高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (9): 2019-2026.doi: 10.7503/cjcu20140326

• 高分子化学 • 上一篇    下一篇

聚谷氨酸为骨架的梳状基因载体的合成及生物学性能评价

何宁(), 孙贺春, 徐欢喜, 邵张章   

  1. 安徽中医药大学药学院, 合肥 230031
  • 收稿日期:2014-04-08 出版日期:2014-09-10 发布日期:2019-08-01
  • 作者简介:联系人简介: 何宁, 男, 博士, 副教授, 主要从事药用高分子材料研究. E-mail: hening826@163.com
  • 基金资助:
    安徽省高等学校省级自然科学研究重点项目(批准号: KJ2012A182)资助

Synthesis and Biological Evaluation of Comblike Non-virus Gene Delivery Vector with the Poly(L-glutamic acid) as Backbone

HE Ning*(), SUN Hechun, XU Huanxi, SHAO Zhangzhang   

  1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, China
  • Received:2014-04-08 Online:2014-09-10 Published:2019-08-01
  • Contact: HE Ning E-mail:hening826@163.com

摘要:

以聚谷氨酸为骨架, 用低分子量聚乙烯亚胺胺解聚谷氨酸苄酯, 得到聚谷氨酸-g-聚乙烯亚胺, 用异佛尔酮二异氰酸酯将聚乙二醇单甲醚偶联到聚谷氨酸-g-聚乙烯亚胺上, 合成了梳状聚阳离子基因载体聚谷氨酸-g-(聚乙烯亚胺-b-聚乙二醇). 利用核磁共振氢谱、 激光粒度分析仪、 Zeta电位仪和凝胶电泳对聚阳离子载体及其与质粒脱氧核糖核酸(pDNA)形成的复合物进行了表征. 通过噻唑蓝(MTT)细胞毒性测试、 绿色荧光蛋白质粒pEGFP-C1及荧光素酶质粒pGL3体外转染实验考察了载体的细胞毒性及基因转染效率. 结果表明, 当聚乙烯亚胺中N原子和DNA中P原子的摩尔比(N/P)大于5时, 载体能很好地包裹DNA, 载体与DNA形成的复合物粒径约为130 nm, Zeta电位约为28 mV; 通过MTT实验和体外质粒转染实验显示出载体在测量范围内具有极低的细胞毒性和较高的转染效率.

关键词: 聚阳离子基因载体, 聚乙烯亚胺, 绿色荧光蛋白, 聚乙二醇, 聚谷氨酸, 基因转染效率

Abstract:

A novelcomblike catiomer, poly(L-glutamic acid)-graft-[polyethylenimine-block-poly(ethylene glycol)][PG-g-(PEI-b-PEG)], was synthesized by coupling of poly(L-glutamic acid)-graft-polyethylenimine(PG-g-PEI) and PEG using isophorone diisocyanate(IPDI). PG-g-PEI was firstly prepared via the ammonolysis of poly(γ-benzyl L-glutamate)(PBLG) with the low molecular weight polyethylenimine. The molecular structural property of PG-g-(PEI-b-PEG) was confirmed by 1H nuclear magnetic resonance spectroscopy(1H NMR) and gel permeation chromatography(GPC). The complexes of PG-g-(PEI-b-PEG)/pDNA were measured by dynamic light scattering and zeta-potential instrument. The results showed that PG-g-(PEI-b-PEG) could efficiently condense pDNA into nanosized particles with positive surface charges. The DNA binding ability of PG-g-(PEI-b-PEG) was further examined by gel retardation assay, and PG-g-(PEI-b-PEG) can retard plasmid DNA completely at N/P ratios above 5 in electrophoresis on agarose gel. The PG-g-(PEI-b-PEG) shows relative lower cytotoxicity against HEK 293T cells even at high N/P ratio demonstrated by MTT assay. The gene transfection evaluation revealed that the higher transfection efficiency for PG-g-(PEI-b-PEG) as a delivery agent was obtained at N/P ratio of 5, which exceeded 20-fold that of PEI 25000. These results suggest that the novel comblike catiomer PG-g-(PEI-b-PEG) might be an excellent biocompatible candidate for gene delivery system.

Key words: Cationic gene delivery vector, Polyethylenimine, Green fluorescent protein, Poly(ethylene glycol), Poly(L-glutamic acid), Gene tramsferction efficiency

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