Decoding extracellular vesicle-biomolecule interactions: How the biomolecular corona shapes extracellular vesicle biodistribution and clearance in vivo.

Extracellular vesicles (EVs) are promising endogenous nanocarriers, yet how biomolecules adsorption shapes their in vivo fate remains unclear. Here, we examine how biomolecular corona formation regulates the biodistribution, cellular recognition, and clearance of extracellular vesicles. Using EVs derived from macrophages and breast cancer cells, together with artificially prepared cell membrane-derived vesicles (CVs) generated from the corresponding parental cells as controls, we observed markedly different circulation and clearance behaviors despite their comparable physicochemical properties. Proteomic analyses revealed distinct and extensive biomolecular adsorption profiles on EVs compared with their corresponding CVs. In vivo imaging shows that EVs undergo faster systemic clearance and preferential accumulation in the liver and spleen. Cellular-level analyses identify Kupffer cells, liver sinusoidal endothelial cells, splenic macrophages, and circulating innate immune cells-especially neutrophils-as key mediators of EVs clearance. In vitro studies further confirm that biomolecular corona formation enhances immune recognition and uptake, with EVs consistently showing higher phagocytosis than CVs. Notably, opsonin proteins such as complement C3 and immunoglobulins are enriched on EVs surfaces and strongly correlate with increased macrophage uptake. Overall, these results highlight biomolecular corona formation as a critical determinant of EVs biological identity and in vivo fate.