Project Summary Intercellular communication based on the delivery of miRNAs from one cell to adjacent or remote cells is now known to be a critical part of normal cellular/organismal function. Recent work has demonstrated that a new class of extracellular nanoparticles, exomeres, may be more important for miRNA-mediated intercellular communication than extracellular vesicles (EVs). Exomeres are small, non-membranous secreted particles that are different from EVs. Compared to EVs, exomeres are smaller, have distinct protein and nucleic acid content, and are not encapsulated by lipid membranes. Also, exomeres contain Argonaute-2 (Ago2), one of the principal proteins of the RNA interference (RNAi) pathway. With respect to intercellular communication, exomeres contain miRNAs, which are likely bound by Ago2, and the EGFR ligand, amphiregulin, which could initiate receptor-mediated endocytosis. Thus, exomeres are a natural RNA-containing nanoparticle evolved to have molecular and physical properties that facilitate efficient endocytosis and intracellular processing of the miRNA cargo by recipient cells. In this work, we will begin to explore whether exomeres could be used for delivery of exogenous miRNAs (or siRNAs) for therapeutic applications. The mechanisms of exomere biogenesis, secretion, endocytosis, and intracellular trafficking remain poorly understood. This is due, in part, to the challenges of isolating exomeres. Current exomere isolation approaches have allowed for preliminary characterization of exomeres, but further study of exomeres, and their critical roles in RNA-mediated intercellular communication, will require approaches for isolating exomeres that yield high concentrations of reasonably homogeneous complexes. In this work, we will develop a novel, scalable exomere isolation approach based on tangential flow filtration (TFF) that will enhance our ability to study exomeres and the endocytosis and intracellular trafficking of exomeres by recipient cells. The overarching goal of this work is to demonstrate the utility of TFF for exomere isolation and to demonstrate that exomeres isolated by TFF are identical to those isolated by current approaches. Given that TFF is compatible with possible industrial production methods for exomeres, our results will provide the foundation for future efforts to use exomeres for RNA delivery in clinical applications.