原子转移自由基聚合(ATRP)阳离子化菊粉作为非病毒基因载体的研究

doi:10.7503/cjcu20140087

摘要/Abstract

摘要：

利用原子转移自由基聚合(ATRP)将聚甲基丙烯酸N,N-二甲氨基乙酯[P(DMAEMA)]偶联到菊粉多糖(Inulin)上, 合成了新型基因载体PDIN. 利用核磁共振仪、动态光散射分析仪、透射电子显微镜和凝胶电泳对PDIN及其与质粒pDNA的复合物进行了表征. 凝胶阻滞实验结果表明, PDIN可以通过静电相互作用稳定结合pDNA. 噻唑蓝(MTT)细胞毒性测试、溶血实验、绿色荧光蛋白表达质粒(pGFP)及β半乳糖苷酶表达质粒(pβgal)转染实验结果表明, PDIN对MCF-7, Hela, COS7和HepG2细胞的毒性较小; 其溶血率低, 具有良好的血液相容性; 载体PDIN能有效将pGFP和pβgal带入COS7细胞并表达, 在N/P为1时转染效率最高, 其β半乳糖苷酶的酶活为(3.36±0.74) U/mg蛋白, 比Lipo2000转染效率[(4.33±0.77) U/mg蛋白]略低. 因此, 所合成的载体PDIN是一种有潜在应用价值的非病毒基因载体.

关键词: 菊粉多糖, 原子转移自由基聚合(ATRP), 绿色荧光蛋白表达质粒(pGFP), β, 半乳糖苷酶表达质粒(pβ, gal), 非病毒基因载体

Abstract:

In order to develop new biocompatible gene delivery system, in the present study, a novel non-virus gene delivery vector PDIN was synthesized by grafting poly(DMAEMA) onto the backbone of inulin oligosaccharide via atom transfer radical polymerization(ATRP). ¹H NMR, particle size analysis and zeta-potential measurements, TEM and gel electrophoresis were performed to characterize PDIN and PDIN/pDNA complexes. At different N/P ratios(2, 1, 0.5 and 0.25), PDIN could condense pDNA into complex nanoparticles of 75.55 nm to 104 nm in sizes. Gel electrophoresis retardation assay demonstrated that PDIN could interact with pDNA via electrostatic interaction excellently even in the presence of high concentration heparin. The results of MTT assay displayed that the PDIN/pDNA complexes showed comparable cytotoxicity against MCF-7, HeLa, COS7 and HepG2 cells with that of Lipofectamine2000/pDNA complex. PDIN showed good biocompatibility due to its low hemolytic rate. The activity of β-galactosidase expressed in COS7 cells revealed that the highest transfection efficiency[(3.36±0.74) U/mg protein] of PDIN as a new gene delivery carrier was obtained at N/P ratio of 1, which was comparable with that of Lipofectamine2000[(4.33±0.77) U/mg]. These results suggested that inulin oligosaccharide-grafted poly(DMAEMA)(PDIN) might be an excellent biocompatible candidate for gene delivery systems.

Key words: Inulin, Atom transfer radical polymerization, pGFP, pβ, gal, Non-virus gene vector

中图分类号:

TrendMD:

王静云, 付雪, 包永明. 原子转移自由基聚合(ATRP)阳离子化菊粉作为非病毒基因载体的研究. 高等学校化学学报, 2014, 35(10): 2124.

WANG Jingyun, Fu Xue, BAO Yongming. Cationization of Inulin via Atom Transfer Radical Polymerization for Gene Delivery^†. Chem. J. Chinese Universities, 2014, 35(10): 2124.

图/表 7

Scheme 1 Synthetic routes of PDIN

Table 1 Particle size and ζ potential of PDIN/pDNA complexes

N/P	Mean diameter/nm(PDI)	ζ potential/mV	N/P	Mean diameter/nm(PDI)	ζ potential/mV
0.25	104±4.08(0.183)	2.36±0.12	1	85.4±5.42(0.236)	5.32±0.14
0.5	96.59±8.51(0.192)	4.24±0.28	2	75.58±0.43(0.232)	6.13±0.37

Fig.1 TEM image of PDIN polymer

Fig.2 Hemolysis test results for PDIN and PDIN/pDNA (A) PDIN at different concentrations. a. 1 mg/mL PDIN; b. 2 mg/mL PDIN; c. 1 mg/mLPEI 25K; d. 2 mg/mL PEI 25K; (B) PDIN/pDNA complex(N/P=2) at 1 mg/mL of PDIN. a. PDIN/pDNA; b. PEI 25K/pDNA.

Fig.3 Cell viability of PDIN/pDNA complexes on different cells Cells were treated with PDIN/pDNA or Lipo2000/pDNA complexes for 48 h at 37 ℃. Cell viability was measured by the MTT method. * P<0.05; ** P<0.01. a. Control; b. N/P=0.25; c. N/P=0.5; d. N/P=1; e. N/P=2; f. Lipo2000.

Fig.4 Transfection efficiency of PDIN/pGFP at different N/P ratios in COS7 cells(200×)

Fig.5 Transfection efficiency of PDIN/pβgal at different N/P ratios a. N/P=0.25; b. N/P=0.5; c. N/P=1; d. N/P=2; e. pDNA; f. Lipo2000/pDNA.

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