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Optimization of pharmacokinetics and intracellular pharmacokinetics of viral and non-viral gene delivery system

病毒和非病毒基因传递系统的药代动力学和细胞内药代动力学优化

基本信息

批准号：
13557217
负责人：
HARASHIMA Hideyoshi
金额：
$ 9.98万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Since endosomal escape and the nuclear delivery of plasmid DNA constitute major barriers to trans-gene expression, a quantitative evaluation of intracellular trafficking of the plasmid DNA would be highly desirable in terms of optimizing a non-viral gene delivery system. In the present study, a novel strategyis proposed for quantifying the intracellular localization of rhodamin-labeled plasmid DNA in endosomes/lysosomes, cytosol and the nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. Using this approach, the intracellular trafficking of plasmid DNA was analyzed after transfection with LipofectAMINE PLUS, steaiylated octaarginine (STR-RS) and R8. In the case of R8, most of the plasmid DNA was trapped by endosomes/lysosomes. STR-R8 enhanced endosomal escape and following nucleartranslocation time dependently. LipofectAMINE PLUS was much effective in rapidly delivering DNA to the nucleus as well as the cytosol, even 1 hr after transfection. These differences in the intracellular trafficking of plasmid DNA correlated well with the transfection activities of these non-viral systems. Therefore, this method has the potential of quantitatively analyzing the intracellular pharmacokinetics of plasmid DNA after transfection by non-viral systems and promises to provide useful information for optimizing non-viral gene delivery systems.

由于质粒DNA的内体逃逸和核递送构成转基因表达的主要障碍，因此就优化非病毒基因递送系统而言，质粒DNA的细胞内运输的定量评价将是高度期望的。在本研究中，提出了一种新的策略来定量罗丹明标记的质粒DNA在胞内体/溶酶体，胞质溶胶和细胞核中的细胞内定位。分别用LysoSensor DND-189和Hoechst 33258染色内体/溶酶体和细胞核，以将其与细胞溶质区分开。使用该方法，在用LipofectAMINE PLUS、硬脂酰化八精氨酸（STR-RS）和R8转染后分析质粒DNA的细胞内运输。在R8的情况下，大部分质粒DNA被内体/溶酶体捕获。STR-R8增强内体逃逸和随后的核转位时间依赖性。LipofectAMINE PLUS在将DNA快速递送至细胞核以及细胞质方面非常有效，甚至在转染后1小时。质粒DNA的细胞内运输的这些差异与这些非病毒系统的转染活性密切相关。因此，该方法具有潜在的定量分析质粒DNA转染后的细胞内药代动力学的非病毒系统，并有望为优化非病毒基因传递系统提供有用的信息。