Tros de Ilarduya C, Arangoa M A, Moreno-Aliaga M J, Düzgüneş N
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, 31080 Pamplona, Spain.
Biochim Biophys Acta. 2002 Apr 12;1561(2):209-21. doi: 10.1016/s0005-2736(02)00348-6.
Cationic liposomes and the complexes they form with DNA (lipoplexes) constitute the most promising alternative to the use of viral vectors for gene therapy. One of the limitations to their application in vivo, however, is the inhibition of gene delivery by serum. In a previous study, we demonstrated that transferrin (Tf)-lipoplexes were superior to plain lipoplexes in transfecting HeLa cells in the presence of high concentrations of serum. With the goal of obtaining efficient gene expression in vivo, we evaluated the efficacy of Tf-lipoplexes (containing DOTAP and cholesterol) in transfecting primary hepatocytes and adipocytes in the presence of high serum concentrations. The association of transferrin with cationic liposomes increased luciferase expression compared to plain lipoplexes in primary cells as well as in HepG2 and 3T3-L1 differentiated adipocytes. The complexes were not cytotoxic and were highly effective in protecting DNA from attack by DNase I. An efficient and reliable method was developed to prepare lipoplexes containing both Tf and protamine sulfate, where the latter was mixed with transferrin, followed by the addition of cationic liposomes and DNA. The resulting protamine-Tf-lipoplexes increased significantly the levels of gene expression in cultured cells and in various tissues in mice following i.v. administration.
阳离子脂质体及其与DNA形成的复合物（脂质体复合物）是基因治疗中使用病毒载体最有前景的替代方案。然而，它们在体内应用的局限性之一是血清对基因传递的抑制作用。在先前的一项研究中，我们证明了转铁蛋白（Tf）-脂质体复合物在高浓度血清存在下转染HeLa细胞方面优于普通脂质体。为了在体内获得有效的基因表达，我们评估了Tf-脂质体复合物（含有DOTAP和胆固醇）在高血清浓度下转染原代肝细胞和脂肪细胞的效果。与普通脂质体复合物相比，转铁蛋白与阳离子脂质体的结合在原代细胞以及HepG2和3T3-L1分化脂肪细胞中增加了荧光素酶的表达。这些复合物没有细胞毒性，并且在保护DNA免受DNase I攻击方面非常有效。开发了一种有效且可靠的方法来制备同时含有Tf和硫酸鱼精蛋白的脂质体复合物，其中将后者与转铁蛋白混合，然后加入阳离子脂质体和DNA。静脉注射后，所得的鱼精蛋白-Tf-脂质体复合物显著提高了培养细胞和小鼠各种组织中的基因表达水平。
