Gene transfer by means of lipo- and polyplexes: Role of clathrin and caveolae-mediated endocytosis
Gene transfer by means of lipo- and polyplexes: Role of clathrin and caveolae-mediated endocytosis
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DOI：
10.1080/08982100600848819
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发表时间：
2006-01-01
影响因子：
4.4
通讯作者：
Hoekstra, Dick
中科院分区：
文献类型：
--
作者：
Rejman, Joanna;Conese, Massimo;Hoekstra, Dick
在本文中，我们解决的贡献不同的内吞途径的细胞内摄取和处理不同大小的乳胶颗粒和质粒DNA复合物通过荧光显微镜和流式细胞仪分析。通过使用网格蛋白依赖性或小窝依赖性内吞作用的一些特异性抑制剂，我们能够区分这两种途径。小于200 nm的乳胶颗粒完全通过网格蛋白介导的内吞作用内化，而较大的颗粒通过小窝依赖的pathway进入细胞。质粒DNA复合物的摄取途径似乎强烈依赖于复合物的性质。因此，含有阳离子脂质DOTAP的脂质复合物仅通过网格蛋白依赖性机制内化，而由阳离子聚合物聚乙烯亚胺（PEI）制备的聚合复合物通过两种途径以大致相等的比例内化。孵育后的细胞与含有荧光素酶基因的lipoplexes丰富的荧光素酶的表达，这是有效地阻止网格蛋白依赖性内吞作用的抑制剂，但不是由小窝依赖性摄取机制的抑制剂。相比之下，荧光素酶转染的细胞与复合物是不受网格蛋白介导的内吞作用的抑制，但几乎完全阻断抑制剂干扰的小窝途径。结果进行了讨论，相对于可能的差异，质粒DNA从lipoplexes和polyplexes释放到胞质溶胶中的机制，并在摄取和处理的复合物的大小的作用。我们的数据表明，非病毒基因转染的改善可以非常受益于控制颗粒大小，这将允许通过非降解途径靶向颗粒内化，涉及小窝介导的内吞作用。
In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.