Improved Accessibility of Extracellular Vesicle Surface Molecules Upon Partial Removal of the Protein Corona by High...

Recent studies have confirmed that a biomolecular corona forms around extracellular vesicles (EVs) in biofluids. However, there is limited data on how this adsorbed corona affects the accessibility of EV surface molecules. Here, we investigated various potential corona-stripping conditions for their ability to affect the immune detection of EVs. First, we artificially formed an EV corona around nascent HEK293T-PalmGFP cell-derived large EVs (lEVs) by incubating them with Cy5-labelled human plasma proteins. The co-localisation rate of plasma proteins and lEVs decreased significantly upon high-salt washing with NaCl, LiCl and KCl solutions, suggesting a considerable removal of the corona components. Additional evidence for corona modification was a significantly increased fluorescent annexin V binding to plasma lEVs and annexin V affinity capture of both THP1- and blood plasma-derived lEVs upon high-salt washing. A similar effect of high ionic strength was observed when THP1 lEVs were separated from a serum-containing medium, which allowed for corona formation, but not when EVs were produced under serum-free conditions. Using a MACSPlex kit and high-salt washing for small EVs from plasma and THP1 conditioned medium, we also demonstrated significantly improved immunodetection of 15 and 9 out of 37 surface markers, respectively. In this Technical Note, we present evidence that modifying the protein corona around EVs can significantly affect the immune detection of specific EV markers.