Project Summary Oral insulin will improve the life quality of diabetes patients but current oral protein formulations face multiple obstacles during their gastrointestinal transport and absorption resulting in low therapeutic functionality. In particular, penetrating the intestinal mucus layer and the epithelial cell layer to reach the blood remained two major challenges for any oral formulation. Despite advancement in mucus-penetrating and absorption-enhancing technologies, current oral delivery of protein drugs remains low in absorption and bioavailability, and/or increases the risk of leaky gut and safety concerns such as autoimmune disease, bacterial infection and inflammatory bowel diseases. There is an urgent need for a safe and efficient oral delivery technology to drastically enhance protein transport through the mucus and intestinal epithelium, and to enable oral insulin with high bioavailability. The long-term research goal of the PI is to develop a platform technology that addresses these barriers altogether and enables oral insulin feasible for clinical translation. The objective of this project is to explore the mechanism for a novel insulin delivery system in addressing the mucus transport, epithelium absorption, and gut- related safety issues and contributing to a high oral bioavailability. Specific aims include the identification of mechanism/key parameters of the delivery system contributing to the preferred properties and performance in addressing these obstacles through in vitro and in vivo studies (involving diabetic mice and rats). Furthermore, this project will validate the feasibility of the oral insulin prototype on diabetic minipigs, which is a more relevant large animal model for predicting oral bioavailability in humans. The success of this project will directly lead to the development of highly promising oral insulin– a life-changing treatment for diabetes patients. This project will also produce foundational knowledge regarding how the particular delivery platform addresses the multiple barriers for oral protein delivery; this will impact a broad range of future development of oral protein drugs.