Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, Guangzhou, 510006, People's Republic of China.
Pharm Res. 2011 Apr;28(4):812-26. doi: 10.1007/s11095-010-0335-z. Epub 2010 Dec 14.
PURPOSE: Inefficient endosomal escape and poor nuclear import are thought to contribute to low gene transfer efficiency of polycations. To overcome these drawbacks, we prepared multiple gene delivery formulations including low cytotoxic polycation, histone containing NLSs and chloroquine as the endosomolytic agent. METHODS: Comb-shaped poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) copolymer was synthesized by aminolysis of poly-γ-benzyl-L-glutamate using low-molecular-weight polyethylenimine (800 Da). The formation of DNA/histone/PLGE terplex was observed by atomic force microscope and gel retardation assay. The particle size and zeta potential of DNA complexes with varying content of histone were also measured to confirm the terplex formation. Cytotoxicity of vectors was assayed by MTT. Multiple gene delivery formulations were optimized to their best transfection efficiency that was monitored by fluorescence microscope and flow cytometry. In vivo gene delivery of the optimal formulation was evaluated by the GFP-expression levels in drosophila melanogaster. RESULTS: The DNA/histone/PLGE terplex was successfully formed. The PLGE and histone together condensed DNA into small, discrete particles (less than 200 nm in diameter) in isotonic solution. Cytotoxicity of PLGE and histone were much lower than that of PEI 25 K. Either histone or chloroquine contributed to enhancing the levels of transfection activity of PLGE polymer. However, chloroquine and histone did not show a synergistic effect on the improvement of transfection efficiency. The optimal formulation was the DNA/histone/PLGE terplex at the N/P ratio of 15 and histone/ DNA weight ratio of 0.8. Compared with Lipofectamine 2000 and PEI 25 K, the optimal formulation showed significantly increased levels of GFP-expression both in vitro and in vivo. CONCLUSION: This formulation provided a versatile approach for preparing high efficiency of the polycation-based gene vectors. It also reinforced the finding of earlier studies that nuclear import and endosomal escape were rate-limiting steps for nonviral gene delivery.
目的：多聚阳离子的基因转染效率低被认为是由于其内在的低效率的内涵体逃逸和较差的核导入。为了克服这些缺点，我们制备了多种基因传递制剂，包括低细胞毒性的聚阳离子、含有核定位信号的组蛋白和作为内涵体溶解剂的氯喹。
方法：通过用低分子量聚乙烯亚胺（800 Da）对聚-γ-苄基-L-谷氨酸进行氨解，合成了梳状聚（L-谷氨酸）接枝的低分子量聚乙烯亚胺（PLGE）共聚物。通过原子力显微镜和凝胶电泳实验观察 DNA/组蛋白/PLGE 三聚体的形成。还测量了具有不同组蛋白含量的 DNA 复合物的粒径和 zeta 电位，以证实三聚体的形成。通过 MTT 法测定载体的细胞毒性。通过荧光显微镜和流式细胞术优化了多种基因传递制剂，以获得最佳的转染效率。通过 GFP 表达水平在果蝇中的表达评估最佳制剂的体内基因传递。
结果：成功地形成了 DNA/组蛋白/PLGE 三聚体。PLGE 和组蛋白一起将 DNA 凝聚成小的、离散的颗粒（直径小于 200nm）在等渗溶液中。PLGE 和组蛋白的细胞毒性明显低于聚乙烯亚胺 25 K。组蛋白或氯喹都有助于提高 PLGE 聚合物的转染活性水平。然而，氯喹和组蛋白在提高转染效率方面没有协同作用。最佳制剂是 DNA/组蛋白/PLGE 三聚体在 N/P 比为 15 和组蛋白/DNA 重量比为 0.8 的情况下。与 Lipofectamine 2000 和 PEI 25 K 相比，最佳制剂在体外和体内均显著提高了 GFP 的表达水平。
结论：该制剂为制备高效多聚阳离子基因载体提供了一种通用的方法。它还加强了早期研究的发现，即核导入和内涵体逃逸是非病毒基因传递的限速步骤。
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