Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.
Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.
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DOI：
10.1002/jev2.12130
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发表时间：
2021-08
影响因子：
16
通讯作者：
Dekker N
中科院分区：
文献类型：
--
作者：
Silva AM;Lázaro-Ibáñez E;Gunnarsson A;Dhande A;Daaboul G;Peacock B;Osteikoetxea X;Salmond N;Friis KP;Shatnyeva O;Dekker N
细胞外囊泡（EV）已被广泛探索用于蛋白质的治疗性递送。然而，缺乏定量装载到工程化EV中的货物蛋白的方法。在这里，我们描述了在单囊泡和单分子水平上进行EV分析的工作流程，以准确量化不同EV分选蛋白在促进货物装载到EV中的效率。Expi 293 F细胞经工程改造以表达与绿色荧光蛋白（GFP）融合的EV分选蛋白。通过针对特定EV分选结构域的蛋白质印迹法证实了高水平的GFP加载到分泌的EV中，但是通过Nanoflow细胞术的定量单囊泡分析在不到一半的分析颗粒中检测到GFP，反映了EV异质性。ExoView中的抗四跨膜蛋白EV免疫染色证实了在CD 63+、CD 81+或CD 9 + EV的不同亚群中的异质性GFP分布。通过单分子定位显微术定量GFP向单个囊泡中的加载。综合结果表明，与PDGFRβ跨膜结构域融合的TSPAN 14、CD 63和CD 63/CD 81是最有效的EV分选蛋白，每个囊泡平均积累50-170个单个GFP分子。总之，我们验证了一套适用于EV制剂高分辨率分析的互补技术，这些技术可以可靠地捕获其异质性，并提出了用于EV工程应用的高效EV分选蛋白。
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single‐vesicle and single‐molecule level to accurately quantify the efficiency of different EV‐sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV‐sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV‐sorting domains, but quantitative single‐vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti‐tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single‐Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV‐sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high‐resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV‐sorting proteins to be used in EV engineering applications.
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影响因子：
46.9
作者：
Alvarez-Erviti, Lydia;Seow, Yiqi;Wood, Matthew J. A.
通讯作者：
Wood, Matthew J. A.
DOI：
10.1126/science.aau6977
发表时间：
2020-02-07
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Kalluri R;LeBleu VS
通讯作者：
LeBleu VS
影响因子：
17.1
作者：
Lee K;Fraser K;Ghaddar B;Yang K;Kim E;Balaj L;Chiocca EA;Breakefield XO;Lee H;Weissleder R
通讯作者：
Weissleder R
DOI：
10.1073/pnas.1521230113
发表时间：
2016-02-23
影响因子：
11.1
作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
通讯作者：
Thery, Clotilde
影响因子：
16
作者：
Gori, Alessandro;Romanato, Alessandro;Cretich, Marina
通讯作者：
Cretich, Marina