Engineering Surface Coatings for Localized Delivery of Therapeutic Extracellular Vesicles

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PROJECT SUMMARY The PI’s long-term research goal is to engineer the cell-material interface for applications in tissue engineering, regenerative medicine, and medical implants, with a particular focus on bone regeneration. Extracellular vesicle (EV) - based therapies have been of increasing interest in the past decade to treat a variety of pathologies. Although EV delivery from various carrier systems has been shown to induce tissue regeneration in preclinical models, there remains a critical gap in efficient and controlled delivery of therapeutic EVs. This proposal aims to engineer a surface coating system that promotes localized EV delivery for tissue engineering and regenerative medicine applications. The central hypothesis underlying this research is that surface-mediated EV delivery modulates cellular uptake and signaling in comparison to bolus or systemic EV delivery. The specific research objectives of this proposal are to: (1) Investigate the roles of electrostatic and receptor- ligand interactions in the adsorption and release of EVs to/from surface coatings; (2) Investigate the effects of surface-based EV delivery in comparison to bolus EV delivery; and, (3) Demonstrate that EV delivery from surface-coated tissue engineering scaffolds improves tissue regeneration in vivo in a rat spinal fusion model. EVs will be derived from mesenchymal stem/stromal cells (MSCs) and characterized for size, surface charge, and protein markers. Interactions between MSC-EVs and surface coatings composed of ECM proteins, polysaccharides and charged polymers will be analyzed under various conditions (pH, R-L inhibitors, salt screening) via fluorescence labeling studies and quartz crystal microbalance with dissipation analyses. EV uptake efficiency and endocytic pathways will be analyzed in several cell types (MSCs, HUVECs, HEK293s) in comparison to bolus EV delivery via fluorescence microscopy and flow cytometry (+/- endocytic pathway inhibitors). Cellular adhesion, proliferation, and angiogenic differentiation and immunomodulation will also be analyzed in vitro. In vivo bone forming capacity and fusion efficacy of EVs delivered from surface-coated scaffolds will be evaluated in the rat posterolateral lumbar fusion model via manual palpation, radiographic scoring, volumetric microcomputed tomography (µCT) and immunohistochemistry, in comparison to uncoated scaffolds and bolus delivery. Results from this work will significantly advance understanding of how material properties and surface-based EV delivery impact cellular EV uptake, adhesion, proliferation and differentiation. Additionally, this proposal will enable development of effective therapeutic EV surface-coatings that can tailored for a wide variety of scaffolds and/or implants for many different therapies, including cartilage regeneration, diabetic wound healing, cardiac infarction, and tendon and muscle repair.