Mao Zhengwei, Ma Lie, Yan Jiang, Yan Ming, Gao Changyou, Shen Jiacong
Key Laboratory of Macromolecular Synthesis and Functionalization, Ministry of Education, and Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China.
Biomaterials. 2007 Oct;28(30):4488-500. doi: 10.1016/j.biomaterials.2007.06.033. Epub 2007 Jul 20.
A thermoresponsive copolymer, trimethyl chitosan-g-poly(N-isopropylacrylamide) (TMC-g-PNIPAAm), was synthesized by coupling PNIPAAm-COOH to TMC. Their molecular structures were characterized by 1HNMR. The lower critical solution temperature (LCST) of TMC-g-PNIPAAm in PBS was measured as 32 degrees C by dynamic light scattering (DLS) and UV-vis spectroscopy, regardless of the grafting ratios. Upon mixing with DNA, TMC/DNA particles were formed, whose size and morphology were investigated by DLS and transmission electron microscopy, respectively. The particle size ranged from 200 to 900 nm depending on the N/P ratio and was less influenced by the temperature variation. The majority of the particles have spherical morphology. The zeta potentials of these particles were increased along with the N/P ratio. At a given N/P ratio, the zeta potentials were almost constant at 25 degrees C regardless of the existence of serum proteins. However, the values were significantly decreased at 37 degrees C in a solution containing serum protein. The affinity between DNA and TMC was examined by ethidium bromide competitive binding assay. TMC-g-PNIPAAm has stronger ability to combine with DNA at 40 degrees C when the PNIPAAm chain is collapsed. Gel electrophoresis results reveal that the vectors/DNA complexes can be formed regardless of the incubation temperature. HEK293 cell line was chosen as a model to study cellular uptake of the TMC-g-PNIPAAm/DNA particles, gene transfection and cytotoxicity. The grafting of PNIPAAm will not affect cellular uptake of the particles at 37 degrees C. The level of gene transfection could be thermally controlled. By using a temperature variation protocol, i.e. incubation of the cultured cells at 25 degrees C for a while, the gene transfection efficiency was significantly improved. Finally, the optimized gene transfection efficiency achieved by TMC-g-PNIPAAm is comparable to Lipofectamine 2000. No obvious cytotoxicity was detected for the TMC-g-PNIPAAm/DNA particles. These results suggest that TMC-g-PNIPAAm is an effective thermoresponsive gene carrier with minimal cytotoxicity, which has great promise for practical applications.
通过将聚（N-异丙基丙烯酰胺）-COOH偶联到三甲基壳聚糖上，合成了一种热响应共聚物，即三甲基壳聚糖-g-聚（N-异丙基丙烯酰胺）（TMC-g-PNIPAAm）。通过1HNMR对其分子结构进行了表征。通过动态光散射（DLS）和紫外可见光谱法测定了TMC-g-PNIPAAm在磷酸盐缓冲盐溶液（PBS）中的低临界溶液温度（LCST）为32℃，与接枝率无关。与DNA混合后，形成了TMC/DNA颗粒，分别通过DLS和透射电子显微镜研究了其大小和形态。颗粒大小根据氮/磷比在200至900nm范围内，受温度变化的影响较小。大多数颗粒具有球形形态。这些颗粒的zeta电位随着氮/磷比的增加而增加。在给定的氮/磷比下，无论血清蛋白是否存在，在25℃时zeta电位几乎恒定。然而，在含有血清蛋白的溶液中，在37℃时其值显著降低。通过溴化乙锭竞争结合试验检测了DNA与TMC之间的亲和力。当PNIPAAm链折叠时，TMC-g-PNIPAAm在40℃时与DNA结合的能力更强。凝胶电泳结果表明，无论孵育温度如何，都能形成载体/DNA复合物。选择人胚肾293（HEK293）细胞系作为模型，研究TMC-g-PNIPAAm/DNA颗粒的细胞摄取、基因转染和细胞毒性。PNIPAAm的接枝不会影响37℃时颗粒的细胞摄取。基因转染水平可以通过热控制。通过使用温度变化方案，即在25℃下培养细胞一段时间，基因转染效率显著提高。最后，TMC-g-PNIPAAm实现的优化基因转染效率与Lipofectamine 2000相当。未检测到TMC-g-PNIPAAm/DNA颗粒有明显的细胞毒性。这些结果表明，TMC-g-PNIPAAm是一种有效的热响应基因载体，细胞毒性最小，具有很大的实际应用前景。
