Lühmann Tessa, Rimann Markus, Bittermann Anne Greet, Hall Heike
Department of Materials, ETH Zurich, Zurich, Switzerland.
Bioconjug Chem. 2008 Sep;19(9):1907-16. doi: 10.1021/bc800206r. Epub 2008 Aug 22.
Polycationic molecules form condensates with DNA and are used for gene therapy as an alternative to viral vectors. As clinical efficacy corresponds to cellular uptake, intracellular stability of the condensates, and bioavailability of the DNA, it is crucial to analyze uptake mechanisms and trafficking pathways. Here, a detailed study of uptake, stability, and localization of PLL-g-PEG-DNA nanoparticles within COS-7 cells is presented, using FACS analysis to assess the involvement of different uptake mechanisms, colocalization studies with markers indicative for different endocytotic pathways, and immunofluorescence staining to analyze colocalization with intracellular compartments. PLL-g-PEG-DNA nanoparticles were internalized in an energy-dependent manner after 2 h and accumulated in the perinuclear region after >6 h. The nanoparticles were found to be stable within the cytoplasm for at least 24 h and did not colocalize with the endosomal pathway. Nanoparticle uptake was approximately 50% inhibited by genistein, an inhibitor of the caveolae-mediated pathway. However, genistein did not inhibit gene expression, and PLL-g-PEG-DNA nanoparticles were not colocalized with caveolin-1 indicating that caveolae-mediated endocytosis is not decisive for DNA delivery. Clathrin-mediated endocytosis and macropinocytosis pathways were reduced by 17 and 24%, respectively, in the presence of the respective inhibitors. When cells were transfected in the presence of double and triple inhibitors, transfection efficiencies were increasingly reduced by 40 and 70%, respectively; however, no differences were found between the different uptake mechanisms. These findings suggest that PLL-g-PEG-DNA nanoparticles enter by several pathways and might therefore be an efficient and versatile tool to deliver therapeutic DNA.
聚阳离子分子可与DNA形成凝聚物，并作为病毒载体的替代品用于基因治疗。由于临床疗效与细胞摄取、凝聚物的细胞内稳定性以及DNA的生物利用度相关，因此分析摄取机制和运输途径至关重要。在此，我们对COS-7细胞内PLL-g-PEG-DNA纳米颗粒的摄取、稳定性和定位进行了详细研究，使用流式细胞术分析评估不同摄取机制的参与情况，用指示不同内吞途径的标记物进行共定位研究，并通过免疫荧光染色分析与细胞内区室的共定位情况。PLL-g-PEG-DNA纳米颗粒在2小时后以能量依赖的方式内化，并在6小时后积聚在核周区域。发现纳米颗粒在细胞质内至少24小时保持稳定，且不与内体途径共定位。染料木黄酮是一种小窝介导途径的抑制剂，它对纳米颗粒摄取的抑制率约为50%。然而，染料木黄酮并不抑制基因表达，且PLL-g-PEG-DNA纳米颗粒不与小窝蛋白-1共定位，这表明小窝介导的内吞作用对DNA递送并非决定性因素。在各自抑制剂存在的情况下，网格蛋白介导的内吞作用和巨胞饮作用途径分别降低了17%和24%。当在双重和三重抑制剂存在的情况下对细胞进行转染时，转染效率分别逐渐降低了40%和70%；然而，不同摄取机制之间未发现差异。这些发现表明，PLL-g-PEG-DNA纳米颗粒通过多种途径进入细胞，因此可能是一种高效且通用的治疗性DNA递送工具。
