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

Antigen-specific immunotherapy (ASI) has been proposed as an alternative treatment strategy for type 1 diabetes (T1D). ASI aims to induce a regulatory, rather than stimulatory, immune response in order to reduce, or prevent, autoimmune mediated 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

Microneedle delivery of antigen-specific immunotherapy for Type 1 diabetes

Antigen-specific immunotherapy (ASI) involves induction of tolerance to autoantigens. An important protein in the development of type 1 diabetes (T1D) is the autoantigen, proinsulin (PI), the precursor of insulin. Microneedles (MNs) are micron-sized needles that penetrate into the upper skin layers. MNs provide advantages for autoantigen delivery including targeted delivery to the skin’s dendritic cells (DCs), with minimal inflammation. The aim of this Thesis was to develop a PI-coated solid MN system and investigate the potential of this system to induce peripheral tolerance in the non-obese diabetic (NOD) mouse model of T1D. A highly concentrated PI MN coating formulation was developed containing the PI, diluent and a surfactant. The formulation enabled uniform and reproducible coating of the PI on to MNs. Delivery of PI from the MN system was investigated in mouse skin. MN application method and duration were optimised and resulted in skin puncture and reproducible delivery of PI to the skin. In vitro studies identified the insulin-reactive G9 CD8+ T cell as an appropriate biological readout for PI delivery. In vivo delivery studies indicated that MNdelivered PI was delivered to the skin and subsequently processed by DCs into PI peptides, which were cross-presented in the skin draining lymph nodes to adoptively transferred G9 CD8+ T cells. This demonstrated that the PI-coated MN system has potential for inducing peripheral tolerance in the NOD mouse. T1D development was significantly delayed in NOD SCID mice that received cells from PI-treated NOD mice and cells from diabetic NOD mice (experimental group). However, no statistically significant difference in time to T1D development was observed between the experimental group and the control NOD SCID mice that received cells from untreated NOD mice and diabetic NOD mice. Further investigation of the dosage and dosing frequency of PI using the coated MN system is, therefore, warranted