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.
