Development of enzymatic depletion methods for preparation of small extracellular vesicles with long blood-circulation half-life.
Development of enzymatic depletion methods for preparation of small extracellular vesicles with long blood-circulation half-life.
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开发用于制备具有长血液循环半衰期的小细胞外囊泡的酶耗竭方法。
DOI：
10.1007/s11095-022-03405-9
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Takakura Y.
中科院分区：
文献类型：
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作者：
Kobayashi Y;Kitamura S;Takahashi Y;Takakura Y.
目的磷脂酰丝氨酸（PS）缺陷的小细胞外囊泡（sEV）亚群（PS（−）sEV）在血液中长期循环，因此，它们有望具有治疗应用。然而，PS（-）sEV的有限生产使得它们的应用变得困难。在这项研究中，使用酶促反应制备这样的人群的方法developed.MethodsBulk sEVs收集从细胞培养上清液通过ultracenthegation进行酶促反应，使用磷脂酰丝氨酸脱羧酶（PSD）。使用与PS（+）sEV结合的磁珠估计PS（-）sEV的产量。然后，的物理性质和药代动力学（PK）的sEVs进行了evaluated.ResultsEnzymatic消耗PS暴露在sEV表面使用PSD的产量增加PS（−）sEVs。PSD处理几乎不改变PS（−）sEV的理化性质。此外，来自PSD处理的散装sEVs的PS（−）sEVs的血清浓度曲线和PK参数表明长的血液循环half-lifes.ConclusionsTreatment的sEVs与PSD成功地降低了表面PS水平，并增加了散装sEVs中的PS（−）sEV亚群的量。这种基于PSD治疗有效制备PS（−）sEV的方案以及关于基本PK的信息可以为sEV的治疗应用奠定基础。
PurposePhosphatidylserine (PS)-deficientsmall 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.MethodsBulk 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.ResultsEnzymatic 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.ConclusionsTreatment 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.