The Biophysical Characteristics of Extracellular Vesicle Production and Transport

Extracellular vesicles (EVs) produced by mesenchymal stromal cells (MSCs) are cell-secreted nanoparticles with broad potential to treat tissue injuries by delivering cargo to program target cells. Understanding fundamental mechanisms by which the extracellular microenvironment regulates EV production and resulting EV transport will lend a significant insight towards translating EVs as therapeutics. To this end, we show that MSCs produce significantly more EVs on softer substrates due to less integrin activation. MSCs produce EVs more rapidly if adhesion time on substrates is limited to a briefer period. Substrate mechanical properties direct EV number per cell without altering EV size, morphology, therapeutic content, or therapeutic efficacy against a murine model of acute lung injury. Mechanistically, intracellular CD63+ multivesicular bodies (MVBs) transport faster within MSCs on softer hydrogels with less adhesion time, leading to an increased frequency of MVB fusion with the plasma membrane to secrete more exosomes. Furthermore, we show that EVs transport through matrix environments despite being larger than the average nanoporous mesh. Water permeation through aquaporin-1 on the surface of EVs mediates their deformability, allowing navigation through the dense matrix. Matrix stress relaxation further facilitates EVs to overcome confinement, and matrix stiffness leads to a fluctuating transport motion. The combination of water permeation, matrix stress relaxation and matrix stiffness results in a greatly enhanced EV transport through matrix. In sum, this thesis elucidates the regulation by the biophysical environment of MSC-EV production and transport and presents novel approaches to understand fundamental mechanisms of EV-matrix interactions and informs potential strategies to improve translation of EVs as promising therapeutics.