药学学报, 2019, 54(5): 919-926
引用本文:
陈英, 胡锦芳, 张长林, 曹端文, 魏筱华. 聚酰胺-胺-聚谷氨酸-聚乙烯亚胺共聚物的制备及作为基因载体的性能研究[J]. 药学学报, 2019, 54(5): 919-926.
CHEN Ying, HU Jin-fang, ZHANG Chang-lin, CAO Duan-wen, WEI Xiao-hua. Synthesis and evaluation of a dendritic poly(L-glutamic acid)-graftpolyethylenimine copolymer as an efficient gene delivery vector[J]. Acta Pharmaceutica Sinica, 2019, 54(5): 919-926.

聚酰胺-胺-聚谷氨酸-聚乙烯亚胺共聚物的制备及作为基因载体的性能研究
陈英1,3, 胡锦芳1, 张长林2, 曹端文1, 魏筱华1,3
1. 南昌大学第一附属医院药学部GCP中心, 江西 南昌 330006;
2. 南昌大学第一附属医院检验科, 江西 南昌 330006;
3. 南昌大学药学院, 江西 南昌 330006
摘要:
本文合成了一种以零"代"聚酰胺-胺(generation 0 polyamidoamine,PAMAM G0)为内核、聚谷氨酸接枝小分子聚乙烯亚胺(polyethylenimine,PEI)为侧链的共聚物(PGLP),并研究了其作为基因载体的性能。应用1H NMR(proton nuclear magnetic resonance spectroscopy)确认结构;采用琼脂糖凝胶电泳考察PGLP对基因的压缩包裹能力和保护能力,动态光散射粒度分析仪测定PGLP/pDNA复合物的粒径及电位;运用细胞毒性检测试剂盒(CellCounting Kit-8,CCK-8)和溶血实验(南昌大学第一附属医院科研伦理委员会批准)考察PGLP的细胞毒性,体外细胞转染实验评估PGLP/pDNA复合物的转染能力。结果表明,PGLP可以有效压缩包裹DNA并保护其免受血清酶的降解;当PGLP/pDNA质量比大于或等于1(w/w ≥ 1)时,复合物平均粒径和电位分别约在105~200 nm和+10~+28 mV之间。毒性实验表明:PGLP及其复合物的细胞毒性均显著低于PEI 25K及其复合物,具有较高的安全性。细胞转染实验表明:PGLP/pDNA复合物在w/w=8时可获得最大转染能力,在有血清和无血清的条件下对多种细胞(HEK 293T、HeLa、BEL 7402和RASMC)的转染效率均显著高于PEI 25K或Lipofectamine 2000在最优转染比的效率。抗血清转染能力分析表明:PGLP/pDNA复合物(w/w=8)在血清中的转染效率与无血清转染效率的比值(转染比)高于PEI 25K/pDNA复合物,具有更强的抗血清转染能力。因此,PGLP是一种有潜力的基因载体。
关键词:    聚酰胺-胺      聚谷氨酸      聚乙烯亚胺      共聚物      基因载体     
Synthesis and evaluation of a dendritic poly(L-glutamic acid)-graftpolyethylenimine copolymer as an efficient gene delivery vector
CHEN Ying1,3, HU Jin-fang1, ZHANG Chang-lin2, CAO Duan-wen1, WEI Xiao-hua1,3
1. Clinical Trial Research Center, Department of Pharmacy, the First Affiliated Hospital, Nanchang University, Nanchang 330006, China;
2. Department of Clinical Lab oratory, the First Affiliated Hospital, Nanchang University, Nanchang 330006, China;
3. School of Pharmacy, Nanchang University, Nanchang 330006, China
Abstract:
The study was designed to synthesize a novel dendritic copolymer composed of polyamidoamine dendrimer G0 as the inner core and poly(L-glutamic acid) grafted low molecular weight polyethylenimine (PGLP) as surrounding arms for gene delivery vector. The molecular structure of PGLP was confirmed by 1H NMR (proton nuclear magnetic resonance spectroscopy). The DNA combination capability of PGLP was examined by gel retarda tion electrophoresis. The particle sizes and zeta potentials of PGLP/pDNA complexes were determined by dynamic light scattering (DLS). The cytotoxicity of PGLP was evaluated by Cell Counting Kit-8 (CCK-8) and hemolysis assays, which was approved by Research Ethics Committee of the First Affiliated Hospital of Nanchang University. The in vitro transfection efficiency of PGLP was measured by a flow cytometry. The results of physicochemical properties suggested that PGLP could self-assemble with DNA to form complexes with average particle sizes of about 105-200 nm and zeta potentials of about +10-+28 mV, which could protect DNA from serum degradation. The results of biological properties suggested that PGLP showed more higher transfection efficiencies but lower cytotoxicity than PEI 25K or Lipofectamine 2000 in various cell lines (HEK 293T, HeLa, BEL 7402, RASMC). Importantly, it was found that PGLP/pDNA complexes at w/w=8 showed more strong serum-resistant capacity than PEI 25K/pDNA complexes. Therefore, PGLP is a promising candidate vector for gene delivery.
Key words:    poly (amidoamine)    poly(L-glutamic acid)    polyethyleneimine    copolymer    gene vector   
收稿日期: 2018-12-24
DOI: 10.16438/j.0513-4870.2018-1143
基金项目: 国家自然科学基金资助项目(81660591);江西省青年科学基金资助项目(S2017QNJJB0922/612080930043);江西省卫健委普通科技计划项目(20191053).
通讯作者: 曹端文,Tel/Fax:86-791-88695125,E-mail:dwyss66@163.com;魏筱华,Tel/Fax:86-791-88696232,E-mail:wxh-hello@163.com
Email: dwyss66@163.com;wxh-hello@163.com
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