Extracellular vesicles (EVs) have been considered to deliver biological cargos between cells and mediate intercellular communication. However, the mechanisms that underlie the biological process of EV uptake and cytoplasmic cargo release in recipient cells are largely unknown. Quantitative and real-time assays for assessment of the cargo delivery efficiency inside recipient cells have not been feasible. In this study, we developed an EV cargo delivery (EVCD) assay using a split luciferase called the NanoBiT system. Recipient cells expressing LgBiT, a large subunit of luciferase, emit luminescence when the EV cargo proteins fused with a small luminescence tag (HiBiT tag) that can complement LgBiT are delivered to the cytoplasm of recipient cells. Using the EVCD assay, the cargo delivery efficiency of EVs could be quantitatively measured in real time. This assay was highly sensitive in detecting a single event of cargo delivery per cell. We found that modification of EVs with a virus-derived fusogenic protein significantly enhanced the cytoplasmic cargo delivery; however, in the absence of a fusogenic protein, the cargo delivery efficiency of EVs was below the threshold of the assay. The EVCD assay could assess the effect of entry inhibitors on EV cargo delivery. Furthermore, using a luminescence microscope, the cytoplasmic cargo delivery of EVs was directly visualized in living cells. This assay could reveal the biological mechanism of the cargo delivery processes of EVs.

中文翻译：

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实时发光测定细胞外小泡胞质货物的运输

细胞外囊泡（EVs）已被认为可以在细胞之间传递生物货物并介导细胞间通讯。然而，在受体细胞中EV摄取和细胞质货物释放的生物学过程的基础机制尚不清楚。用于评估受体细胞内部货物递送效率的定量和实时测定尚不可行。在这项研究中，我们使用称为NanoBiT系统的拆分萤光素酶开发了EV货物交付（EVCD）分析。当将可与LgBiT互补的小发光标签（HiBiT标签）融合的EV货物蛋白递送到受体细胞的细胞质中时，表达LgBiT（荧光素酶的大亚基）的受体细胞会发光。使用EVCD分析，电动汽车的货运效率可以实时定量测量。该测定法对检测每个细胞的单个货物递送事件非常敏感。我们发现用病毒衍生的融合蛋白修饰电动汽车可显着增强细胞质货物的运输。然而，在不存在融合蛋白的情况下，电动汽车的货物运输效率低于测定的阈值。EVCD分析可以评估进入抑制剂对EV货物运输的影响。此外，使用发光显微镜，可在活细胞中直接观察到EV的胞质货物输送。该测定法可以揭示电动汽车的货运过程的生物学机制。我们发现用病毒衍生的融合蛋白修饰电动汽车可显着增强细胞质货物的运输。然而，在不存在融合蛋白的情况下，电动汽车的货物运输效率低于测定的阈值。EVCD分析可以评估进入抑制剂对EV货物运输的影响。此外，使用发光显微镜，可在活细胞中直接观察到EV的胞质货物递送。该测定法可以揭示电动汽车的货运过程的生物学机制。我们发现用病毒衍生的融合蛋白修饰电动汽车可显着增强细胞质货物的运输。然而，在不存在融合蛋白的情况下，电动汽车的货物运输效率低于测定的阈值。EVCD分析可以评估进入抑制剂对EV货物运输的影响。此外，使用发光显微镜，可在活细胞中直接观察到EV的胞质货物输送。该测定法可以揭示电动汽车的货运过程的生物学机制。使用发光显微镜，可以在活细胞中直接观察到EV的胞质货物运输。该测定法可以揭示电动汽车的货运过程的生物学机制。使用发光显微镜，可以在活细胞中直接观察到EV的胞质货物运输。该测定法可以揭示电动汽车的货运过程的生物学机制。