Gene delivery by dendrimers operates via a cholesterol dependent pathway
Gene delivery by dendrimers operates via a cholesterol dependent pathway
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DOI：
10.1093/nar/gkh595
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发表时间：
2004-05-01
影响因子：
14.9
通讯作者：
George, AJT
中科院分区：
文献类型：
--
作者：
Manunta, M;Tan, PH;George, AJT
了解树突- dna复合物的细胞摄取和细胞内运输是提高非病毒载体介导的基因传递转染效率的重要前提。树状大分子是用于基因转移的合成聚合物。尽管这些阳离子分子显示出作为多功能DNA载体的希望，但对基因传递的机制知之甚少。本文以内皮细胞系为模型，研究了树突的摄取是如何发生的，并评估了树突的内化是在细胞表面随机发生还是在膜筏等优先位置发生。在提取质膜胆固醇后，树状大分子传递的基因转染效率急剧下降。补充膜胆固醇可恢复基因表达。在转染前，由于胆固醇的消耗，树突的结合尤其是内化被强烈地减少。然而，在转染后去除胆固醇并没有抑制所传递基因的表达。在免疫沉淀试验和共聚焦显微镜研究中，荧光树突与存在于膜筏中的神经节苷脂GM1共定位。这些数据有力地表明，膜胆固醇和筏完整性在生理上与树突- dna复合物的细胞摄取有关。因此，这些发现提供了证据，证明膜筏对于基因治疗中非病毒载体的内化是重要的。
Understanding the cellular uptake and intracellular trafficking of dendrimer-DNA complexes is an important prerequisite for improving the transfection efficiency of non-viral vector-mediated gene delivery. Dendrimers are synthetic polymers used for gene transfer. Although these cationic molecules show promise as versatile DNA carriers, very little is known about the mechanism of gene delivery. This paper investigates how the uptake occurs, using an endothelial cell line as model, and evaluates whether the internalization of dendriplexes takes place randomly on the cell surface or at preferential sites such as membrane rafts. Following extraction of plasma membrane cholesterol, the transfection efficiency of the gene delivered by dendrimers was drastically decreased. Replenishment of membrane cholesterol restored the gene expression. The binding and especially internalization of dendriplexes was strongly reduced by cholesterol depletion before transfection. However, cholesterol removal after transfection did not inhibit expression of the delivered gene. Fluorescent dendriplexes co-localize with the ganglioside GM1 present into membrane rafts in both an immunoprecipitation assay and confocal microscopy studies. These data strongly suggest that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer-DNA complexes. Hence these findings provide evidence that membrane rafts are important for the internalization of non-viral vectors in gene therapy.