刺激响应双硒交联聚乙烯亚胺基因微载体

doi:10.7503/cjcu20130926

摘要/Abstract

摘要：

以丙酸二硒醚为交联剂, 通过调节交联剂的用量及反应时间, 制备了4种双硒交联聚乙烯亚胺(PEISeSe), 研究了其对脱氧核糖核酸(DNA)缔合能力、质子缓冲能力和转染效率的影响. 结果表明, 随着交联程度的增大, 质子缓冲能力降低. PEISeSe能有效诱导DNA的缔合, 当聚合物与DNA的质量比≥8时, PEISeSe/DNA组装体可形成150 nm的粒子. 在模拟细胞内的还原性环境下, 双硒键能有效断裂, 显示出很好的响应特性. 将氯喹与PEISeSe/DNA组装体同时加入到HEK293T细胞中, 氯喹的存在有利于PEISeSe/DNA组装体逃离溶酶体. 在细胞内高浓度还原型谷胱甘肽的作用下, PEISeSe交联聚合物可被降解为低分子量的PEI片段, 有利于释放出DNA并进攻细胞核, 提高转染率并降低毒性.

关键词: 双硒交联, 聚乙烯亚胺, 刺激响应, 非病毒基因载体

Abstract:

Four different diselenide-crosslinked polyethylenimine(PEISeSe) were successfully prepared by changing the dosage of cross-linker and reaction time. The influence of cross-linking level on the DNA condensation, proton buffer capacity and transfection efficiency were investigated. The results indicated that the proton buffer capacity of PEISeSe was reduced with cross-linking level. All of the polycations could efficiently condense DNA into spherical particles with diameter of 150 nm at m(PEISeSe)/m(DNA)=8. Moreover, diselenide bonds were easily cleaved in the presence of DTT. Cell culture results indicated that by adding chloroquine to exclude the limitation of lysosome escape, PEISeSe showed decreased cell cytotoxicity and efficient transfection. The proper reason was that the intracellular cleavage of diselenide bonds facilitated DNA release and nuclear entry. The diselenide-crosslinked polycations had promising potential in redox-sensitive gene delivery.

Key words: Diselenide crosslinked, Polyethylenimine, Stimulus-response, Non-viral gene vector

中图分类号:

O631.3
Q782

TrendMD:

孔韵娜, 李文宇, 杜建委, 汤建国, 胡巧玲, 王幽香. 刺激响应双硒交联聚乙烯亚胺基因微载体. 高等学校化学学报, 2014, 35(4): 881.

KONG Yunna, LI Wenyu, DU Jianwei, TANG Jianguo, HU Qiaoling, WANG Youxiang. Stimulus-responsive Diselenide-crosslinked Polyethyleneimine as Gene Vector^†. Chem. J. Chinese Universities, 2014, 35(4): 881.

图/表 10

Scheme 1 Synthesis routes of PEISeSe

Fig.1 1H NMR spectrum of PEISeSe2.7 in D2O

Fig.2 Buffer capacity(BC) of different polymer by acid-base titration Polymer concentration was fixed at 0.2 mg/mL; c(HCl)=0.1 mol/L.

Fig.3 Agarose gel electrophoresis retardation of different polyplexes with various m(PEISeSe)/m(DNA)

Fig.4 TEM images of different polyplexes at m(polymer)/m(DNA)=16 after treatment with 10 mmol/L DTT (A) PEI1800/DNA; (B) PEISeSe2.7/DNA; (C) PEISeSe2.7/DNA after treament with 10 mmol/L DTT.

Fig.5 Zeta potential of different polyplexes with various m(polymer)/m(DNA)Error bars represent mean±SD for n=3. a.PEI1800; b. PEISeSe2.3; c. PEISeSe2.7; d. PEISeSe3.4; e. PEISeSe4.1.

Fig.6 Ethidium bromide exclusion assay of PEISeSe2.7/DNA polyplexes with various m(PEISeSe)/m(DNA)

Fig.7 Cytotoxicity of HEK293T cells exposed to different polymer complexed with 1 μg DNA after incubation for 48 h Error bars represent mean±SD for n=5. a. Control; b.PEI25000; c. PEI1800; d. PEISeSe2.3; e. PEISeSe2.7; f. PEISeSe3.4; g. PEISeSe4.1.

Fig.8 Transfection efficiency of HEK293T cells exposed to different polyplexes in the presence of chloroquine detected by flow cytometryPEI25000 at N/P ratio of 10 is used as compared. Error bars represent mean±SD for n=3.a.PEI25000; b. PEI1800; c. PEISeSe2.3; d. PEISeSe2.7; e. PEISeSe3.4; f. PEISeSe4.1.

Fig.9 Intracellular distribution of PEISeSe2.7/Cy3-DNA polyplexes at m(PEISeSe)/m(DNA)=16The polyplexes were incubated with HEK293T cells for 4 h in the absence(A) and presence of chloroquine(B), followed by incubation in the absence of polyplexes and chloroquine for another 12 h(A3, B3). Cy3-DNA presented as red fluorescence(A1, B1) and DAPI-stained nuclei showed blue color(A2, B2).

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