Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands.
Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands.
J Control Release. 2017 Nov 28;266:100-108. doi: 10.1016/j.jconrel.2017.09.019. Epub 2017 Sep 14.
Recent evidence has established that extracellular vesicles (EVs), including exosomes and microvesicles, form an endogenous transport system through which biomolecules, including proteins and RNA, are exchanged between cells. This endows EVs with immense potential for drug delivery and regenerative medicine applications. Understanding the biology underlying EV-based intercellular transfer of cargo is of great importance for the development of EV-based therapeutics. Here, we sought to characterize the cellular mechanisms involved in EV uptake. Internalization of fluorescently-labeled EVs was evaluated in HeLa cells, in 2D (monolayer) cell culture as well as 3D spheroids. Uptake was assessed using flow cytometry and confocal microscopy, using chemical as well as RNA interference-based inhibition of key proteins involved in individual endocytic pathways. Experiments with chemical inhibitors revealed that EV uptake depends on cholesterol and tyrosine kinase activity, which are implicated in clathrin-independent endocytosis, and on Na/H exchange and phosphoinositide 3-kinase activity, which are important for macropinocytosis. Furthermore, EV internalization was inhibited by siRNA-mediated knockdown of caveolin-1, flotillin-1, RhoA, Rac1 and PAK1, but not clathrin heavy chain. Together, these results suggest that EVs enter cells predominantly via clathrin-independent endocytosis and macropinocytosis. Identification of EV components that promote their uptake via pathways that lead to functional cargo transfer might allow development of more efficient therapeutics through EV-inspired engineering.
最近的证据表明，细胞外囊泡（EVs），包括外泌体和微泡，形成了一种内源性的运输系统，通过该系统，生物分子（包括蛋白质和 RNA）在细胞之间进行交换。这使得 EVs 在药物输送和再生医学应用方面具有巨大的潜力。了解基于 EV 的细胞间货物转移的生物学基础对于基于 EV 的治疗方法的发展非常重要。在这里，我们试图表征参与 EV 摄取的细胞机制。使用荧光标记的 EV 评估 HeLa 细胞中的内化，在二维（单层）细胞培养以及 3D 球体中进行。使用化学方法和针对参与各个内吞途径的关键蛋白的 RNA 干扰抑制来评估摄取，使用流式细胞术和共聚焦显微镜。化学抑制剂实验表明，EV 的摄取取决于胆固醇和酪氨酸激酶活性，这与网格蛋白非依赖性内吞作用有关，以及 Na/H 交换和磷酸肌醇 3-激酶活性，这对巨胞饮作用很重要。此外，EV 的内化通过小干扰 RNA 介导的窖蛋白 1、Flotillin-1、RhoA、Rac1 和 PAK1 的敲低而被抑制，但不是网格蛋白重链。总之，这些结果表明，EV 主要通过网格蛋白非依赖性内吞作用和巨胞饮作用进入细胞。鉴定通过导致功能性货物转移的途径促进其摄取的 EV 成分可能允许通过基于 EV 的工程开发更有效的治疗方法。
