The rote of dextran conjugation in transifection mediated by dextran-grafted polyethylenimine
The rote of dextran conjugation in transifection mediated by dextran-grafted polyethylenimine
复制标题
DOI：
10.1002/jgm.572
复制
发表时间：
2004-08-01
影响因子：
3.5
通讯作者：
Fang, TY
中科院分区：
文献类型：
--
作者：
Tseng, WC;Tang, CH;Fang, TY
背景通过伯胺的缀合是修饰用于基因递送的聚阳离子载体的最常用方法之一。方法将分子量为1500的右旋糖酐接枝到聚乙烯亚胺（PEI）上，形成不同程度的接枝，研究偶联对转基因表达机制的影响。流式细胞仪检测质粒进入细胞的量，并分别用单叠氮乙锭标记质粒和绿色荧光蛋白（GFP）检测转基因表达水平。接枝PEI的缓冲能力通过滴定测定，和DNA-聚合物复合物的完整性进行了检查，通过暴露于heparin.Results葡聚糖接枝到PEI被发现显着减少的细胞毒性，缓冲能力，细胞进入，和DNA-聚合物复合物的完整性。随着接枝率从0增加到1.84%，下降幅度增大;然而，在最佳的接枝程度下，葡聚糖接枝的PEI将GFP阳性细胞的百分比提高到CHO和MDA-MB-231细胞的未修饰PEI介导的水平的3倍和1.3倍，结论葡聚糖与PEI伯胺的偶联抑制了质粒的跨膜进入，但当结合度低于0.64%时，DNA-聚合物复合物结构的改变能够促进转基因表达。版权所有（C）2004约翰威利父子有限公司。
Background Conjugation through primary amines is one of the most commonly used methods to modify polycationic vectors for gene delivery. A better understanding of the effect of the conjugation on the mechanisms of transgene expression can help design efficient polycationic vectors.Methods Dextran with a molecular weight of 1500 was grafted onto polyethylenimine (PEI) to produce various degrees of grafting in an effort to investigate how the conjugation affected the mechanisms of transgene expression. Flow cytometry was employed to quantitate the cellular entry of plasmid and the level of transgene expression, which were measured using ethidium monoazide labeled plasmid and green fluorescent protein (GFP), respectively. The buffering capacity of the grafted PEI was determined by titration, and the integrity of the DNA-polymer complexes were examined by exposure to heparin.Results Grafting of dextran onto PEI was found to significantly diminish the cytotoxicity, buffering capacity, cellular entry, and the integrity of the DNA-polymer complexes. The reductions enlarged as the degree of grafting increased from 0 to 1.84%; however, at an optimal degree of grafting, the dextran-grafted PEI enhanced the percentages of GFP-positive cells to a level 3 times and 1.3 times of those mediated by unmodified PEI for CHO and MDA-MB-231 cells, respectively.Conclusions These results demonstrated that the conjugation of dextran onto the primary amines of PEI inhibited the entry of plasmid across the cell membrane, but the change in the structures of the DNA-polymer complexes was able to promote transgene expression when the degrees of conjugation fell below 0.64%. Copyright (C) 2004 John Wiley Sons, Ltd.