Determining The Role of Surface Glycans in The Pharmacokinetics of Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication and are thereby expected to be promising carriers for drug delivery. Understanding the factors that affect sEV pharmacokinetics is crucial for its application as a drug delivery carrier. In this study, the role of sEV surface glycans was investigated by evaluating the effects of enzymatic deglycosylation treatment on sEV pharmacokinetics. First, control glycoprotein fetuin was used to optimize the glycosidase treatment conditions. B16-BL6-derived sEVs labeled with fusion proteins comprising Gag protein and Gaussia luciferase (gLuc) (Gag-gLuc) were then treated with glycosidases, Peptide-N-Glycosidase F or O-glycosidase, which cleaves N- and O-glycans, respectively. Glycosidase-treated sEVs showed physicochemical characteristics comparable to those of the untreated sEVs. However, removal of N-glycans from B16-BL6 sEVs enhanced cellular uptake by the peritoneal macrophages, while the removal of O-glycans had minimal impact, as evaluated by flow cytometry. To determine the effect of surface glycans on the sEV pharmacokinetics, Gag-gLuc labeled B16-BL6 sEVs treated with or without glycosidases were then intravenously administered to mice. Glycosidase-treated sEVs showed almost identical clearance from the blood circulation as that of the untreated sEVs. These results suggest minimal impact of surface glycans on sEV pharmacokinetics, despites its effect on cellular uptake.