3D Printed Microheater Sensor-Integrated, Drug-Encapsulated Microneedle Patch System for Pain Management.

Microneedle patch devices have been widely utilized for transdermal drug delivery in pain management, but is challenged by accurate control of drug release and subsequent diffusion to human body. The recent emerging wearable electronics that could be integrated with microneedle devices offer a facile approach to address such a challenge. Here a 3D-printed microheater integrated drug-encapsulated microneedle patch system for drug delivery is presented. The ink solution comprised polydimethylsiloxane (PDMS) and multiwalled carbon nanotubes (MWCNTs) with a mass concentration of up to 45% (≈10 times higher of existing ones) is prepared and used to print crack-free stretchable microheaters on substrates with a broad range of materials and geometric curves. The adhesion strength of the printed microheater on the microneedle patch in elevated temperatures is measured to evaluate their integration performance. Assessments of encapsulated drug release into rat's skin are confirmed by examining degradation of microneedles, skin morphologies, and released fluorescent signals. Results and demonstrations established here creates a new opportunity for developing sensor controlled smart microneedle patch systems by integrating with wearable electronics, potentially useful in clinical and biomedical research.

Skin immunisation activates an innate lymphoid cell-monocyte axis regulating CD8+ effector recruitment to mucosal tissues.

CD8+ T cells provide a critical defence from pathogens at mucosal epithelia including the female reproductive tract (FRT). Mucosal immunisation is considered essential to initiate this response, however this is difficult to reconcile with evidence that antigen delivered to skin can recruit protective CD8+ T cells to mucosal tissues. Here we dissect the underlying mechanism. We show that adenovirus serotype 5 (Ad5) bio-distributes at very low level to non-lymphoid tissues after skin immunisation. This drives the expansion and activation of CD3- NK1.1+ group 1 innate lymphoid cells (ILC1) within the FRT, essential for recruitment of CD8+ T-cell effectors. Interferon gamma produced by activated ILC1 is critical to licence CD11b+Ly6C+ monocyte production of CXCL9, a chemokine required to recruit skin primed CXCR3+ CD8+T-cells to the FRT. Our findings reveal a novel role for ILC1 to recruit effector CD8+ T-cells to prevent virus spread and establish immune surveillance at barrier tissues.

Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays.

The generation of tissue resident memory (TRM) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8+ T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8+ T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8+ T cell expression of CXCR3+, CD103+, CD49a+, CD69+, CD127+ homing, retention and survival markers. Furthermore, memory CD8+ T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8+ T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces.

Skin vaccination with live virus vectored microneedle arrays induce long lived CD8(+) T cell memory.

A simple dissolvable microneedle array (MA) platform has emerged as a promising technology for vaccine delivery, due to needle-free injection with a formulation that preserves the immunogenicity of live viral vectored vaccines dried in the MA matrix. While recent studies have focused largely on design parameters optimized to induce primary CD8(+) T cell responses, the hallmark of a vaccine is synonymous with engendering long-lasting memory. Here, we address the capacity of dried MA vaccination to programme phenotypic markers indicative of effector/memory CD8(+) T cell subsets and also responsiveness to recall antigen benchmarked against conventional intradermal (ID) injection. We show that despite a slightly lower frequency of dividing T cell receptor transgenic CD8(+) T cells in secondary lymphoid tissue at an early time point, the absolute number of CD8(+) T cells expressing an effector memory (CD62L(-)CD127(+)) and central memory (CD62L(+)CD127(+)) phenotype during peak expansion were comparable after MA and ID vaccination with a recombinant human adenovirus type 5 vector (AdHu5) encoding HIV-1 gag. Similarly, both vaccination routes generated CD8(+) memory T cell subsets detected in draining LNs for at least two years post-vaccination capable of responding to secondary antigen. These data suggest that CD8(+) T cell effector/memory generation and long-term memory is largely unaffected by physical differences in vaccine delivery to the skin via dried MA or ID suspension.

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays.

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

Dissolvable microneedle patches for the delivery of cell-culture-derived influenza vaccine antigens.

Microneedle patches are gaining increasing attention as an alternative approach for the delivery of vaccines. In this study, a licensed seasonal influenza vaccine from 2007 to 2008 was fabricated into dissolvable microneedles using TheraJect's microneedle technology (VaxMat). The tips of the microneedles were made of antigens mixed with trehalose and sodium carboxymethyl cellulose. The patches containing 15 µg per strain of the influenza antigen were characterized extensively to confirm the stability of the antigen following fabrication into microneedles. The presence of excipients and very low concentrations of the vaccine on the microneedle patches made it challenging to characterize using the conventional single radial immunodiffusion analysis. Novel techniques such as capture enzyme-linked immunosorbent assay and enzyme digestion followed by mass spectroscopy were used to characterize the antigens on the microneedle patches. The in vivo studies in mice upon microneedle administration show immunogenicity against monovalent H1N1 at doses 0.1 and 1 µg and trivalent vaccine at a dose of 1 µg. The initial data from the mouse studies is promising and indicates the potential use of microneedle technology for the delivery of influenza vaccine.