Development of Enzymatic Depletion Methods for Preparation of Small Extracellular Vesicles with Long Blood-Circulation Half-Life

Abstract Purpose Phosphatidylserine (PS)-deficient small extracellular vesicle (sEV) subpopulations (PS(−) sEVs) circulate in blood for long periods; hence, they are expected to have therapeutic applications. However, limited production of PS(−) sEVs makes their application difficult. In this study, a method for the preparation of such populations using an enzymatic reaction was developed. Methods Bulk sEVs collected from a cell culture supernatant via ultracentrifugation were subjected to an enzymatic reaction using phosphatidylserine decarboxylase (PSD). The yield of PS(−) sEVs was estimated using magnetic beads that bind to PS(+) sEVs. Then, the physical properties and pharmacokinetics (PK) of the sEVs were evaluated. Results Enzymatic depletion of PS exposed on sEV surfaces using PSD increased the yield of PS(−) sEVs. PSD treatment hardly changed the physicochemical properties of PS(−) sEVs. Moreover, the serum concentration profile and PK parameters of the PS(−) sEVs derived from PSD-treated bulk sEVs indicated a long blood-circulation half-life. Conclusions Treatment of sEVs with PSD successfully reduced surface PS levels and increased the amount of the PS(−) sEV subpopulation among bulk sEVs. This protocol of efficient preparation of PS(−) sEVs based on PSD treatment, as well as information on the basic PK, can be foundational for the therapeutic application of sEVs.