Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy
Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy
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DOI：
10.1016/j.ymthe.2004.01.005
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发表时间：
2004-03-01
影响因子：
12.4
通讯作者：
Harashima, H
中科院分区：
文献类型：
--
作者：
Akita, H;Ito, R;Harashima, H
由于内体逃逸和质粒 DNA (pDNA) 的核递送构成转基因表达的主要障碍，因此在优化非病毒基因递送系统方面非常需要对 pDNA 的细胞内运输进行定量评估。在本研究中，提出了一种定量内体/溶酶体、细胞质和细胞核中罗丹明标记的 pDNA 的新策略。内体/溶酶体和细胞核分别用 LysoSensor DND-189 和 Hoechst 33258 染色，以将它们与胞质溶胶区分开。将源自罗丹明的簇的像素面积用作pDNA量的指标。该方法适用于分析由 LipofectAMINE PLUS、硬脂酰化八精氨酸 (STR-R8) 和八精氨酸 (R8) 转染的 pDNA 的细胞内运输。就 R8 而言，大部分 pDNA 被内体/溶酶体捕获。 STR-R8 表现出内体逃逸，随后以时间依赖性方式发生核转位。 LipofectAMINE PLUS 在将 pDNA 快速递送至细胞核和细胞质中最为有效。 pDNA 细胞内运输的这些差异与转基因表达密切相关。因此，该方法能够定量分析 pDNA 的细胞内药代动力学，并有望为优化非病毒基因传递系统提供有用的信息。
Since endosomal escape and the nuclear delivery of plasmid DNA (pDNA) constitute major barriers for transgene expression, a quantitative evaluation of intracellular trafficking of pDNA would be highly desirable in terms of optimizing a nonviral gene delivery system. In the present study, a novel strategy is proposed for the quantification of rhodamine-labeled pDNA in endosomes/lysosomes, cytosol, and nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. The pixel areas of the clusters derived from the rhodamine were used as an index for the amount of pDNA. This approach was applied to the analysis of the intracellular trafficking of pDNA transfected by LipofectAMINE PLUS, stearylated octaarginine (STR-R8), and octaarginine (R8). In the case of R8, most of the pDNA was trapped by endosomes/lysosomes. STR-R8 exhibited endosomal escape followed by nuclear translocation in a time-dependent manner. LipofectAMINE PLUS was the most effective in rapidly delivering pDNA to the nucleus as well as the cytosol. These differences in the intracellular trafficking of pDNA correlated well with the transgene expression. Therefore, this method enables the quantitative analysis of the intracellular pharmacokinetics of pDNA and promises to provide useful information for optimizing nonviral gene delivery systems.