Nanostructured Affinity Membrane to Isolate Extracellular Vesicles from Body Fluids for Diagnostics and Regenerative Medicine

Electrospun regenerated cellulose (RC) nanofiber membranes were prepared starting from cellulose acetate (CA) with different degrees of substitution. The process was optimized to obtain continuous and uniformly sized CA fibers. After electrospinning, the CA membranes were heat-treated to increase their tensile strength before deacetylation to obtain regenerated cellulose (RC). Affinity membranes were obtained by functionalization, exploiting the hydroxyl groups on the cellulose backbone. 1,4-Butanediol-diglycidyl ether was used to introduce epoxy groups onto the membrane, which was further bioconjugated with the anti-CD63 antibody targeting the tetraspanin CD63 on the extracellular vesicle membrane surface. The highest ligand density was obtained with an anti-CD63 antibody concentration of 6.4 µg/mL when bioconjugation was performed in carbonate buffer. The resulting affinity membrane was tested for the adsorption of extracellular vesicles (EVs) from human platelet lysate, yielding a very promising binding capacity above 10 mg/mL and demonstrating the suitability of this approach.