da Cruz M T, Simões S, Pires P P, Nir S, de Lima M C
Department of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, Portugal.
Biochim Biophys Acta. 2001 Feb 9;1510(1-2):136-51. doi: 10.1016/s0005-2736(00)00342-4.
We investigated the mode of interaction of lipoplexes (DOTAP:DOPE/DNA) with HeLa cells, focusing on the analysis of the initial steps involved in the process of gene delivery. We evaluated the effect of different factors, namely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin into the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were performed upon cell treatment with inhibitors of endocytosis. Our results indicate that a decrease of the net charge of the complexes (upon addition of DNA) generally leads to a decrease in the extent of binding, cell association and fusion, except for the neutral complexes. Association of transferrin to the lipoplexes resulted in a significant enhancement of the interaction processes referred to above, which correlates well with the promotion of transfection observed under the same conditions. Besides triggering internalization of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was reduced upon their incubation with cells in the presence of serum, suggesting that serum components limit the transferrin fusogenic properties. Results were analyzed by using a theoretical model which allowed to estimate the kinetic parameters involved in lipoplex--cell interactions. The deduced fusion and endocytosis rate constants are discussed and compared with those obtained for other biological systems. From the kinetic studies we found a twofold enhancement of the fusion rate constant (f) for the ternary lipoplexes. We also concluded that HeLa cells yield a relatively low rate of endocytosis. Overall, our results estimate the relative contribution of fusion of lipoplexes with the plasma membrane, endocytosis and fusion with the endosomal membrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies.
我们研究了脂质体（DOTAP:DOPE/DNA）与HeLa细胞的相互作用模式，重点分析了基因递送过程中涉及的初始步骤。我们评估了不同因素的影响，即阳离子脂质与DNA的化学计量比、细胞培养基中血清的存在以及配体转铁蛋白掺入脂质体对脂质体与细胞的结合、缔合和融合（脂质混合）程度的影响。在用内吞作用抑制剂处理细胞时进行了平行实验。我们的结果表明，复合物净电荷的减少（加入DNA后）通常会导致结合、细胞缔合和融合程度的降低，但中性复合物除外。转铁蛋白与脂质体的缔合导致上述相互作用过程显著增强，这与在相同条件下观察到的转染促进作用密切相关。除了引发复合物的内化外，转铁蛋白还被证明介导与内体膜的融合。在血清存在下，这种类型的复合物与细胞孵育时，融合程度降低，这表明血清成分限制了转铁蛋白的融合特性。通过使用理论模型分析结果，该模型允许估计脂质体与细胞相互作用中涉及的动力学参数。讨论了推导的融合和内吞速率常数，并与其他生物系统获得的常数进行了比较。从动力学研究中，我们发现三元脂质体的融合速率常数（f）提高了两倍。我们还得出结论，HeLa细胞的内吞速率相对较低。总体而言，我们的结果估计了脂质体与质膜融合、内吞作用以及与内体膜融合对其与细胞相互作用的相对贡献，这些信息对于基因治疗策略的发展至关重要。
