Isolation and purification of extracellular vesicles based on dual mechanisms of charge/size exclusion by quaternized...

Extracellular vesicles (EVs), as key mediators of intercellular communication, are widely present in various biological samples. In this study, chitosan (CS) and polyethyleneimine (PEI) with different molecular weights were grafted onto Fe₃O₄ microspheres, followed by quaternization to yield two series of multifunctional brush-like materials denoted as Fe₃O₄@QCS and Fe₃O₄@QPEI. The quaternary ammonium groups efficiently capture EVs via electrostatic binding to membrane phosphate groups. Meanwhile, the grafted CS and PEI chains of different molecular weights form brush layers with tunable sieving properties, enabling size‑selective enrichment of EVs via the size exclusion effect. With moderate chain length and an extended polymer brush conformation, Fe₃O₄@QCS (200 kDa) captured EVs from fetal bovine serum (FBS) with the broadest size distribution. To isolate EVs from biological samples with high protein interference, we developed a charge/size synergy strategy. First, Fe₃O₄@PAA, a polyacrylic acid (PAA)-coated anionic magnetic nanoparticle, was used to adsorb positively charged contaminant proteins. Subsequently, Fe₃O₄@QCS (200 kDa) was employed to capture EVs and residual proteins, followed by salt-gradient elution to remove negatively charged contaminant proteins and release EVs. This strategy successfully achieved a protein removal efficiency of 96.9% and an EV recovery rate of 87.8% for milk samples, identifying 236 EV-characteristic proteins. This method was further applied to the enrichment of human plasma EVs, and downstream proteomic analysis identified 484 EV proteins.