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

由于内体逃逸和质粒DNA的核传递构成了转基因表达的主要障碍，因此在优化非病毒基因传递系统方面，质粒DNA细胞内运输的定量评估将是非常可取的。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在核内体/溶酶体、细胞质和细胞核中的细胞内定位。内溶酶体和细胞核分别用LysoSensor DND-189和Hoechst 33258染色，与细胞质区分开。使用这种方法，分析转染LipofectAMINE PLUS、精氨酸（STR-RS）和R8后质粒DNA的细胞内运输。在R8中，大部分质粒DNA被核内体/溶酶体捕获。STR-R8增强核内体逃逸和核易位的时间依赖性。LipofectAMINE PLUS在将DNA快速传递到细胞核和细胞质中非常有效，甚至在转染后1小时。质粒DNA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，该方法具有定量分析非病毒系统转染后质粒DNA细胞内药代动力学的潜力，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 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.