N-亚甲基磷酸化壳聚糖与DNA相互作用的研究

Interaction of N -methylene phosphonic chitosan and plasmid DNA

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 possibility of self-assembling gene nanoparticles between the amphiphilic chitosan derivative and plasmid DNA,and study the interaction mechanism between N -methylene phosphonic chitosan （NMPCS） and plasmid DNA,which might provide a new strategy for developing safe and efficient non-viral vectors. Methods NMPCS was synthesized and analyzed by Fourier-transform infrared spectroscopy（FT-IR） and 13C nuclear magnetic resonance（NMR）. NMPCS/ DNA nanoparticles were prepared and characterized by agarose gel electrophoresis retardation assay,dynamic light scattering （DLS）,atomic force microscopy（AFM） and circular dichroism（CD）. Results FT-IR and NMR results indicated that N-methylene phosphonic group was successfully incorporated with the backbone of chitosan. NMPCS could self assemble with plasmid DNA to form spherical nanoparticles with mean size of 80 ～ 210 nm. Conclusion NMPCS can form gene nanoparticles with plasmid DNA via self-assembling,which displayed great potential in the further application used as safe and efficient non-viral vectors.