Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences , Kyoto University , Sakyo-ku , Kyoto 606-8501 , Japan.
Department of Biopharmaceutics , Kyoto Pharmaceutical University , Misasagi, Yamashina-ku , Kyoto 607-8414 , Japan.
Mol Pharm. 2018 Mar 5;15(3):1073-1080. doi: 10.1021/acs.molpharmaceut.7b00950. Epub 2018 Feb 6.
Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin αβ, an integrin known to be involved in lung targeting, was degraded after ProK treatment. The ProK treatment significantly reduced the lung distribution of EVs after intravenous injection. These results indicate that the surface proteins of EVs such as integrin αβ play some roles in pharmacokinetics in terms of reducing Vd and their distribution to the lung.
细胞外囊泡 (EVs) 是由细胞分泌的小膜囊泡，具有作为药物递送载体的巨大潜力。EV 膜表面蛋白可能在药代动力学中发挥作用。一种可以用于研究 EV 膜表面作用的方法是修饰 EV 的内部空间。在本研究中，我们构建了一个表达来自 Moloney 鼠白血病病毒 (Gag) 和 Gaussia 荧光素酶 (gLuc) 的融合蛋白的质粒 DNA (Gag-gLuc)，以修饰 EV 的内部空间。从 B16BL6 黑色素瘤细胞中收集 EV，用质粒转染，并通过差速超速离心法分离。Gag-gLuc EV 是带负电荷的球形囊泡，直径约为 100nm。gLuc 标记的 Gag-gLuc EV 在血清中稳定。Gag-gLuc EV 经蛋白酶 K (ProK) 处理后 gLuc 活性最小降低，表明 gLuc 被修饰在 EV 的内部空间中。然后，为了评估 EV 表面蛋白对其药代动力学的影响，用 ProK 处理 Gag-gLuc EV 并静脉注射给小鼠。经 ProK 处理的 EV 的分布容积 (Vd) 明显小于未处理的 EV。此外，已知参与肺靶向的整合素 αβ 在 ProK 处理后被降解。ProK 处理后显著降低了 EV 经静脉注射后的肺分布。这些结果表明，EV 表面蛋白（如整合素 αβ）在降低 Vd 和其向肺分布方面在药代动力学中发挥一定作用。
