pH响应的PEG化基因传递体系

doi:10.7503/cjcu20150907

摘要/Abstract

摘要：

制备了苯甲酰亚胺键偶联的聚乙二醇化(PEG化)聚乙烯亚胺(mPEG-CH═N-PEI), 并以还原无pH响应特性mPEG-PEI作为对照. 研究结果表明, PEG链段的引入并未影响聚乙烯亚胺与脱氧核糖核酸(DNA)分子的缔合, 形成了尺寸为80 nm, 表面电位约为10 mV的基因超分子组装体, 具有很好的生理盐稳定性. 在模拟溶酶体的酸性环境下, 苯甲酰亚胺键有效断裂, 显示出很好的pH响应特性. HepG2细胞培养结果表明, 由于PEG链段有效屏蔽了组装体表面的正电荷, PEG化组装体的细胞毒性和内吞效率显著降低, 但溶酶体酸性条件使苯甲酰亚胺键断裂, 有利于组装体逃离溶酶体, 因此mPEG-CH═N-PEI依然具有很高的基因转染效率, 实现了基因载体细胞外稳定传递、细胞内响应解离并高效转染的功能.

关键词: 非病毒基因载体, 聚乙烯亚胺, pH响应, 聚乙二醇化

Abstract:

The pH-sensitive polyethylene glycol-grafted polyethylenimine(mPEG-CH═N-PEI) was synthesized via benzoic imine linker according to the acidic environment of endosomal. The corresponding stable PEGylated polyethylenimine(mPEG-PEI) without pH-sensitivity was synthesized as control. The results showed that both PEGylated polycations could condense DNA into tight spherical nanoparticles about 80 nm in size and 10 mV in ξ-potential, which showed excellent stability under physiological conditions. The benzoic imine linkers were easily cleaved under the pH of 5, which was similar with the endosomal pH. Human hepatoblastoma cell line(HepG2) cell culture results indicated that the cytotoxicity and cellular uptake efficiency of both PEGylated gene nanoparticles decreased remarkably because of the shielding effect of PEG shell. Due to the cleavage of benzoic imine linker at acidic environment of endosomal, the PEG shell of mPEG-CH═N-PEI/DNA polyplexes was detachable and lead to quick endosomal escape. So mPEG-CH═N-PEI/DNA polyplexes showed higher gene expression than the mPEG-PEI/DNA polyplexes. Based on these results, we concluded that PEGylated polyplexes via benzoic imine linkers showed high extracellular stability, intracellular DNA release and effective gene transfection.

Key words: Non-viral vector, Polyethylenimine, pH-Sensitive, PEGylation

中图分类号:

O631.3
Q782

TrendMD:

刘亚杰, 张鹏, 杜建委, 王幽香. pH响应的PEG化基因传递体系. 高等学校化学学报, 2016, 37(5): 1003.

LIU Yajie, ZHANG Peng, DU Jianwei, WANG Youxiang. pH-Responsive PEGylated Gene Delivery System^†. Chem. J. Chinese Universities, 2016, 37(5): 1003.

图/表 8

Fig.1 1H NMR spectra of mPEG-CHO in CDCl3(a) and mPEG-CH═N-PEI in D2O(b)

Fig.2 Agarose gel electrophoresis assay of mPEG-CH═N-PEI/DNA(A), mPEG-PEI/DNA(B), PEI/DNA(C) in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl) with various N/P ratios

Fig.3 Particle sizes(A) and ζ-potentials(B) of different polyplexes in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl)Error bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.4 Typical TEM images of gene polyplexes at different N/P ratiosAll the polyplexes were prepared in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl). (A) mPEG-CH═N-PEI/DNA, N/P=10; (B) mPEG-CH═N-PEI/DNA, N/P=20; (C) mPEG-PEI/DNA, N/P=10; (D) mPEG-PEI/DNA, N/P=20.

Fig.5 Time-dependent change of the particle sizes under physiological salt(150 mmol/L NaCl) conditions(A) or culture media with 10% FBS(B)(t=0 means at prepared condition) and ξ-potentials of various polyplexes at pH=5.0 and 7.4(C)(A) a. PEI/DNA, pH=7.4; b. mPEG-CH═N-PEI/DNA, pH=7.4; c. mPEG-PEI/DNA, pH=7.4; d. mPEG-CH═N-PEI/DNA, pH=5.0; e. mPEG-PEI/DNA, pH=5.0. (B) a. mPEG-CH═N-PEI/DNA in DMEM with 10%(volume fraction) FBS; b. mPEG-PEI/DNA in DMEM with 10%(volume fraction) FBS; c. PEI/DNA in DMEM with 10%FBS. (C) a. mPEG-CH═N-PEI/DNA; b. mPEG-PEI/DNA. Error bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.6 Cell cytotoxicity of HepG2 cells exposed to different nanoparticles with 1 μg DNA after incubation for 48 h(A) and in vitro transfection of the luciferase gene into HepG2 cells mediated by different nanoparticles for 48 h(B)(A) Error bars represent mean± SD for n=5; (B) error bars represent mean ±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.7 Cellular uptake efficiency of different nanoparticles by HepG2 cells for incubation of 4 hError bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.8 Intracellular distribution of Cy3-DNA polyplexed with mPEG-CHN-PEI(A1—A3), mPEG-PEI(B1—B3) and PEI(C1—C3) at an N/P ratio of 10(A1—C1) Cy3; (A2—C2) Lyso-Tracker; (A3—C3) Cy3+Lyso-Tracker.

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