Rojanarata Theerasak, Petchsangsai Maleenart, Opanasopit Praneet, Ngawhirunpat Tanasait, Ruktanonchai Uracha, Sajomsang Warayuth, Tantayanon Supawan
Faculty of Pharmacy, Silpakorn University, Nakhonpathom, Thailand.
Eur J Pharm Biopharm. 2008 Sep;70(1):207-14. doi: 10.1016/j.ejpb.2008.04.022. Epub 2008 May 1.
The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan, TM(47)-Bz(42)-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells), in comparison to quaternized chitosan (TM(43)-CS) and chitosan (CS). Factors affecting the transfection efficiency, such as the carrier/DNA weight ratio, the pH of the culture medium, and the presence of serum, have been investigated. The results revealed that TM(47)-Bz(42)-CS was able to condense with pDNA. As illustrated by the agarose gel electrophoresis, the complete complexes of TM(47)-Bz(42)-CS/DNA were formed at a weight ratio of above 0.5, whereas those of TM(43)-CS/DNA and CS/DNA were formed at a ratio of above 1. TM(47)-Bz(42)-CS showed superior transfection efficiency to TM(43)-CS and CS at all weight ratios tested. Higher transfection efficiency and gene expression were observed when the carrier/DNA weight ratios increased. The highest transfection efficiency was found at a weight ratio of 8. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on CS, which promoted the interaction and condensation with DNA, as well as N-quaternization, which increased the CS water solubility. During cytotoxicity studies, it was found that high concentrations of TM(47)-Bz(42)-CS and TM(43)-CS could decrease the Huh7 cell viability. In conclusion, this novel CS derivative, TM(47)-Bz(42)-CS, shows promising potential as a gene carrier by efficient DNA condensation and a mediated higher level of gene transfection in Huh7 cells.
本研究的目的是研究季铵化的N-(4-N,N-二甲基氨基苄基)壳聚糖（TM(47)-Bz(42)-CS）与编码绿色荧光蛋白的质粒DNA（pEGFP-C2）对人肝癌细胞系（Huh7细胞）的转染效率，并与季铵化壳聚糖（TM(43)-CS）和壳聚糖（CS）进行比较。研究了影响转染效率的因素，如载体/DNA重量比、培养基的pH值和血清的存在。结果表明，TM(47)-Bz(42)-CS能够与pDNA凝聚。如琼脂糖凝胶电泳所示，TM(47)-Bz(42)-CS/DNA的完整复合物在重量比高于0.5时形成，而TM(43)-CS/DNA和CS/DNA的完整复合物在重量比高于1时形成。在所有测试的重量比下，TM(47)-Bz(42)-CS的转染效率均优于TM(43)-CS和CS。当载体/DNA重量比增加时，观察到更高的转染效率和基因表达。在重量比为8时发现最高转染效率。结果表明，基因转染的改善归因于壳聚糖上的疏水基团（N,N-二甲基氨基苄基）取代，其促进了与DNA的相互作用和凝聚，以及N-季铵化，其增加了壳聚糖的水溶性。在细胞毒性研究中，发现高浓度的TM(47)-Bz(42)-CS和TM(43)-CS会降低Huh7细胞活力。总之，这种新型壳聚糖衍生物TM(47)-Bz(42)-CS通过有效的DNA凝聚和介导Huh7细胞中更高水平的基因转染，显示出作为基因载体的潜在前景。
