Penacho Nuno, Simões Sérgio, de Lima Maria C Pedroso
Department of Vectors and Gene Therapy at the Center for Neuroscience and Cell Biology, Faculty of Science and Technology, University of Coimbra, Portugal.
Mol Membr Biol. 2009 May;26(4):249-63. doi: 10.1080/09687680902766716. Epub 2009 Mar 10.
Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.
在过去几年中，阳离子脂质体介导的非病毒基因递送取得了显著进展。然而，就转染效率而言，所获得的结果仍远不能令人满意，特别是与使用病毒载体所达到的转染效率相比。我们之前已经证明，转铁蛋白与阳离子脂质体的结合在体外和体内的多种细胞中均能显著提高转染效率。在这项工作中，我们探索了几种策略，以进一步提高转铁蛋白相关脂质体复合物介导的转染效率。为此，研究了低分子量（2.7、2.0和0.8 kDa）聚乙烯亚胺在不同N/P比下对DNA预凝聚、脂质组成、阳离子脂质/DNA（+/-）电荷比以及脂质体复合物中表面活性剂的存在对转染的影响。测试了两种不同的脂质体制备模式，并评估了它们的复合物与DNA的细胞结合程度以及保护所携带DNA的能力。我们的结果表明，通过乙醇注入法制备的阳离子脂质体所产生的复合物，其中携带的DNA用低分子量聚乙烯亚胺预凝聚，在介导转染方面非常高效。转铁蛋白与各种脂质体制剂结合时观察到的对聚乙烯亚胺复合物介导的转染的差异调节作用表明，这些复合物通过不同途径（涉及网格蛋白与小窝蛋白）进入细胞，很可能是利用它们固有的生物物理性质从内体逃逸到细胞质中。
