Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
J Pharm Sci. 2021 Sep;110(9):3261-3267. doi: 10.1016/j.xphs.2021.05.021. Epub 2021 Jun 4.
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.
小细胞外囊泡 (sEVs) 是细胞间通讯的重要介质，因此有望成为药物递送的有前途的载体。了解影响 sEV 药代动力学的因素对于将其作为药物递送载体的应用至关重要。在这项研究中，通过评估酶去糖基化处理对 sEV 药代动力学的影响，研究了 sEV 表面糖基的作用。首先，使用控制糖蛋白胎球蛋白来优化糖苷酶处理条件。然后用糖苷酶，即肽-N-糖苷酶 F 或 O-糖苷酶处理标记有融合蛋白（包括 Gag 蛋白和 Gaussia 荧光素（gLuc）的 B16-BL6 衍生的 sEV），分别切割 N-和 O-聚糖。糖苷酶处理的 sEV 表现出与未处理的 sEV 相当的物理化学特性。然而，从 B16-BL6 sEV 上去除 N-聚糖增强了腹腔巨噬细胞的摄取，而通过流式细胞术评估，去除 O-聚糖的影响最小。为了确定表面糖基对 sEV 药代动力学的影响，然后将用或不用糖苷酶处理的 Gag-gLuc 标记的 B16-BL6 sEV 静脉注射到小鼠体内。糖苷酶处理的 sEV 从血液循环中的清除速度与未处理的 sEV 几乎相同。这些结果表明，尽管表面糖基对细胞摄取有影响，但对 sEV 药代动力学的影响很小。
