Vandenbroucke Roosmarijn E, De Smedt Stefaan C, Demeester Joseph, Sanders Niek N
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
Biochim Biophys Acta. 2007 Mar;1768(3):571-9. doi: 10.1016/j.bbamem.2006.11.006. Epub 2006 Nov 16.
Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.
几份报告显示，经TAT修饰的脂质体和颗粒能快速高效地转运至细胞质中。然而，TAT修饰的脂质体的摄取机制和生物学效应均未得到详细研究。在本报告中，我们表明TAT修饰的脂质体基因转移的增加取决于脂质体中阳离子脂质的量以及TAT与脂质体偶联的方式。我们证明，TAT修饰和未修饰的脂质体的细胞摄取都非常迅速，并且与先前的报道相反，是温度依赖性的。此外，内化后，TAT修饰和未修饰的脂质体最终都存在于溶酶体囊泡中，表明网格蛋白介导的内吞作用参与其中。此外，氯丙嗪是网格蛋白依赖性内吞作用的特异性抑制剂，它能强烈抑制TAT修饰和未修饰的脂质体的细胞摄取和生物学活性。我们还发现，当细胞膜中的胆固醇被制霉菌素（一种脂质筏介导途径的抑制剂）结合时，这些脂质体的摄取和生物学活性会降低。考虑到这些数据，我们得出结论，TAT修饰和未修饰的脂质体主要通过胆固醇依赖性网格蛋白介导的途径内化。
