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目的探讨双亲性壳聚糖衍生物与质粒DNA通过自组装的方式形成基因纳米粒子的可行性,考察载体与质粒DNA的相互作用机制,为开发安全高效的非病毒载体提供新的途径。方法合成了一种双亲性壳聚糖衍生物N-亚甲基磷酸化壳聚糖(NMPCS),利用复凝聚的方法制备NMPCS/DNA纳米粒子复合物。用傅立叶变换红外光谱、核磁共振谱等手段对NMPCS进行了表征,通过凝胶电泳阻滞实验、动态光散射、原子力显微镜、圆二色谱等实验对NMPCS与质粒DNA之间的相互作用进行研究。结果经傅立叶变换红外光谱及核磁共振谱分析表明,改性后的壳聚糖的特征基团有显著变化,证明了亚甲基磷酸基团成功的引入。NMPCS与DNA能自组装形成类似球形的基因纳米粒子。结论壳聚糖基双亲性分子可能与细胞膜中的双亲性分子具有潜在相容性,使得载体/DNA复合物通过一种膜的去组装过程跨越细胞膜而最终表达,从而有望研制出高效的非病毒载体。
OBJECTIVE: To investigate the feasibility of gene chitosan derivatives and plasmid DNA by self-assembly to explore the mechanism of interaction between vector and plasmid DNA, and to provide a new approach for the development of safe and efficient non-viral vector. Methods A novel amphiphilic chitosan derivative N-methylene phosphorylated chitosan (NMPCS) was synthesized and the NMPCS / DNA nanoparticle complex was prepared by complex coacervation. NMPCS was characterized by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The interaction between NMPCS and plasmid DNA was studied by means of gel electrophoresis, dynamic light scattering, atomic force microscopy and circular dichroism. Results Fourier Transform Infrared Spectroscopy and Nuclear Magnetic Resonance Spectroscopy analysis showed that the modified chitosan has significant changes in the characteristic groups, indicating the successful introduction of methylene phosphoric acid groups. NMPCS and DNA self-assemble to form spheroidal gene-like nanoparticles. Conclusion Chitosan-based amphiphilic molecules may have potential compatibility with amphipathic molecules in the cell membrane, so that the carrier / DNA complex can eventually be expressed across the cell membrane through the de-assembly process of a membrane, so that it is expected to develop an efficient non-virus Vector.