PROJECT SUMMARY While nanoparticle-based platforms have become a leading drug delivery platform for treating various diseases, several challenges remain in current nanomedicine, including toxicity, inefficient endothelial barrier crossing, rapid elimination, and nonspecific accumulation in the body. Extra- cellular vesicles (EVs) provide a natural delivery system that can transfer various cellular cargo to adjacent and distant cells. EVs offer unique advantages for engineering while possessing in- herent immune evasion capability and tissue penetrating characteristics. However, inefficient therapeutic cargo packaging and insufficient EV production from cells limit the current EV-based drug delivery approach. Chronic inflammatory disease (CID) imposes health and economic burdens on communi- ties worldwide. Current therapy of CID is neither sufficient nor disease-modifying. Our ultimate goal is to develop a safe and effective EV-based therapy for CID patients. The overall objectives in this application are to (i) develop an ultrasound (US)-based platform termed EVEiR (Extracel- lular Vesicle Engineering and induced Release) for delivering anti-inflammatory cytokine IL10, and (ii) determine their therapeutic efficacy using an in vitro intestinal model. The central hypoth- esis is that externally applied mechanical cues using US stimulation and the mechanical proper- ties of the cell microenvironment may increase the production and function of engineered EVs (eEVs) derived from mesenchymal stem cells (MSCs) in 3D cultures. We will test the central hypothesis by pursuing two Specific Aims: 1) Develop US-assisted EVEiR for efficient production of anti-inflammatory IL10-carrying eEVs (IL10+ eEVs); and 2) Demonstrate the feasibility of IL10+ eEVs derived from MSCs in 3D cultures for targeted delivery of therapeutics. Aim 1 will determine the efficiency of IL10+ eEV production using the novel US-based techniques. Aim 2 will charac- terize the properties of IL10+ eEVs and their anti-inflammatory effect using an in vitro intestinal model. The innovation of this proposal is to utilize the synergy with the impact of non-viral intra- cellular IL10 packaging in EVs, the unique concept of pulsed US stimulation of MSCs in 3D hy- drogel constructs for efficient eEV production. In addition, an in vitro intestinal model allows for efficient characterization of anti-inflammatory IL10+ eEVs in a physiologically relevant environ- ment. The proposed research is significant because the successful completion of this project will develop a rapid, cost-effective, and scalable platform to generate MSC-derived therapeutic EVs for treating CID and facilitate the translation of MSC-derived EVs to the clinic.