Project Summary Extracellular vesicles (EVs) are released by cells and are thought to play important roles in cell- cell communication, including protective and pathogenic roles in disease. The objective of this proposal is to develop a straightforward and scalable separation technology that effectively fractionates extracellular vesicle (EV) subpopulations with high purity and high speed. Heterogeneity of biophysical characteristics and composition of EVs introduces an extra level of complexity when studying their diverse functions. The lack of ability to fractionate EVs into subpopulations hampers efforts to understand EV function in cell-cell communication and realize the potential of EVs in diagnostic and therapeutic applications. Ultracentrifugation remains the gold standard for isolating EVs, but serial and density gradient approaches require large equipment that cannot be multiplexed, necessitates high skill and many hours of processing. Size exclusion chromatography (SEC) can isolate EV subpopulations by size, but results in significant dilution and suffers from contamination with lipoproteins, particularly VLDLs, which are the same size as small EVs and common in plasma. We propose to address these limitations by developing a novel nanopocket membrane and using a modified tangential flow filtration (TFF) approach that effectively captures and releases EV subpopulations based on specific physical properties, while eliminating lipoprotein contaminants. In Aim 1, we will adapt the use of nanosphere lithography to regularly place polystyrene nanospheres across a substrate to be used as templates for nanopockets on the surface of the membrane. Using different bead sizes and etching times, we will create nanopocket membranes of varying physical attributes (pocket radius, depth, pore size) to capture EV subpopulations. In Aim 2, nanopocket membranes will be integrated into devices where conditions for EV capture and release (fluid shear, transmembrane pressure, release conditions) will be optimized. Using media, plasma and urine spiked with known concentrations of pre-purified EV subpopulations, we will target capture and release of small-EVs as well as medium and large EVs in series with increasing size nanopockets. Contaminating LDLs and VLDLs, common in plasma, will be removed with negatively-charged dextran sulfate beads added to the TFF circuit. This work will be successful if membranes with nanopockets of tunable size can capture and release small, medium and large EVs from cell culture media, plasma and urine with the precision of SEC (at higher concentration), while exceeding the purity and yield of ultracentrifugation in <1 hour.

Project Narrative Extracellular vesicles (EVs) are released by cells and are thought to play important roles in cell- cell communication, including protective and pathogenic roles in disease. Isolating and purifying EVs will not only enable research to better understand their function, but also potentially use them in therapeutic and diagnostic applications. The purpose of this project is to develop a precise and rapid purification technology to isolate EV subpopulations.