中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design

目的采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒,选择壳聚糖浓度和质粒基因浓度作为实验考察因素,应用两因素五水平中心组合设计优化最佳转染制备区域,优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态;通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位;通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性;通过倒置荧光显微镜观察质粒基因在细胞内的表达;通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形,纳米粒平均粒径为217.6 nm,粒径多分散系数为0.241,表明粒径分布较窄。纳米粒zeta电位为+22.4 m V,表明纳米粒表面带有正电荷,可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高,能够高效地将绿色荧光蛋白质粒基因递送到细胞内,并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。

Objective This study aimed to optimize the preparation condition of DNA-chitosan nanoparticles with high transfec-tion efficency through a central composition design .Methods The DNA-chitosan nanoparticles were prepared by complex coacervation between pEGFP and chitosan .We selected the concentrations of chitosan and plasmid as two experimental factors , and a central compos-ite design with two factors and five levels was used to optimize the preparation condition of DNA-chitosan nanoparticles for high transfec-tion efficency .The concentrations of chitosan and plasmid were selected as the independent variables , respectively .The dependent varia-bles included average particle size and transfection efficiency .The morphology of DNA-chitosan nanoparticles was observed using a trans-mission electron microscope .The size and zeta potential of nanoparticles were measured by dynamic light scattering （ DLS） and electro-phoretic light scattering （ ELS ） , respectively .The stability of plasmids in the process of nanoparticles preparation was investigated through the agrose gel electrophoresis .The expression of plasmids delivered by nanoparticles was observed under an inverted fluorescence microscope .The transfection efficiency of DNA-chitosan nanoparticles was assayed by flow cytometry .Results The preparation condition of DNA-chitosan nanoparticles with high transfection efficency was optimized successfully .Under the optimum preparative conditions , the DNA-chitosan nanoparticles were almost spherical .The average size of nanoparticles was 217.6nm, and distributed in a narrow range with a polydispersity index of 0.241.The zeta potential was ＋22.4 mV, which suggested that a den-sity of positive charge exist onto the surface of nanoparticles and consequently enhanced the stability of nanoparticles suspension .The results of gel electrophoresis showed that plasmids were not destroyed in the process of nanoparticles preparation .The cell transfection of nanoparticles was very highly efficient .The nanoparticles could effectively deliver the pEGFP plasmids into cells to express the green fluorescent protein at a high level.Conclusion The established mathematic models have the good predictive function .Under the optimum preparative condi-tions, the DNA-chitosan nanoparticles have the high potential of cell transfection .