Department of Chemical Biology, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China.
Anal Bioanal Chem. 2023 Mar;415(7):1287-1298. doi: 10.1007/s00216-022-04248-4. Epub 2022 Aug 10.
Extracellular vesicles (EVs) have emerged as an attractive drug delivery system owing to their natural roles in intercellular communication. On account of the large intrinsic heterogeneity of EVs, it is highly desirable to evaluate not only the encapsulation efficiency but also the alteration of biological functionality after the drug-loading process at the single-particle level. However, the nanoscale size of EVs poses a great challenge. Taking advantage of nano-flow cytometry (nFCM) in the multiparameter analysis of single EVs as small as 40 nm, six commonly used drug-loading strategies (coincubation, electroporation, extrusion, freeze-thawing, sonication, and surfactant treatment) were exploited by employing doxorubicin (Dox) as the model drug. Encapsulation ratio, EV concentration, drug content, and membrane proteins of Dox-loaded EVs were measured at the single-particle level. Our data indicated that coincubation and electroporation outperformed other methods with an encapsulation ratio of approximately 45% and a higher Dox content in single EVs. Interestingly, the labeling ratios of membrane proteins indicated that varying degrees of damage to the surface proteins of EVs occurred upon extrusion, freeze-thawing, sonication, and surfactant treatment. Confocal fluorescence microscopy and flow cytometry analysis revealed that Dox-loaded EVs prepared by electroporation induced the strongest apoptosis followed by coincubation. These results correlated well with their cellular uptake rate and fundamentally with the Dox encapsulation efficiency of single EVs. nFCM provides a rapid and sensitive platform for single-particle assessment of drug-loading strategies for incorporating drugs into EVs.
细胞外囊泡 (EVs) 因其在细胞间通讯中的天然作用而成为一种有吸引力的药物传递系统。由于 EVs 的内在异质性很大，因此非常需要在单颗粒水平上不仅评估封装效率，而且还评估药物加载过程后生物功能的变化。然而，EVs 的纳米级尺寸带来了巨大的挑战。利用纳米流式细胞术 (nFCM) 对小至 40nm 的单个 EV 进行多参数分析，可以利用六种常用的药物加载策略（共孵育、电穿孔、挤出、冻融、超声处理和表面活性剂处理），并用阿霉素 (Dox) 作为模型药物。在单颗粒水平上测量载药 EV 的包封率、EV 浓度、药物含量和膜蛋白。我们的数据表明，共孵育和电穿孔的包封率约为 45%，且单 EV 中的 Dox 含量更高，优于其他方法。有趣的是，膜蛋白的标记比率表明，在挤出、冻融、超声处理和表面活性剂处理过程中，EV 表面蛋白发生了不同程度的损伤。共聚焦荧光显微镜和流式细胞术分析显示，电穿孔制备的载 Dox EV 诱导的细胞凋亡最强，其次是共孵育。这些结果与它们的细胞摄取率以及与单 EV 的 Dox 封装效率密切相关。nFCM 为评估将药物纳入 EV 的药物加载策略的单颗粒提供了快速而灵敏的平台。
