Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles
Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles
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DOI：
10.1016/j.jconrel.2013.08.014
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发表时间：
2013-11-28
影响因子：
10.8
通讯作者：
Vader, Pieter
中科院分区：
文献类型：
--
作者：
Kooijmans, Sander A. A.;Stremersch, Stephan;Vader, Pieter
细胞外囊泡（EV）是蛋白质和核酸的特化内源性载体，参与细胞间通讯。因此，EVs被提议作为用于递送核酸和其他大分子的候选药物递送系统。然而，由于缺乏用核酸装载囊泡的技术，基于 EV 的药物递送系统的制备受到阻碍。在这项工作中，我们现在详细描述了为此目的使用电穿孔方法的特征。当使用荧光标记的 siRNA 对 EV 进行电穿孔时，siRNA 保留与之前发表的结果相当（基于荧光光谱和荧光波动光谱的 20-25%），并且使用未标记的 siRNA 进行电穿孔导致 EV 颗粒中的 siRNA 保留显着（通过 RT-PCR 测量）。值得注意的是，当 siRNA 在不存在 EV 的情况下进行电穿孔时，测量到类似甚至更高的 siRNA 保留。纳米颗粒跟踪分析和共聚焦显微镜显示，电穿孔后大量形成不溶性 siRNA 聚集体，通过添加 EDTA 可以显着减少这种聚集体。其他减少聚集体形成的策略，包括使用带有导电聚合物电极的比色皿和使用酸性柠檬酸盐电穿孔缓冲液，比 EDTA 更有效地减少 siRNA 沉淀。然而，在这些条件下，siRNA 保留低于 0.05%，并且在存在或不存在 EV 的情况下电穿孔的样品之间没有测量到 siRNA 保留的显着差异。我们的结果表明，用 siRNA 电穿孔 EV 伴随着广泛的 siRNA 聚集体形成，这可能会导致高估实际加载到 EV 中的 siRNA 量。此外，我们的数据清楚地表明电穿孔的效率远低于之前描述的效率，并强调需要采用替代方法来制备负载 siRNA 的 EV。 (C) 2013 Elsevier B.V. 保留所有权利。
Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20-25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in siRNA retention could be measured between samples electroporated in the presence or absence of EVs. Our results show that electroporation of EVs with siRNA is accompanied by extensive siRNA aggregate formation, which may cause overestimation of the amount of siRNA actually loaded into EVs. Moreover, our data clearly illustrate that electroporation is far less efficient than previously described, and highlight the necessity for alternative methods to prepare siRNA-loaded EVs. (C) 2013 Elsevier B. V. All rights reserved.