›› 2013, Vol. 33 ›› Issue (1): 1-.doi: 10.3969/j.issn.1674-8115.2013.01.001

• 论著(基础研究) •    下一篇

新型纳米非病毒基因输送体系PCL-PEG-chitosan的构建与特性

王 斌1, 金才益1, 童海骏2, 戴尅戎2,3, 张晓玲2,3   

  1. 1.武警浙江总队医院骨二科, 嘉兴 314000; 2.上海交通大学 医学院健康科学研究所, 骨科细胞与分子生物学实验室, 上海 200025; 3.上海交通大学 医学院附属第九人民医院骨科, 上海市骨科内植物重点实验室, 上海 200011
  • 出版日期:2013-01-28 发布日期:2013-02-06
  • 通讯作者: 张晓玲, 电子信箱: xlzhang@sibs.ac.cn。
  • 作者简介:王 斌(1973—), 男, 副主任医师;电子信箱: monarchy@sina.com。
  • 基金资助:

    国家科技部国际合作项目(2011DFA30790);上海市教委曙光计划(10SG22);上海教委重点学科建设基金(J50206);武警浙江总队医院横向合作项目(0536J1A181)

Construction and characteristics of novel PCL-PEG-chitosan nano non-viral gene delivery system

WANG Bin1, JIN Cai-yi1, TONG Hai-jun2, DAI Ke-rong2,3, ZHANG Xiao-ling2,3   

  1. 1.Second Department of Orthopaedics, Armed Police Corps Hospital of Zhejiang, Jiaxing 314000, China;2.Laboratory of Orthopedic Cellular and Molecular Biology, Institute of Health Sciences, Shanghai Jiaotong University School of Medicine &|Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China;3.Shanghai Key Laboratory of Orthopaedic Implant, Department of Orthopaedics, the Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China
  • Online:2013-01-28 Published:2013-02-06
  • Supported by:

    Foundation of the Ministry of Science and Technology of China, 2011DFA30790;Shanghai Municipal Education Committee Foundation, 10SG22, J50206;Armed Police Corps Hospital of Zhejiang, 0536J1A181

摘要:

目的 优化新型纳米非病毒载体聚己内酯(PCL)-聚乙二醇(PEG)-壳聚糖(chitosan)(PCL-PEG-chitosan)在体外与质粒的复合条件,并进行转染活性测定。方法 在体外利用凝胶电泳方法确定非病毒载体与携带绿色荧光蛋白基因的质粒(pEGFP)复合的最小氮磷比。利用动态光散射粒径测量仪确定复合物在不同氮磷比、不同缓冲溶液体系下粒径的变化情况。将pEGFP与PCL-PEG-chitosan进行复合后转染293T细胞,荧光显微镜观察转染效率。结果 凝胶电泳结果显示,非病毒载体PCL-PEG-chitosan与pEGFP复合的最小氮磷比为4∶1,PCL-chitosan与pEGFP复合的最小氮磷比也为4∶1,chitosan与pEGFP复合的最小氮磷比为1∶1。动态光散射实验结果显示:非病毒载体PCL-PEG-chitosan与pEGFP复合而成的复合物的粒径随着氮磷比的增大而减小;在醋酸盐缓冲体系与DMEM缓冲体系中,复合物的粒径基本一致,相对稳定。荧光显微镜观察发现,经过接枝改性的纳米载体PCL-PEG-chitosan的转染效率有所提高。结论 PCL修饰可减弱chitosan对pEGFP的复合能力,提高最低复合氮磷比。经PCL和PEG修饰的纳米载体PCL-PEG-chitosan的细胞转染活性有所提高,但尚需进一步改进。

关键词: PCL-PEG-chitosan, 修饰, DNA, 转染, 氮磷比

Abstract:

Objective To optimize the complexation condition of the novel nano non-viral gene vectors of polycaprolactone (PCL)-polyethylene glycol (PEG)-chitosan with plasmid in vitro, and evaluate the transfection activity of the complexes. Methods The minimum N/P ratio of the complexation of non-viral gene vector with plasmid carrying green fluorescent protein gene (pEGFP) was measured by gel electrophoresis in vitro. The particle sizes of the complexes under different N/P ratios and different buffering conditions were determined by dynamic light scattering method. The 293T cells were transfected with complexes of pEGFP with PCL-PEG-chitosan, and the transfection efficiency of complexes was observed under fluorescence microscope. Results The results of gel electrophoresis demonstrated that the minimum N/P ratio of complexation of PCL-PEG-chitosan with pEGFP was 4∶1, the minimum N/P ratio of complexation of PCL-chitosan with pEGFP was also 4∶1, and the minimum N/P ratio of complexation of chitosan with pEGFP was 1∶1. Dynamic light scattering test revealed that the particle sizes of the complexes of PCL-PEG-chitosan with pEGFP decreased with the increase of N/P ratios, and the particle sizes in acetate buffer system and DMEM buffer system were similar and maintained stable. Fluorescence microscopic observation indicated that the transfection efficiency of PCL-PEG-chitosan improved to some extent. Conclusion PCL modification can attenuate the complexation ability of chitosan with pEGFP, and increase the minimum complexation N/P ratio. The transfection efficiency of PCL-PEG-chitosan improves after PCL and PEG modification, and needs to be further improved.

Key words: PCL-PEG-chitosan, modification, DNA, gene transfection, N/P ratio