Optimizing plasma-derived small extracellular vesicle purification to enhance downstream applications

Abstract

Small extracellular vesicles (sEVs) are functional carriers of biomolecules from host cells, making them highly suitable for early disease diagnosis and monitoring disease progression. However, the purification of sEVs from plasma is complicated by the presence of high levels of proteins, particularly lipoproteins. We have developed an original pretreatment method that effectively removes lipoproteins and fibrinogen, thereby facilitating subsequent sEV purification. Sequential application of ultracentrifugation and optical density gradient on pretreated plasma significantly enhances the purity of sEVs, achieving the removal of over 99% of contaminants and increasing mass spectrometry protein detections by 13.3 and 6.3 times compared to traditional ultracentrifugation and size exclusion chromatography methods, respectively. Furthermore, proteomic analysis verified the validity of sEVs isolated using our approach. Overall, our optimized method substantially improves the purity of plasma sEVs without compromising yield, thereby greatly enhancing their potential for clinical testing and basic biochemical research.