Abstract
Antifreeze proteins (AFPs) from cold-adapted organisms excel at modifying ice crystal growth and inhibiting ice recrystallization, making them highly attractive compounds for cryopreservation and frostbite prevention. Traditional drug delivery methods are inadequate for AFPs because their large molecular weight and charge prevent them from crossing cellular membranes and from reaching target tissues through oral, intramuscular, or intravenous routes. Transdermal microneedle (MN) patches offer a promising alternative for delivering AFPs through capillary networks just beneath the epidermis or into specific cellular compartments. However, current materials and protocols are inadequate for encapsulating, storing, and delivering functional AFPs. Here, we demonstrate that silk MN patches, manufactured using a novel heat-free fabrication method, can encapsulate and deliver AFPs into porcine skin, an ex vivo model that closely resembles human tissue. We show that the silk MN patches preserve AFP activity, achieve effective penetration and degradation in the tissue, and successfully deliver functional proteins into the porcine epidermis. These results highlight the potential of encapsulating AFPs as a first step toward their use for frostbite prevention and targeted long-term tissue and organ cryostorage applications. Our findings support the use of silk fibroin as a raw material for manufacturing transdermal microneedle patches to deliver emerging therapeutics.