PROJECT SUMMARY Post-traumatic osteoarthritis (PTOA) is an insidious consequence of joint injury, ~50% of patients with knee- injuries exhibit PTOA within 10-years of injury. Presently, no cure for PTOA exists, but the acute nature of the precipitating injuries provides for a unique approach to PTOA treatment: targeted prophylactic pharmaceutical intervention to mitigate/prevent the initiation of disease post-injury. Many pre-clinical investigations for targeted treatment have been conducted. However, due to incredibly rapid intra-articular (i.a.) drug clearance, disease- modifying drug efficiency is highly limited, requiring repeated high-dose administration of free drug for efficacy. Give the inefficiencies of i.a. administration of free drug, delivery approaches that extend drug-residence time by targeting the tissues of the injured joint could represent a cost-effective method of increasing therapeutic efficacy. We propose a novel and versatile platform for the thermally responsive, localized delivery of candidate PTOA drugs to injured joints to limit initiation/progression of PTOA. Our approach relies on our pioneering development of elastin-collagen-peptide conjugates that uniquely form cargo-laden nanovesicles that facilitate long-term passive release at body temperature and accelerated-/burst-delivery at mildly hypothermic temperatures. In addition, the collagen-like peptides comprising the vesicle’s outer ‘shell’ can target denatured collagens, allowing accumulation in tissues with elevated collagen damage/remodeling. In this proposal, we will evaluate the loading of candidate PTOA disease-modifying drugs (with a focus on dexamethasone (Dex)) in refined elastin-collagen nano-vesicles (ECnV) and monitor their stability, as well as passive and hypothermally-triggered drug release. Studies on naïve and ‘injured/activated’ chondrocytes, synovial fibroblasts, and monocyte/macrophages, and articular cartilage and synovial tissue explants, will confirm the cyto-/biocompatibility and quantify the suppression of ‘injury’ markers by Dex-loaded ECnVs. We will conduct in vivo experiments using a non-invasive repeated joint loading (overuse) model of PTOA to demonstrate the selective retention of ECnVs within injured joints after intra-articular (i.a.) injection. Multi-scale in vivo, in situ, and histological/immunohistochemical analyses will be employed to evaluate the pharmacokinetics of passively and hypothermally-triggered cargo release, tissue localization/biodistribution, and the local and systemic biocompatibility/safety of ECnVs delivered to both healthy and early-PTOA joints. Finally, we will characterize the ability of ECnV-based delivery of Dex to improve disease-modifying physiology and PTOA outcomes prophylactically in the aforementioned non-invasive, joint injury model, with standard i.a. liposomal and free-Dex treatments serving as comparators. Although the proposed work focuses on increasing PTOA therapy effectiveness, it will also la...