Tseng Wen-Chi, Fang Tsuei-Yun, Su Ling-Yu, Tang Chien-Hsiang
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan.
Mol Pharm. 2005 May-Jun;2(3):224-32. doi: 10.1021/mp050007t.
Branched polyethylenimine (PEI) is a cationic polymer capable of forming self-assembly complexes with DNA to become a highly efficient agent used in gene delivery. Conjugation through the primary amines of PEI is a most commonly used approach further to enable the targeting delivery or to improve the stability of the DNA-polymer complexes. An understanding of how the conjugation affects the transfection mechanisms can help in the design of efficient polycationic vectors. In order to investigate the effects of conjugation, folate and the dextrans of molecular weight 1500 (dex-1500) and 10 000 (dex-10000) were used to prepare three different types of PEI conjugates: dextran-PEI, folate-PEI, and folate-dextran-PEI, which were subsequently employed to form complexes with DNA. These conjugates were found to cause less cytotoxicity than the unmodified PEI as revealed by the MTT method, and to be able to deliver an approximate amount of ethidium monoazide labeled plasmid into the cells. The efficiencies of green fluorescent protein (GFP) expression mediated by these conjugates, however, were less efficient than those mediated by the unmodified PEI. A titration experiment suggested that conjugation through the primary amines of PEI resulted in the loss of relative buffering capacity, a major factor aiding the release of plasmid from the endosomes, presumably because the conjugated molecules hindered the protonation of the PEI conjugates. When a quantitative relationship between relative buffering capacity and transfection efficiency was examined, a threshold of relative buffering capacity, around 50% of the unmodified PEI, was noted to be required for minimal detection of GFP positive cells. In addition, the cytotoxicity could be also related to the relative buffering capacity in an approximately linear trend. It is thus concluded that the severe loss of relative buffering capacity by conjugation might be attributed to the inefficiency of transgene expression mediated by the dextran-PEI conjugates.
支化聚乙烯亚胺（PEI）是一种阳离子聚合物，能够与DNA形成自组装复合物，成为基因递送中使用的高效试剂。通过PEI的伯胺进行共轭是一种最常用的方法，可进一步实现靶向递送或提高DNA-聚合物复合物的稳定性。了解共轭如何影响转染机制有助于设计高效的聚阳离子载体。为了研究共轭的影响，使用叶酸以及分子量为1500（dex-1500）和10000（dex-10000）的葡聚糖制备了三种不同类型的PEI共轭物：葡聚糖-PEI、叶酸-PEI和叶酸-葡聚糖-PEI，随后将它们用于与DNA形成复合物。MTT法显示，这些共轭物的细胞毒性比未修饰的PEI小，并且能够将大致等量的单叠氮溴化乙锭标记质粒递送至细胞中。然而，这些共轭物介导的绿色荧光蛋白（GFP）表达效率低于未修饰的PEI介导的效率。滴定实验表明，通过PEI的伯胺进行共轭导致相对缓冲能力丧失，这是帮助质粒从内体释放的一个主要因素，推测是因为共轭分子阻碍了PEI共轭物的质子化。当检查相对缓冲能力与转染效率之间的定量关系时，发现检测GFP阳性细胞的最小值需要约为未修饰PEI 50%的相对缓冲能力阈值。此外，细胞毒性也可能与相对缓冲能力呈近似线性趋势相关。因此得出结论，共轭导致的相对缓冲能力严重丧失可能是葡聚糖-PEI共轭物介导的转基因表达效率低下的原因。
