In vitro evaluation of endothelial exosomes as carriers for small interfering ribonucleic acid delivery.
In vitro evaluation of endothelial exosomes as carriers for small interfering ribonucleic acid delivery.
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DOI：
10.2147/ijn.s64267
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发表时间：
2014
影响因子：
8
通讯作者：
He J
中科院分区：
文献类型：
--
作者：
Banizs AB;Huang T;Dryden K;Berr SS;Stone JR;Nakamoto RK;Shi W;He J
外泌体是来自多泡体的纳米尺寸细胞外囊泡的一个亚群，尺寸在30至150 nm范围内，成为用于小干扰核糖核酸（siRNA）递送的有前途的载体，因为它们能够通过携带的小非编码RNA、信使RNA、脱氧核糖核酸和蛋白质在细胞之间传递分子信息。内皮细胞参与许多重要的生物学过程，并且是循环外泌体的主要来源。在这项研究中，我们从内皮细胞中制备了外泌体，并评估了它们将siRNA递送到原代内皮细胞中的能力。通过连续离心和超离心从培养的小鼠主动脉内皮细胞中分离和纯化外泌体。与来自其他细胞来源的外泌体颗粒类似，内皮外泌体是纳米大小的囊泡，通过NanoSight仪器和透射电子显微镜检查。酶联免疫吸附测定分析证实了两种外泌体标志物：CD9和CD63的表达。流式细胞术和荧光显微镜研究表明，内皮外泌体在细胞内的分布是不均匀的。在一项对表达荧光素酶的内皮细胞进行的基因沉默研究中，装载有siRNA的外泌体抑制荧光素酶表达超过40%。相比之下，单独的siRNA和对照siRNA仅抑制荧光素酶表达小于15%。总之，我们证明了内皮外泌体具有容纳和递送短的外源核酸到内皮细胞中的能力。
Exosomes, one subpopulation of nanosize extracellular vesicles derived from multivesicular bodies, ranging from 30 to 150 nm in size, emerged as promising carriers for small interfering ribonucleic acid (siRNA) delivery, as they are capable of transmitting molecular messages between cells through carried small noncoding RNAs, messenger RNAs, deoxyribonucleic acids, and proteins. Endothelial cells are involved in a number of important biological processes, and are a major source of circulating exosomes. In this study, we prepared exosomes from endothelial cells and evaluated their capacity to deliver siRNA into primary endothelial cells. Exosomes were isolated and purified by sequential centrifugation and ultracentrifugation from cultured mouse aortic endothelial cells. Similar to exosome particles from other cell sources, endothelial exosomes are nanometer-size vesicles, examined by both the NanoSight instrument and transmission electron microscopy. Enzyme-linked immunosorbent assay analysis confirmed the expression of two exosome markers: CD9 and CD63. Flow cytometry and fluorescence microscopy studies demonstrated that endothelial exosomes were heterogeneously distributed within cells. In a gene-silencing study with luciferase-expressing endothelial cells, exosomes loaded with siRNA inhibited luciferase expression by more than 40%. In contrast, siRNA alone and control siRNA only suppressed luciferase expression by less than 15%. In conclusion, we demonstrated that endothelial exosomes have the capability to accommodate and deliver short foreign nucleic acids into endothelial cells.