Faculté de Pharmacie, Univ Paris-Sud, 5 Rue J.B. Clément, 92296 Châtenay-Malabry Cedex, France.
J Control Release. 2012 Sep 28;162(3):545-52. doi: 10.1016/j.jconrel.2012.07.015. Epub 2012 Jul 20.
The mechanism by which hyaluronic acid (HA)-bearing lipoplexes target the A549 lung cancer cell line was evaluated. For this purpose, cationic liposomes targeting the CD44 receptor were designed thanks to the incorporation in their composition of a conjugate between high molecular weight HA and the lipid DOPE (HA-DOPE). Liposomes containing HA-DOPE were complexed at different lipids:DNA ratios with a reporter plasmid encoding the green fluorescent protein (GFP). Diameter, zeta potential, lipoplex stability and DNA protection from nucleases have been determined. Lipids:DNA ratios of 2, 4 and 6 provided a diameter around 250 nm with a zeta potential of -30 mV. The strength of lipids:DNA interaction and the fraction of DNA protected from enzymatic degradation increased with the lipids:DNA ratio. 2D-immunoelectrophoresis demonstrated the low capacity to activate the C3 fraction of the complement system of any of these three ratios, with and without HA-DOPE. Transfection efficiency in the presence of 0, 10 and 15% of HA-DOPE or unconjugated HA, was determined on the CD44-expressing A549 cells by flow cytometry. Lipoplexes at a lipids:DNA ratio of 2 containing 10% (w/w) of HA-DOPE were the most efficient for transfection. The maximal level of GFP expression was obtained after 6h of incubation demonstrating a slow transfection kinetics of lipoplexes. Finally, lipoplex cellular uptake, measured indirectly by the level of transfection using flow cytometry and validated by fluorescence microscopy, was shown to be mediated by the CD44 receptor and caveolae. These results demonstrate the strong specificity of DNA targeting through the CD44 receptor using HA of high molecular weight as a ligand.
研究了含透明质酸（HA）的脂质体靶向 A549 肺癌细胞系的机制。为此，设计了靶向 CD44 受体的阳离子脂质体，方法是在其组成中加入高分子量 HA 与脂质 DOPE（HA-DOPE）的缀合物。将含有 HA-DOPE 的脂质体在不同的脂质：DNA 比与编码绿色荧光蛋白（GFP）的报告质粒复合。测定了粒径、Zeta 电位、脂质体的稳定性以及对核酸酶的 DNA 保护。脂质：DNA 比为 2、4 和 6 时，粒径约为 250nm，Zeta 电位为-30mV。脂质：DNA 相互作用的强度和受酶降解保护的 DNA 分数随脂质：DNA 比的增加而增加。二维免疫电泳表明，在有或没有 HA-DOPE 的情况下，这三种比例中的任何一种都能以低能力激活补体系统的 C3 部分。在存在 0、10 和 15%HA-DOPE 或未结合的 HA 的情况下，通过流式细胞术测定 CD44 表达的 A549 细胞的转染效率。脂质：DNA 比为 2 且含有 10%（w/w）HA-DOPE 的脂质体对于转染最有效。孵育 6 小时后 GFP 表达水平达到最大值，表明脂质体的转染动力学较慢。最后，通过流式细胞术间接测量转染水平（通过转染效率来衡量）并通过荧光显微镜验证，表明脂质体的细胞摄取是由 CD44 受体和小窝介导的。这些结果表明，使用高分子量 HA 作为配体通过 CD44 受体进行 DNA 靶向具有很强的特异性。
