聚氰基丙烯酸正丁酯纳米粒基因载体的制备及相关性质的体外研究(英文)

Preparation of polybutylcyanoacrylate nanoparticles gene delivery system and study on its characteristics in vitro

目的 优选制备可载带质粒DNA的聚氰基丙烯酸正丁酯纳米粒 (PBCA -NPs)的工艺条件 ,并研究其体外生物学特性。方法 选用乳化聚合法制备PBCA -NPs,采用正交实验法 ,以激光粒度分析仪及原子力显微镜 (AFM )综合分析实验结果来确定最优化的制备条件。用阳离子表面活性剂十六烷基三乙基溴化铵(CTAB)对纳米粒进行表面修饰后连接质粒DNA ,并分析PBCA -NPs及CTAB修饰的PBCA -NPs的细胞毒性效应、DNA装载效率、PBCA -NPs -DNA抗超声破坏和抗DNaseI的降解作用及其在体外的基因转染能力。结果 激光粒度分析仪及原子力显微镜结果表明 ,PBCA -NPs粒形圆整 ,大小均匀 ,平均粒径 10 6nm。琼脂糖凝胶电泳及紫外分光光度计测定DNA装载量达 93%。该纳米粒能显著提高质粒DNA抵抗核酸酶降解和超声波剪切的能力。细胞转染实验表明 ,该纳米粒传递质粒DNA进入HepG2和 3T3细胞的效率较高。结论 利用该制备工艺生产出的具备良好生物学特性的PBCA -NPs是基因转运和基因治疗中极具应用潜力的非病毒载体。

Objective: To optimize the preparation of the gene delivery system, plasmid DNA loaded-polybutycyanocrylate nanoparticles (PBCA-NPs-DNA), its biological characteristics were simultaneously investigated in vitro.Methods:The emulsion polymerization method was employed to prepare PBCA-NPs. The formula and preparation conditions were optimized by the orthogonality experiment design and by Master Sizer 2000 laser granule size instrument and atomic force microscope (AFM) analysis. Through surface modification of PBCA-NPs with the cationic surfactant cetyltrimethylammonium bromide (CTAB), plasmid DNA (pEGFP-N1) was loaded on the PBCA-NPs by adsorption. The cytotoxic effect of PBCA-NPs and CTAB modified PBCA-NPs were studied. The loading efficiency and the effects of sonication and DNase I on PBCA-NPs-DNA were determined. The cells, permeability of PBCA-NPs-DNA was demonstrated by cell uptake experiment in vitro. Results:The mean diame-ter of optimal PBCA-NPs was 106 nm by laser granule size instrument and atomic force microscope (AFM) analysis, and the DNA loading ratio reached to 93% by agarose gel analysis and UV spectrophotometry analysis. The PBCA-NPs can protect plasmid DNA from digestion by deoxyribonuclease I (DNase I) and sonication-induced shearing in vitro. The cellular uptake by different cell lines (HepG2 and 3T3) was enhanced.Conclusions:The technology of preparation is reasonable and can be used to provide PBCA-NPs of satisfactory quality. PBCA-NPs generated have good biological characteristics as a potential promising non-viral vector for gene transfer and gene therapy.