PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles
PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles
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DOI：
10.1078/0171-9335-00363
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发表时间：
2004-04-01
影响因子：
6.6
通讯作者：
Davis, ME
中科院分区：
文献类型：
--
作者：
Mishra, S;Webster, P;Davis, ME
非病毒基因递送载体的体外研究通常不在生理条件下进行，因此可能无法为体内研究提供有意义的结果。我们通过检查其细胞摄取和运输来确定用于体外研究的聚阳离子-质粒 DNA 复合物 (polyplex) 的行为是否与更适用于体内使用的变体相似。支链聚乙烯亚胺 (25 kDa) 或含有线性 β-环糊精的聚合物均用于配制复合物，该复合物可进行 PEG 化（PEG：聚（乙二醇））以产生在生理盐浓度下稳定的颗粒。报告了聚合复合物及其聚乙二醇化变体的粒径、细胞摄取、细胞内运输和报告基因表达。聚乙二醇化赋予颗粒盐稳定性，但导致荧光素酶表达减少。通过透射电子显微镜对体外颗粒内化的检查表明，未修饰的聚合复合物作为大聚集体进入细胞，而聚乙二醇化颗粒在细胞外部和内部都保持小且离散。未修饰的和聚乙二醇化的颗粒通过内吞途径进入细胞并积聚在核周区域。免疫标记揭示了细胞质和细胞核中未包装的外源 DNA。似乎所有颗粒类型都在囊泡内流向细胞核，并在囊泡和/或细胞质中经历降解，最终一些外源 DNA 进入细胞核，并在细胞核中进行转录。在比较复合物及其聚乙二醇化变体时，颗粒形态、细胞摄取和所得表达的显着差异表明，如果结果与体内性能相关，则应使用为生理条件制备的颗粒进行体外研究。
In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear beta-cyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.