Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells
Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells
复制标题
影响多药耐药乳腺癌细胞中 Pluronic P123 修饰的聚丙烯亚胺/pDNA 复合物转染效率和细胞摄取机制的因素
DOI：
10.1016/j.colsurfb.2015.12.023
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发表时间：
2016-04-01
影响因子：
5.8
通讯作者：
Sha, Xianyi
中科院分区：
文献类型：
--
作者：
Gu, Jijin;Hao, Junguo;Sha, Xianyi
通常，有效基因递送的主要障碍是核酸如质粒DNA（pDNA）或小干扰RNA（siRNA）的细胞内化和内体逃逸。我们先前开发了Pluronic P123修饰的聚丙烯亚胺（PPI）/pDNA（P123-PPI/pDNA）聚合复合物作为基因递送系统。结果表明，P123-PPI/pDNA复合物在多药耐药乳腺癌细胞中显示出比PPI/pDNA复合物更高的转染效率。作为一个持续的努力，本调查的影响因素的转染效率，细胞摄取机制，和细胞内命运的P123-PPI/pDNA复合物的报告。P123的存在是影响P123-PPI/pDNA复合物在MCF-7/ADR细胞中转染效率的主要因素，但其他参数如N/P比、FBS浓度、孵育时间和温度也是重要的。在体外研究了网格蛋白介导的内吞作用（CME）、小窝介导的内吞作用（CvME）和巨胞饮作用的内吞抑制剂对P123-PPI/pDNA复合物的细胞摄取和转染效率的影响。数据显示P123-PPI/pDNA复合物的内化从CME和CvME两者获得。用TRITC-转铁蛋白（CME指示剂）、Alexa Fluor 555-CTB（CvME指示剂）、单克隆抗α-微管蛋白（微管指示剂）和LysoTracker绿色（内体/溶酶体指示剂）进行共定位实验以确认内化途径。结果表明，CME和CvME在P123-PPI/pDNA复合物的有效转染中起重要作用。内体/溶酶体系统和骨架，包括肌动蛋白丝和微管，是内化后运输所必需的。(C)2015 Elsevier B. V.版权所有。
Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-a-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization. (C) 2015 Elsevier B.V. All rights reserved.