Permeabilization and Analysis of Brain-Derived Extracellular Vesicles Isolated from Human Blood

Summary: Extracellular Vesicles (EVs) are nanoscale cellular packages found in all bodily fluids that carry complex biomolecules (e.g., protein, lipids, RNA). The growing interest in EV research has explored their use in assessing diseases of the central nervous system (CNS) and as a vehicle for treatment delivery to the CNS. However, with the current technology, only surface biomolecules can be analyzed in intact vesicles. No established methods exist for examining intravesicular contents in intact EVs. We describe a novel method to characterize the contents of intact and tissue-specific EVs. EVs were isolated from human plasma by serial centrifugation. The size and concentration of the isolated EVs were confirmed by nanoparticle tracking analysis. For cargo analysis, EVs were first immobilized and fixed on a biotin-coated chip purchased from Oxford Nanoimaging (ONI). EVs were then permeabilized with a detergent solution followed by incubation with fluorescent- conjugated antibodies against surface tetraspanins (CD63, CD9, CD81), intracellular proteins (Alix, Syntenin), and proteins specific for their tissue of origin (TMEM119 – CNS/microglia, CD230 – Neurons). Imaging analysis was then performed by direct stochastic optical reconstruction microscopy (dSTORM) technique. Density-based cluster analysis was utilized to identify EV subpopulations based on the co-localization of up to three markers. The efficacy of permeabilization was further confirmed by harvesting EVs from GFP-expressing cells and demonstrating co-localization of GFP with EV surface markers. Currently, we are employing this approach to study Post-Acute Sequalae of COVID-19 (PASC). We hypothesized that EVs carry inflammatory proteins and mRNAs, SARS-CoV-2 Spike protein, and viral genetic material from the infected cells and tissues to the brain and contribute to the pathogenesis of PASC. Preliminary data shows an increased co-localization of SARS-CoV-2 spike protein with potential inflammatory genes within CNS-derived EVs in PASC patients compared to control patients.