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

doi:10.7503/cjcu20140326

摘要/Abstract

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

中图分类号:

O631.1⁺1

TrendMD:

何宁, 孙贺春, 徐欢喜, 邵张章. 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. 高等学校化学学报, 2014, 35(9): 2019.

HE Ning, SUN Hechun, XU Huanxi, SHAO Zhangzhang. Synthesis and Biological Evaluation of Comblike Non-virus Gene Delivery Vector with the Poly(L-glutamic acid) as Backbone^†. Chem. J. Chinese Universities, 2014, 35(9): 2019.

图/表 10

Scheme 1 Schematic illustration of PG-g-(PEI-b-PEG)/pDNA complex formation

Scheme 2 Synthetic routes of comblike PG-g-(PEI-b-PEG) catiomer

Fig.1 1H NMR spectra of PBLG in CDCl3(A), PG-g-PEI(B) and PG-g-(PEI-b-PEG)(C) Peaks a—g are corresponding to the structures in Scheme 2, respectively.

Fig.2 GPC curves of PG-g-PEI(a) and PG-g-(PEI-b-PEG)(b)

Fig.3 Percentages of cell viability at various polymer concentrations for PG-g-PEI(a), PG-g-(PEI-b-PEG)(b), PEI 25000(c) and PEI 800(d)

Fig.4 Gel retardation assay of PG-g-(PEI-b-PEG)/pDNA complexes prepared at different N/P ratios Lane 1: free DNA; lanes 2—7: PG-g-(PEI-b-PEG)/pDNA complexes with N/P ratios of 0.5, 1, 3, 5, 10 and 20; lane 8: PG-g-(PEI-b-PEG) polymer solution.

Fig.5 Particle size of PG-g-PEI/pDNA and PG-g-(PEI-b-PEG)/pDNA complexes

Fig.6 Zeta potential of PG-g-PEI/pDNA and PG-g-(PEI-b-PEG)/pDNA complexes

Fig.7 Cell morphologies(A1—F1) and pEGFP-C1 expression under the observation of the fluorescence microscopies(A2—F2) using PG-g-(PEI-b-PEG) as the vector (A1, A2)—(F1, F2) are N/P ratio of 0.5, 3, 5, 10, 40 and 0, respectively.

Fig.8 Transfection efficiency of PG-g-(PEI-b-PEG)/pGL3 complexes using various N/P ratios in HEK 293T cells

参考文献 20

[1]	Thomas W., Nigel P., Seppo Y., Gene,2013, 525(2), 162—169
[2]	Park T. G., Jeong J. H., Kim S. W., Adv. Drug Deliv. Rev. ,2006, 58(4), 467—486
[3]	Melanie G., Jens L., Anastasia M., Daniela G., Wolfgang K., Achim A., European Journal of Pharmaceutics and Biopharmaceutics,2011, 77(3), 438—449
[4]	Godbey W. T., Wu K. K., Mikos A. G., Journal of Controlled Release,1999, 60(2/3), 149—160
[5]	Young H. K., Jeong H. P., Lee M., Journal of Controlled Release,2005, 103(1), 209—219
[6]	Chen L. N., Wang Y., Zhu Y., Sun Y. X., Wang Y. X., Chem. J. Chinese Universities,2013, 34(3), 720—725
	(陈丽娜, 王颖, 朱莹, 孙一新, 王幽香. 高等学校化学学报, 2013, 34(3), 720—725)
[7]	Zhang W., Liu X.L., Zhang M., Shi Q. H.,Acta Polymerica Sinica, 2013, (8), 1025—1032
	(张维, 刘相丽, 张猛, 史清洪. 高分子学报, 2013, (8), 1025—1032)
[8]	Wang W., Nan W., Sun L., Reactive and Functional Polymers,2013, 73(8), 993—1000
[9]	Kong Y. N., Li W. Y., Du J. W., Tang J. G., Hu Q. L., Wang Y. X., Chem. J. Chinese Universities,2014, 35(4), 881—887
	(孔韵娜, 李文宇, 杜建委, 汤建国, 胡巧玲, 王幽香. 高等学校化学学报, 2014, 35(4), 881—887)
[10]	Xun M. M., Liu Y. H., Guo Q., European Journal of Medicinal Chemistry,2014, 78, 118—125
[11]	Wang J. Y., Dou B. R., Bao Y. M., Li S. W., Chem. J. Chinese Universities,2012, 33(8), 1757—1764
	(王静云, 窦柏蕊, 包永明, 李森武. 高等学校化学学报, 2012, 33(8), 1757—1764)
[12]	Yuan R. X., Zuo Y. F., Xiao Z. P., Huang W. L., Shuai X. T., Acta Polymerica Sinica,2009, 1(2), 104—110
	(苑仁旭, 左瑜芳, 肖中鹏, 黄文林, 帅心涛. 高分子学报, 2009, 1(2), 104—110)
[13]	Liu J., Xu L., Jiang X. L., Hennink W. E., Wang X., Zhuo R., Journal of Controlled Release,2011, 152(S1), e157—e159
[14]	Wang Y. M., Zhang Z. F., Yu Q., Chem. J. Chinese Universities,2008, 29(10), 2011—2014
	(王燕铭, 张振方, 于琦. 高等学校化学学报, 2008, 29(10), 2011—2014)
[15]	Xie J., Li X., Jiang C. J., Lee R. J., Zhou Y. L., Teng L. S., Chem. Res. Chinese Universities,2013, 29(5), 1003—1005
[16]	Javier S. N., Peter F., Daniel P., European Journal of Medicinal Chemistry,2014, 76, 43—52
[17]	Zhang X. M., Zhang J. W., Wei G. Y., Liu D. X., Yu D. H., Yao W. G., Dong W. F., Chem. J. Chinese Universities,2012, 33(9), 2085—2091
	(张鑫淼, 张佳伟, 魏光耀, 刘东旭, 于达慧, 姚卫国, 董文飞. 高等学校化学学报, 2012, 33(9), 2085—2091)
[18]	Huang F. W., Wang H. Y., Li C., Wang H. F., Sun Y. X., Feng J., Zhang X. Z., Zhuo R. X., Acta Biomaterialia,2010, 6(11), 4285—4295
[19]	Guo J. S., Huang Y. B., Jing X. B., Chen X. S., Polymer,2009, 50(13), 2847—2855
[20]	Cheon J. B., Jeong Y., Cho C. S., Polymer,1999, 40(8), 2041—2050