壳聚糖盐作为非病毒基因载体在CHO-K1细胞中的评估。
Evaluation of chitosan salts as non-viral gene vectors in CHO-K1 cells.
作者信息
Weecharangsan Wanlop, Opanasopit Praneet, Ngawhirunpat Tanasait, Apirakaramwong Auayporn, Rojanarata Theerasak, Ruktanonchai Uracha, Lee Robert J
机构信息
Faculty of Pharmacy, Silpakorn University, Nakhonpathom 73000, Thailand.
出版信息
Int J Pharm. 2008 Feb 4;348(1-2):161-8. doi: 10.1016/j.ijpharm.2007.07.011. Epub 2007 Jul 17.
The aim of this study was to investigate chitosan/DNA complexes formulated with various chitosan salts (CS) including chitosan hydrochloride (CHy), chitosan lactate (CLa), chitosan acetate (CAc), chitosan aspartate (CAs) and chitosan glutamate (CGl). They were assesed for their DNA complexing ability, transfection efficiency in CHO-K1 (Chinese hamster ovary) cells and their effect on cell viability. CHy, CLa, CAc, CAs and CGl, MW 45kDa formed a complex with pcDNA3-CMV-Luc at various N/P ratios. CGl/DNA complexes were formulated with various chitosan molecular weights (20, 45, 200 and 460kDa). The CS/DNA complexes were characterized by agarose gel electrophoresis and investigated for their transfection efficiency in CHO-K1 cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in CHO-K1 cells. Gel electrophoresis illustrated that complete complexes formed at N/P ratios above 2 in all CS of MW 45kDa. The transfection efficiency of CS/DNA complexes was dependent on the salt form and MW of chitosan, and the N/P ratio of CS/DNA complexes. Of different CS, the maximum transfection efficiency was found in different N/P ratios. CHy/DNA, CLa/DNA, CAc/DNA, CAs/DNA and CGl/DNA complexes showed maximum transfection efficiencies at N/P ratios of 12, 12, 8, 6 and 6, respectively. Cytotoxicity results showed that all CS/DNA complexes had low cytotoxicity. This study suggests CS have the potential to be used as safe gene delivery vectors.
本研究的目的是研究用各种壳聚糖盐(CS)配制的壳聚糖/DNA复合物,包括盐酸壳聚糖(CHy)、乳酸壳聚糖(CLa)、醋酸壳聚糖(CAc)、天冬氨酸壳聚糖(CAs)和谷氨酸壳聚糖(CGl)。评估了它们的DNA复合能力、在CHO-K1(中国仓鼠卵巢)细胞中的转染效率及其对细胞活力的影响。分子量为45kDa的CHy、CLa、CAc、CAs和CGl在不同的N/P比下与pcDNA3-CMV-Luc形成复合物。用不同分子量(20、45、200和460kDa)的壳聚糖配制CGl/DNA复合物。通过琼脂糖凝胶电泳对CS/DNA复合物进行表征,并研究其在CHO-K1细胞中的转染效率。通过在CHO-K1细胞中进行3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑(MTT)测定来确定复合物的细胞毒性。凝胶电泳表明,在所有分子量为45kDa的CS中,N/P比高于2时形成了完全复合物。CS/DNA复合物的转染效率取决于壳聚糖的盐形式和分子量以及CS/DNA复合物的N/P比。在不同的CS中,在不同的N/P比下发现了最大转染效率。CHy/DNA、CLa/DNA、CAc/DNA、CAs/DNA和CGl/DNA复合物分别在N/P比为12、12、8、6和6时显示出最大转染效率。细胞毒性结果表明,所有CS/DNA复合物的细胞毒性都很低。本研究表明,CS有潜力用作安全的基因递送载体。