透明质酸修饰壳聚糖／pDNA纳米微球的制备及体外转染软骨细胞

Preparation of hyaluronic acid modified chitosan/pDNA nanoparticles and transfection of chondrocytes in vitro

目的以透明质酸（HA）修饰基因载体壳聚糖（CS）纳米微球，制作一种新型的HA／CS／pDNA基因转染系统，观察其结构特征及体外对软骨细胞的转染能力。方法将不同比例的HA和CS与负载增强型绿色荧光蛋白基因（EGFP）的质粒DNA（pDNA）以复凝聚法制成纳米微球，以扫描电镜检测纳米微球形态，Zeta电位粒度分析仪测定其粒径、表面电位；凝胶电泳阻滞实验观察CS和pDNA的结合力；体外转染兔关节软骨细胞，以流式细胞仪及荧光显微镜检测转染效率。结果HA／CS／pDNA纳米微球多呈球形，粒径为（223±51）mm，表面Zeta电位为（17．4±6．1）mV，可有效保护pDNA免受DNaseⅠ的降解。体外转染实验证明HA／CS／pDNA纳米微球能介导pEGFP转染软骨细胞并在细胞内表达绿色荧光蛋白，转染率达（15．450±0．404）％，比裸pDNA组和CS／pDNA组有更高的转染效率（P〈0．05）。结论复凝聚法制备的HA／CS／pDNA纳米微球是一种有效的新型非病毒基因转染系统，对软骨细胞有着潜在的靶向基因转染能力。

Objective To prepare hyaluronic acid （HA） -modified ehitosan （ CS）/pDNA （ HA/ CS/pDNA） nanoparticles as novel gene vectors, study their structural characteristics and gene transfection efficiency for chondrocytes in vitro in rabbits. Methods The HA/CS/pDNA nanoparticles were prepared by a complex coacervation method with different ratios of HA： CS mixed with plasmid DNA （pDNA） , which load enhanced green fluorescent protein （EGFP） gene. The morphology of the nanoparticles was observed under the transmission electron microscopy. The sizes and zeta-potentials of the nanoparticles were measured by a Marven-nano laser diffractometer. The binding of pDNA was evaluated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed with the chondrocytes of rabbits. The gene transfection efficiency was measured by using flow eytometry and under the fluorescence microscopy. Results HA/CS/pDNA nanoparticles were mainly spherical, with an average size of （223 ± 51 ）nm, and zeta-potential of （ 17.4 ± 6. 1 ） mV. The agarose gel electrophoresis analysis confirmed that they could effectively protect pDNA from degradation against DNase Ⅰ. Gene transfection in vitro proved that HA/CS/pDNA nanoparticles could be efficiently transfected into chondrocytes of rabbits, the expression of green fluorescent proteins was observed under the fluorescent microscopy, and the transfection efficiency reached as high as （ 15. 450 ±0. 404 ） % , significantly higher than that of the naked pDNA or the CS/pDNAnanoparticles （P 〈 0. 05 ）. Conclusion HA/CS/pDNA nanoparticles were an effective novel non-viral gene transfer vector, which possessed the potential targeting transfection ability on chondrocytes.