Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro - A quantitative study
Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro - A quantitative study
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DOI：
10.1016/j.jconrel.2009.09.008
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发表时间：
2010-01-25
影响因子：
10.8
通讯作者：
Molema, Grietje
中科院分区：
文献类型：
--
作者：
Asgeirsdottir, Sigridur A.;Talman, Eduard G.;Molema, Grietje
小干扰RNA（siRNA）的应用要求siRNA特异性和有效地递送到特定细胞类型中。我们开发了一种新型的非病毒靶向系统，将siRNA特异性地递送到炎症激活的内皮细胞中。这是通过将阳离子两亲性脂质SAINT与识别炎性细胞粘附分子E-选择素的抗体缀合来实现的。这些抗-E-选择素-SAINT脂质复合物（SAINTarg）在体外和离体在经受炎性条件的人肾组织切片中维持亲本抗体的抗原识别能力。常规SAINT介导的转染导致人微血管内皮细胞（HMEC-1）和条件永生化肾小球内皮细胞（ciGEnC）中的有效基因沉默。然而，原代人脐静脉内皮细胞（HUVEC）转染差，我们可以定量相关的细胞类型的特定能力，以促进siRNA的摄取的现象。重要的是，SAINTarg增加了活化内皮细胞的siRNA摄取和转染特异性。与用非靶向SAINT转染相比，用SAINTarg转染将显著更多的siRNA递送到活化的HUVEC中。siRNA摄取的增强通过激活的HUVEC中VE-钙粘蛋白的基因和蛋白表达的沉默的改善得到证实，表明SAINTarg将功能活性siRNA递送到内皮细胞中。所获得的结果证明了小核苷酸载体系统的成功设计，其具有改善的和特异性的siRNA递送到否则难以抑制的原代内皮细胞中，这另外显著减少了基因沉默所需的siRNA的量。(C)2009 Elsevier B. V.保留所有权利。
Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was Corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. (C) 2009 Elsevier B.V. All rights reserved.