Silk Microneedles for Targeted Epidermal Delivery of Antifreeze Proteins

Antifreeze proteins (AFPs) can inhibit ice recrystallization and reshape ice growth, but their size/charge make systemic delivery ineffective for reaching skin layers relevant to frostbite prevention or localized cryoprotectant delivery. This study developed silk fibroin (SF) microneedle (MN) patches fabricated using a heat-free process designed to preserve protein structure and activity during encapsulation. High-resolution inverse molds were 3D-printed and translated into PDMS molds, then AFP-loaded SF was centrifuged into the needle cavities across repeated short cycles to concentrate cargo in the needles; a second SF solution formed the patch backing. Patches (20×20 needle arrays; ~620–660 µm needle height) encapsulated fluorescently labeled AFP-III at doses up to 500 µg without altering MN morphology or bulk mechanics. Functional assays showed rapid release (≈50% within 15 min; ≈70% within 400 min in water) and preserved thermal hysteresis activity (~0.3 °C) after dissolution, confirming retained antifreeze function. Ex vivo porcine-skin testing demonstrated reliable epidermal penetration (100–300 µm) and AFP deposition into the epidermis with fluorescence extending beyond the immediate needle tracks after a 30 min application, consistent with diffusion and local dispersion. Overall, the work supports silk MN patches as a practical platform for transdermal delivery of fragile protein therapeutics, with near-term relevance to frostbite mitigation and longer-term potential for targeted cryoprotectant delivery to tissues and organs.