Agitation during lipoplex formation improves the gene knockdown effect of siRNA
Agitation during lipoplex formation improves the gene knockdown effect of siRNA
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DOI：
10.1016/j.ijpharm.2011.03.001
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发表时间：
2011-05-30
影响因子：
5.8
通讯作者：
Kiwada, Hiroshi
中科院分区：
文献类型：
--
作者：
Barichello, Jose Mario;Kizuki, Shinji;Kiwada, Hiroshi
治疗性siRNA成功递送到指定的靶细胞及其在细胞内作用位点的可用性是RNAi治疗成功的关键要求。在本研究中，我们重点研究了siRNA-脂质体的制备工艺及其对siRNA基因敲除效率的影响。研究了siRNA-lipoplex （vo - ltsir）制备过程中的搅拌（旋涡混合）及其对稳定表达的细胞GFP基因敲低效率的影响，并将其与自发形成的lipoplex （spoo - ltsir）的效率进行了比较。在N/P比为7.62 （siRNA剂量为30 nM）时，尽管两种脂质体都带正电，但它们的大小却存在显著差异。在siRNA剂量为30 nM时，vor-LTsiR完成了50%的基因敲低，而spot - ltsir在120 nM水平上取得了类似的敲低效果，这表明制备过程显著影响siRNA的基因敲低效果。vor-LTsiR的摄取主要通过网格蛋白介导的内吞作用，而spot - ltsir的摄取主要通过膜融合。此外，通过抑制网格蛋白介导的内吞作用，基因敲低效率显著降低。制备过程所促进的脂质体的大小很可能定义了进入途径，导致细胞内化siRNA的数量增加，并增强了基因敲低的功效。本研究的结果明确表明，适当的siRNA脂质体制备程序对细胞摄取效率有重大贡献，从而对siRNA的基因敲除效率有重大贡献。(C) 2011 Elsevier B.V.版权所有
The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA. (C) 2011 Elsevier B.V. All rights reserved.