Intercellular communication is essential to proper functioning of many biological processes. Recent evidence has now established that one form of intercellular communication occurs via the secretion of lipid vesicles known as extracellular vesicles, or EVs. EVs are capable of transferring proteins and RNAs from the cytoplasm of an EV-producing cell to the cytoplasm of a recipient cell where these RNAs and proteins have an effect on recipient cell behavior. Potentially, this mechanism of intercellular communication could be harnessed for therapeutic biomolecule delivery. To realize this potential, the rules governing EV-mediated intercellular communication must be elucidated. Many open questions exist regarding how EVs are produced, loaded with biomolecular cargo, taken up by recipient cells, and how each of these processes could be modulated for therapeutic purposes.;Here we investigate several properties of EV-mediated intercellular communication. First, we develop a Targeted and Modular EV Loading (TAMEL) platform to investigate how biophysical properties impact RNA loading into EVs and delivery of RNA cargo by EVs to recipient cells. Second, we investigate rules that limit the display of targeting peptides on the surface of EVs for targeted EV delivery to recipient cells. We demonstrate that targeting peptides are degraded by endosomal proteases and develop a glycosylation strategy for stabilizing targeting peptides on the surface of EVs. Third, we leverage EV peptide display to investigate (a) whether specific EV subsets can be enriched by affinity chromatography and (b) whether this enables enhanced cargo molecule delivery. As a whole, this work fills an important gap in understanding of EV-mediated intercellular communication and may enable novel therapeutic strategies harnessing EVs as biomolecular delivery vehicles.
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