Isolation of small extracellular vesicles by interacting with inorganic surface.

UNLABELLED: Extracellular vesicles (EVs) have great potential as diagnostic and therapeutic tools because they are important mediators of intercellular communication. Although several EV isolation methods have been developed, efficient and selective isolation remains challenging owing to the presence of coexisting proteins and other EV subtypes. Herein, we report an EV purification method using inorganic materials composed of calcium phosphate and calcium carbonate combining with magnetic particles, wherein EVs are captured to phosphate and carbonate groups via surface calcium residues through metal coordinate bonds with their negatively charged phospholipids of EVs.

METHODS

Cultured supernatants from three cell lines (HEK293T and cancer cells, MCF7 and PC3) were subjected to sequential centrifugation and filtration to remove cell debris and components smaller than 100 kDa, followed by purification using our inorganic material-based method. For comparison, conventional approaches, including polymer precipitation and phosphatidylserine (PS)-specific binding protein-based purification, were used. Purified EVs were characterized based on total protein content, surface marker expression (CD63 and CD81), and miRNA levels.

RESULTS

The results revealed that our method enriched EVs with higher surface marker expression and miRNA content more efficiently than other approaches, while maintaining EV integrity and minimizing protein contamination. Although EVs were isolated from the same cell line, their compositions differed, indicating that the purification method should be carefully selected.

CONCLUSION

Thus, our inorganic material-mediated approach provides an effective platform for small extracellular vesicle (sEV) isolation, with potential applications in basic research and clinical diagnostics.