School of Pharmacy, Department of Health Sciences, University of Eastern Finland, PO Box 1627 FIN-70211 Kuopio, Finland.
J Control Release. 2012 Aug 20;162(1):167-75. doi: 10.1016/j.jconrel.2012.06.013. Epub 2012 Jun 16.
The low transfection efficacy of non-viral gene delivery systems limits the therapeutic application of these vectors. Besides the inefficient release of the complexes or pDNA from endolysosomes into the cytoplasm or poor nuclear uptake, the nuclear and post-nuclear processing might unfavorably affect the transgene expression. Positively charged amphiphilic 1,4-dihydropyridine (1,4-DHP) derivatives were earlier proposed as a promising tool for the delivery of DNA into target cells in vitro and in vivo. However, the structure/activity relationship of these carriers is poorly understood as yet. In this work we studied the intracellular processing of complexes, composed of three structurally related 1,4-DHP derivatives, in a retinal pigment epithelial (ARPE-19) cell line. The pre- and post-nuclear processing of the complexes was quantified on the nuclear, mRNA and transgene expression level. Here we show that the interaction of 1,4-DHP complexes with the cell membrane temporarily increases the permeability of the ARPE-19 cell membrane for small molecular compounds. However, the main mechanism for internalization of 1,4-DHP complexes is endocytosis. We found that all examined derivatives are able to destabilize endosomal membranes by lipid exchange upon acidification. In addition, the buffering capacity of some of the compounds may contribute to the endosomal escape of the complexes as well through the proton sponge effect. Previously we reported that cellular uptake of 1,4-DHP complexes does not correlate with transgene expression. In this study we surprisingly revealed that there is no correlation between the amount of plasmids taken up by the cell and the amount of plasmids found in the cell nucleus. Furthermore, it was found that a high amount of plasmid in the nucleus does not ensure high mRNA expression, likely due to remaining interactions of the carrier with the plasmids. Neither did the expression of mRNA always result in the production of a functional protein, possibly due to the interaction of free carrier with intracellular components which are involved in the post-translational modification of protein and folding process. Overall, our data suggest that succeeding of both the pre- and the post-nuclear intracellular processes is equally essential for successful transgene expression.
非病毒基因传递系统的低转染效率限制了这些载体的治疗应用。除了复合物或 pDNA 从内涵体到细胞质的释放效率低下或核摄取不良外，核内和核后加工也可能不利于转基因表达。带正电荷的两亲 1,4-二氢吡啶（1,4-DHP）衍生物早期被提议作为一种有前途的工具，用于将 DNA 递送到体外和体内的靶细胞中。然而，这些载体的结构/活性关系目前还了解甚少。在这项工作中，我们研究了三种结构相关的 1,4-DHP 衍生物组成的复合物在视网膜色素上皮（ARPE-19）细胞系中的细胞内处理。复合物的核前和核后处理在核、mRNA 和转基因表达水平上进行了定量。在这里，我们表明 1,4-DHP 复合物与细胞膜的相互作用暂时增加了 ARPE-19 细胞膜对小分子化合物的通透性。然而，1,4-DHP 复合物内化的主要机制是内吞作用。我们发现，所有被检查的衍生物都能够通过酸化时的脂质交换来破坏内涵体膜的稳定性。此外，一些化合物的缓冲能力也可能通过质子海绵效应有助于复合物的内涵体逃逸。此前我们报道，1,4-DHP 复合物的细胞摄取与转基因表达不相关。在这项研究中，我们令人惊讶地发现，细胞摄取的质粒数量与细胞核中发现的质粒数量之间没有相关性。此外，还发现细胞核中大量质粒并不保证高 mRNA 表达，这可能是由于载体与质粒之间的相互作用所致。mRNA 的表达也不一定会产生功能性蛋白质，这可能是由于游离载体与参与蛋白质翻译后修饰和折叠过程的细胞内成分相互作用所致。总的来说，我们的数据表明，核内和核后细胞内过程的成功都同等重要，对于成功的转基因表达是必要的。
