays contacting and impacting hyperelastic layered skin

The Nanopatch™ is a silicon array of microprojections for epidermal and dermal delivery of vaccines, resulting in enhanced immunogenicity in comparison to intramuscular injection. Achieving this requires the fracture of skin superficial barriers and penetration to the targeted depth, reliant upon negotiating the complex non-linear elastic and failure properties of skin-a multilayer composite biomaterial. In this work, computational models of projection-skin mechanical interaction are developed and applied to investigate the mechanical stress generated to fracture skin. Our analytical results on a homogenous linear-elastic skin model suggest that the array projections exert an uneven force distribution on the skin surface, leading to a non-homogeneous stress across the loaded skin region. In addition, the creation of high localized tensile stress is sensitive to a precise trade-off between projection spacing and tip diameter. Numerical simulations are further performed using a layered hyperelastic skin representation and compared with the analytical findings. The resulting deformation and stresses are significantly increased due to, respectively, the compliant top skin layers and their non-linear elastic properties. This underlines the importance of accounting for the stratified structure of the skin as well as the strain-hardening properties of its strata when assessing the achievement of failure criteria

Dynamic application of microprojection arrays to skin induces circulating protein extravasation for enhanced biomarker capture and detection

Surface modified microprojection arrays are a needle-free alternative to capture circulating biomarkers from the skin in\ua0vivo for diagnosis. The concentration and turnover of biomarkers in the interstitial fluid, however, may limit the amount of biomarker that can be accessed by microprojection arrays and ultimately their capture efficiency. Here we report that microprojection array insertion induces protein extravasation from blood vessels and increases the concentration of biomarkers in skin, which can synergistically improve biomarker capture. Regions of blood vessels in skin were identified in the upper dermis and subcutaneous tissue by multi-photon microscopy. Insertion of microprojection array designs with varying projection length (40–190\ua0μm), density (5000–20,408 proj.cm) and array size (4–36\ua0mm) did not affect the degree of extravasation. Furthermore, the location of extravasated protein did not correlate with projection penetration to these highly vascularised regions, suggesting extravasation was not caused by direct puncture of blood vessels. Biomarker extravasation was also induced by dynamic application of flat control surfaces, and varied with the impact velocity, further supporting this conclusion. The extravasated protein distribution correlated well with regions of high mechanical stress generated during insertion, quantified by finite element models. Using this approach to induce extravasation prior to microprojection array-based biomarker capture, anti-influenza IgG was captured within a 2\ua0min application time, demonstrating that extravasation can lead to rapid biomarker sampling and significantly improved microprojection array capture efficiency. These results have broad implications for the development of transdermal devices that deliver to and sample from the skin

Repeat Offenders: If They Learn, We Punish Them More Severely

Many legal systems are designed to punish repeat offenders more severely than first time offenders. However, existing economic literature generally offers either mixed or qualified results regarding optimal punishment of repeat offenders. This paper analyzes optimal punishment schemes in a two period model, where the social planner announces possibly-different sanctions for offenders based on their detection history. When offenders learn how to evade the detection mechanism employed by the government, escalating punishments can be optimal. The contributions of this paper can be listed as follows: First, it identifies and formalizes a source which may produce a marginal effect in the direction of punishing repeat offenders more severely, namely learning. Next, it identifies conditions under which the tendency in legal systems to punish repeat offenders more severely is justified. Overall, the findings suggest that the traditional variables identified so far in the literature are not the only relevant ones in deciding how repeat offenders should be punished, and that learning dynamics should also be taken into account

Depth-resolved characterization of diffusion properties within and across minimally-perturbed skin layers

We examine by both experimental and computational means the diffusion of macromolecules through the skin strata (both the epidermis and dermis). Using mouse skin as a test case, we present a novel high-resolution technique to characterize the diffusion properties of heterogeneous biomaterials using 3D imaging of fluorescent probes, precisely-deposited in minimally-perturbed in vivo skin layers. We find the diffusivity of the delivered macromolecules (70 kDa and 2 MDa rhodamine-dextrans) low within the packed cellular arrangement of the epidermis, while gradually increasing (by ~ an order of magnitude) through the dermis - as pores in the fibrillar network enlarge from the papillary to the reticular dermis. Our experimental and computational approaches for investigating the diffusion through skin strata help in the assessment and optimization of controlled delivery of drugs (e.g. vaccines) to specific sites (e.g. antigen presenting cells)

CXCL1 gene silencing in skin using liposome-encapsulated siRNA delivered by microprojection array

The barrier morphology of skin provides major obstacles for the application of siRNA for gene silencing, which current delivery technologies do not effectively overcome. Emerging technologies utilise microprojection array devices to penetrate into the skin epidermis and dermis for delivery of drug payloads

Colocalization of cell death with antigen deposition in skin enhances vaccine immunogenicity

Vaccines delivered to the skin by microneedles - with and without adjuvants - have increased immunogenicity with lower doses than standard vaccine delivery techniques such as intramuscular or intradermal injection. However, the mechanisms underlying this skin-mediated "adjuvant" effect are not clear. Here, we show that the dynamic application of a microprojection array (the Nanopatch) to skin generates localized transient stresses invoking cell death around each projection. Nanopatch application caused significantly higher levels (∼65-fold) of cell death in murine ear skin than i.d. injection using a hypodermic needle. Measured skin cell death is associated with modeled stresses ∼1-10 MPa. Nanopatch-immunized groups also yielded consistently higher anti-immunoglobulin G endpoint titers (up to 50-fold higher) than i.d. groups after delivery of a split virion influenza vaccine. Importantly, colocalization of cell death with nearby live skin cells and delivered antigen was necessary for immunogenicity enhancement. These results suggest a correlation between cell death caused by the Nanopatch with increased immunogenicity. We propose that the localized cell death serves as a "physical immune enhancer" for the adjacent viable skin cells, which also receive antigen from the projections. This natural immune enhancer effect has the potential to mitigate or replace chemical-based adjuvants in vaccines

Cellular metabolism and pore lifetime of human skin following microprojection array mediation

Skin-targeting microscale medical devices are becoming popular for therapeutic delivery and diagnosis. We used cryo-SEM, fluorescence lifetime imaging microscopy (FLIM), autofluorescence imaging microscopy and inflammatory response to study the puncturing and recovery of human skin ex vivo and in vivo after discretised puncturing by a microneedle array (Nanopatch®). Pores induced by the microprojections were found to close by ~25% in diameter within the first 30 min, and almost completely close by ~6 h. FLIM images of ex vivo viable epidermis showed a stable fluorescence lifetime for unpatched areas of ~1000 ps up to 24 h. Only the cells in the immediate puncture zones (in direct contact with projections) showed a reduction in the observed fluorescence lifetimes to between ~518-583 ps. The ratio of free-bound NAD(P)H (α1/α2) in unaffected areas of the viable epidermis was ~2.5-3.0, whereas the ratio at puncture holes was almost double at ~4.2-4.6. An exploratory pilot in vivo study also suggested similar closure rate with histamine administration to the forearms of human volunteers after Nanopatch® treatment, although a prolonged inflammation was observed with Tissue Viability Imaging. Overall, this work shows that the pores created by the microneedle-type medical device, Nanopatch®, are transient, with the skin recovering rapidly within 1-2 days in the epidermis after application