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

由于内质粒逃逸和核传递是外源基因表达的主要障碍，从优化非病毒基因传递系统的角度出发，定量评价质粒DNA在细胞内的传递是非常必要的。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/溶酶体、胞浆和细胞核中的定位。分别用LysoSensorDND-189型和Hoechst189型染色内体/溶酶体和胞核，以区别于胞浆。用这种方法分析了脂联胺、硬脂酸化的辛精氨酸(STR-RS)和R8在细胞内转运的情况。在R8的情况下，大部分的质粒DNA被内体/溶酶体捕获。STR-R8依赖地增加内体逃逸和随后的核转位时间。即使在转染后1小时，LipofetAMINE plus在将DNA快速运送到细胞核和胞浆中也非常有效。质粒DNA在细胞内运输的这些差异与这些非病毒系统的转染活性很好地相关。因此，该方法有可能定量分析非病毒系统转染质粒DNA后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 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.