(493e) Development of Nanopocket Membranes for the Purification and Fractionation of Extracellular Vesicle Subpopulations

The use of nanoscale extracellular vesicles (EV) for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. Currently, the “gold standard” method for isolating EVs from biofluids is ultracentrifugation, which requires large volumes of biofluid, long processing times, expensive instrumentation, and trained technicians. Gel precipitation and size exclusion chromatography have been developed that remove the need for ultracentrifugation, but these methods suffer from low yield and/or contamination with co-precipitated proteins.

We have recently demonstrated a new method termed tangential flow for analyte capture (TFAC)¹ using ultrathin silicon-based nanomembranes to purify extracellular vesicles from complex biological fluids such as blood plasma and cell culture media (Figure 1A-D). In this method, EVs and similarly sized analytes are captured on the pores of an ultrathin membrane where they can be washed and released. TFAC resembles bind and elute purification strategies although it distinguishes itself from affinity chromatography because the binding is purely physical. TFAC does not require engineered surface chemistries for capture or chemical treatments for elution.

In order to increase the specificity of EV capture including separation of different EV sub-populations based on size and surface markers, we are developing a series of nanopocket membranes (Figure 1E). We use nanosphere lithography (NSL) to pattern and etch nanoscale (50-500 nm) pockets, each with a pore, on the surface of a membrane. Nanopockets can be fabricated with varying radius and depth as well as surface properties akin to size and affinity chromatography. These properties can be tailored to sizes of different EV subpopulations, where a series of different nanopocket sizes could capture, fractionate and purify EV populations from complex biofluids such as plasma.

Figure 1. Small extracellular vesicles (sEV) captured from undiluted blood plasma. A) SEM images showing the thinness and high porosity of nanoporous silicon nitride (NPN). B) In normal flow filtration (NFF) a protein cake of ~8 μm cake rapidly builds up on the membrane surface. C) In tangential flow filtration for analyte capture (TFAC) with the same sample, small vesicles are captured on the membrane surface with minimum fouling. D) Nanogold conjugated anti-CD63 antibody labels an EV captured in a pore. Scale bar = 200 nm for A, B and C. Scale bar = 50 nm for D. E) Cross-section schematic of a nanopocket membrane. Pocket radius and depth can be tuned to target EVs of specific sizes. Smaller pockets will be placed upstream of larger pockets. Pocket walls will also be modified with switchable biochemical groups for affinity capture.

¹ Dehghani M, Luca K, Flax J, McGrath J and Gaborski TR. Tangential flow microfluidics for the capture and release of nanoparticles and extracellular vesicles on conventional and ultrathin membranes. Advanced Materials Technologies. 2019 4(11): 1900539.