壳聚糖纳米粒作为基因载体的研究:粒径对转染效率的影响

研究粒径对壳聚糖(chitosan，CS)纳米粒介导的转染效率的影响。通过调整CS溶液加入质粒基因(plasmid DNA，pDNA)溶液的速度和涡旋时间制备250，580和1 300 nm粒径pDNA/CS纳米粒，研究粒径对CS介导的细胞转染效率的影响。为深入探讨粒径对转染效率的影响，考察了3种粒径pDNA/CS纳米粒的药剂学性质，对抗核酸酶作用和细胞对纳米粒的吸附和摄取行为。结果表明：本文制备的3种粒径纳米粒的药剂学性质和凝聚pDNA的能力等特性基本无差别，均能有效保护pDNA免受核酸酶降解；在HEK293细胞中的转染效率无显著差异；与细胞共孵育4 h，流式细胞仪测定的三者细胞摄取率与摄取量相似；荧光显微图像显示3种粒径纳米粒均以小聚集体形式吸附于细胞表面，激光扫描共聚焦显微图像显示直径约为2 μm小聚集体较易被细胞内吞入胞。因此粒径在250～1 300 nm中对壳聚糖纳米粒介导的细胞转染率基本无影响。

Chitosan nanoparticles as gene vector: effect of particle size on transfection efficiency

The aim was to investigate the effect of particle size on transfection efficiency of chitosan (CS)-based nanoparticles. Nanoparticles were synthesized through complex coacervation CS with plasmid DNA (pDNA). Three kinds of pDNA/CS nanoparticles with different sizes (250, 580 and 1300 nm) were prepared by altering the adding rate and the vortexing time. The particle size, zeta potential and the stability in cultural medium were evaluated by zetasizer. The association efficiency was determined by spectrofluorophotometer. The combination of chitosan with pDNA as well as the ability to protect pDNA from nuclease degradation was analyzed by gel electrophoresis. The transfection efficiency of pDNA/CS nanoparticles in HEK293 cells was investigated by flow cytometry. Using CS grafted fluorescein isothiocyanate as a fluorescent marker, the adsorption features of the nanoparticles were visualized by fluorescence microscopy and the cellular uptake percent was quantitated by flow cytometry. The internalization process of the nanoparticles was visualized by confocal laser scanning microscopy (CLSM) using nanoparticles of the size of 250 nm. Results showed that the three kinds of pDNA/CS nanoparticles had no differences in zeta potential, association efficiency, protection ability, stability and transfection efficiency in HEK293. The nanoparticles were all adsorbed on cell surface in the form of aggregates, and similar cellular uptake percent as well as quantities were observed 4 h post-incubation with HeLa cells. CLSM images showed that the aggregates below 2 microm could be internalized by endocytosis. These results suggest that the transfection efficiency of pDNA/CS nanoparticles does not depend on particle size in the range from 250 nm to 1300 nm.