Poly(ethylene imine)-g-chitosan using EX-810 as a spacer for nonviral gene delivery vectors
Poly(ethylene imine)-g-chitosan using EX-810 as a spacer for nonviral gene delivery vectors
复制标题
DOI：
10.1002/jbm.a.31961
复制
发表时间：
2009-03-15
影响因子：
4.9
通讯作者：
Leong, Kam W.
中科院分区：
文献类型：
--
作者：
Lou, Yu-Lun;Peng, Yu-Shiang;Leong, Kam W.
聚电解质复合物作为基因载体近年来得到了广泛的研究。本研究将聚乙烯亚胺接枝到壳聚糖上作为非病毒基因载体，以提高壳聚糖的水溶性和固有的转染效率。我们提出了一种新的方法，通过打开乙二醇二缩水甘油醚（EX-810）的环氧环，将壳聚糖（CHI）的胺或羟基与PEI的胺基团缀合，这也带来了PEG化化学中提到的优点。通过NMR表征PEI在CHI上的取代度。通过DNA-聚合物复合物的理化性质、修饰聚合物的缓冲能力、细胞毒性和转基因表达效率之间的相关性，初步研究了从细胞进入到内体释放的细胞机制。通过MTT测定的细胞毒性显示PEI-g-CHI的细胞活力高于CHI，尤其是在使用人肾293 T细胞的高浓度下显著。用绿色荧光蛋白（GFP）监测转基因表达的效率和细胞内质粒的量，并通过荧光显微镜观察。当使用高于2.5的重量比时，PEI-g-CHI/DNA多聚物的转染效率显著优于CHI/DNA多聚物。(c)2008 Wiley Periodicals，Inc. J Biomed Mater Res 88A：1058-1068，2009
Polyelectrolyte complexes have been widely studied as gene carriers in recent years. In this study, poly (ethylene imine) was grafted onto chitosan (PEI-g-CHI) as a nonviral gene carrier in order to improve the water solubility as well as the inherent transfection efficiency of chitosan. We present a novel method to conjugate the amine or hydroxyl groups of chitosan (CHI) and the amine groups of PEI through opening the epoxide rings of ethylene glycol diglycidyl ether (EX-810), which also brings the merits as mentioned in PEGylation chemistry. The degree of substitution of PEI onto CHI was characterized by NMR. The preliminarily cellular mechanisms, from the cellular entry to the endosomal release, were investigated by the correlations among the physicochemical properties of the DNA-polymer complexes, the buffering capacity of the modified polymer, the cytotoxicity, and the efficiency of the transgene expression. The cytotoxicity assayed by MTT shows that cell viability of PEI-g-CHI is higher than CHI especially noticeable at high concentrations using human kidney 293T cells. The efficiency of transgene expression and the amount of intracellular plasmid were monitored using green fluorescent protein (GFP) and visualized by fluorescence microscopy. The transfection efficiency of PEI-g-CHI/DNA polyplex is significantly better than CHI/DNA polyplex when using the weight ratios higher than 2.5. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 88A: 1058-1068, 2009