An Affinity-based Method For Nondestructive Isolation Of SEVs

Small extracellular vesicles(sEVs)are nano-sized vesicles with lipid bilayer structure that can be secreted by most cells,which play an important role in cell-cell communication and cancer metastasis.However,due to its small particle size and low density,all the existing methods for extracting sEVs share different defects,and the effective separation and non-destructive release of sEVs still faces great challenges.Two Annexin V-based affinity magnetic beads were designed to address the current problems in the separation and purification of sEVs.One is to use FITC as an intermediate and attach Annexin V-FITC to anti-FITC magnetic beads.The other one is to couple Annexin V directly to carboxyl magnetic beads.These two kinds of magnetic beads share the same principle for the separation and purification of small extracellular vesicles,and both use Annexin V labeling magnetic beads,so that the beads can bind to phosphatidylserine(PS)on the outer surface of sEVs in a calcium dependent manner to accumulate PS positive sEVs.After magnetic separation,impurities other than the sEVs can be removed.More importantly,by adding chelating agent EDTA,these sEVs can be released and isolated non-destructively.This method was applied to isolate and purify sEVs from the culture supernatant of gastric cancer cell MGC-803.After detection using NTA,TEM and Western blot,the particle size,morphology and marker proteins of sEVs were found to be consistent with the standard.Compared with the sEVs extracted by the classical ultracentrifugation method,the size of the sEVs extracted by the two methods is basically the same,and the size distribution of the sEVs extracted by the affinity magnetic bead method is narrower.In terms of morphology,small-cell extracellular vesicles extracted by affinity magnetic bead methods can maintain their intact vesicle structure.In terms of marker proteins,affinity magnetic bead method is more inclined to get sEVs with CD63 positive.In addition,sEVs isolated using affinity magnetic bead methods maintained high biological activity in both the cell uptake assay and the transwell migration assay.Besides,this method is widely applicable and can also be applied to isolate sEVs from diverse biological samples of different species such as plasma,saliva,urine,cow milk,E.coli.culture supernatant quickly and efficiently.Hence,this highly efficient,nondestructive,economical and widely applicable method for sEVs isolation provides a more convenient strategy for the study of their biological functions and an additional option for clinical study of sEVs.