Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, P.O. Box 56, FI-00014, University of Helsinki, Finland.
J Control Release. 2012 Nov 10;163(3):385-95. doi: 10.1016/j.jconrel.2012.09.016. Epub 2012 Oct 4.
Detailed understanding of the uptake mechanisms and intracellular processing of nonviral gene delivery systems will allow design of more effective carriers. This work gets insight into the intracellular kinetics of pDNA delivered by polyethyleneimine (PEI), cationic lipid DOTAP and calcium phosphate (CaP) precipitates. Amount of cell- and nuclear-associated pDNA was quantified by qRT-PCR at multiple time points after transfection. Moreover, the impact of specific endocytic pathways on the cell entry and intracellular kinetics of pDNA was studied by inhibition (blockage) of either clathrin- or dynamin-mediated endocytosis by using both genetically manipulated cell lines and chemical inhibitors of endocytosis. Quantitative analysis of defined kinetic parameters revealed that neither cellular nor nuclear uptake of pDNA correlated with transgene expression, emphasizing the importance of the post-nuclear processes in overall transfection efficacy. Changes in transgene expression observed upon blockage of endocytosis was carrier dependent and correlated relatively well with the changes at the cellular and nuclear uptake levels but not with the amount of cell-associated pDNA. Due to low specificity of chemical inhibitors and activation of alternative endocytosis pathways after genetic blockage of endocytosis neither of these methods is optimal for studying the role of endocytosis. Therefore, one should be careful when interpreting the obtained results from such studies and not to trust the data obtained only from one method.
详细了解非病毒基因传递系统的摄取机制和细胞内处理过程，将有助于设计更有效的载体。本工作深入了解了聚乙烯亚胺（PEI）、阳离子脂质 DOTAP 和磷酸钙（CaP）沉淀递送的 pDNA 的细胞内动力学。转染后多个时间点通过 qRT-PCR 定量细胞和核相关 pDNA 的量。此外，通过使用基因改造的细胞系和内吞作用的化学抑制剂来抑制（阻断）网格蛋白或动力蛋白介导的内吞作用，研究了特定内吞途径对 pDNA 的细胞内进入和细胞内动力学的影响。对定义的动力学参数的定量分析表明，pDNA 的细胞内和核摄取均与转基因表达不相关，这强调了核后过程在整体转染效率中的重要性。在阻断内吞作用时观察到的转基因表达变化与摄取水平的变化相对较好地相关，但与细胞相关 pDNA 的量无关。由于化学抑制剂的特异性低，并且在网格蛋白介导的内吞作用被基因阻断后激活替代内吞途径，因此这两种方法都不是研究内吞作用作用的最佳方法。因此，在解释此类研究中获得的结果时应小心谨慎，不要仅依赖于一种方法获得的数据。
