泊洛沙姆188修饰聚乙烯亚胺作为基因载体的体外评价
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摘要
目的使用泊洛沙姆188(P188)修饰聚乙烯亚胺(PEI),评价其作为体外基因载体的相关性质。方法将P188一端的羟基通过碳酸酯法连接在PEI的氨基上,得到新的聚合物,通过1H-NMR确定新聚合物的结构,该聚合物与DNA形成复合物后,测定复合物的粒径和Zeta电位,噻唑蓝(MTT)法考察复合物对MCF-7细胞、He La细胞、Hep G2细胞的毒性,使用质粒p GL3-lus作为报告基因,测定虫荧光素酶活性评价复合物对He La细胞的转染效率。结果1H-NMR结果表明合成的聚合物具有较高的纯度。复合物的粒径随N/P值有减小的趋势;复合物的Zeta电位随N/P值的增加而增高。复合物的细胞毒性随着N/P值的增加而增大,新聚合物其细胞毒性显著低于未修饰的PEI。新聚合物在高N/P值时仍能保持较高的转染效率,特别是(P188)1-PEI其最佳转染效率(N/P=24)显著高于PEI的最佳转染效率(N/P=6)。结论泊洛沙姆P188修饰的PEI可以作为一种有效的非病毒基因载体转染Hela细胞。
Objective To modify polyethyleneimine( PEI) by using Poloxamer 188( P188),and evaluate its related feature as carriers of genes in vitro. Methods PEI was modified through conjugating one hydroxyl group of P188 to the amino group of PEI by carbonate method. Structural analysis of synthesized polymer was performed by using1H-NMR. The particle size and Zeta potential of synthesized polymer/DNA complexes were measured. The cytotoxicity of the complexes was evaluated using MTT method in MCF-7 cells,He La cells and Hep G2 cells. The p GL3-lus was used as a reporter gene,and the transfection efficiency of complexesat He La cells was evalutated by measuring activity of luciferase. Results The result of1H-NMR showed the purity of these synthesized polymers was high. The particle size of complexes were decreased with the increment of N/P ratios.The Zeta potential of complexes increased with the increment of N/P ratios. The cytotoxicity of the complexes increased with the increment of N/P ratios. The synthesized polymers showed lower cytotoxicity than unmodified PEI. The new synthesized polymers had maintained the high transfection efficiency at high N/P ratios. In particular,the optimal transfection efficiency of( P188)1-PEI( N/P = 24) was significantly higher than that of PEI( N/P = 6). Conclusion The P188 modifed PEI can serve as a effective non-viral gene carriers to transfect Hela cells.
Objective To modify polyethyleneimine( PEI) by using Poloxamer 188( P188),and evaluate its related feature as carriers of genes in vitro. Methods PEI was modified through conjugating one hydroxyl group of P188 to the amino group of PEI by carbonate method. Structural analysis of synthesized polymer was performed by using1H-NMR. The particle size and Zeta potential of synthesized polymer/DNA complexes were measured. The cytotoxicity of the complexes was evaluated using MTT method in MCF-7 cells,He La cells and Hep G2 cells. The p GL3-lus was used as a reporter gene,and the transfection efficiency of complexesat He La cells was evalutated by measuring activity of luciferase. Results The result of1H-NMR showed the purity of these synthesized polymers was high. The particle size of complexes were decreased with the increment of N/P ratios.The Zeta potential of complexes increased with the increment of N/P ratios. The cytotoxicity of the complexes increased with the increment of N/P ratios. The synthesized polymers showed lower cytotoxicity than unmodified PEI. The new synthesized polymers had maintained the high transfection efficiency at high N/P ratios. In particular,the optimal transfection efficiency of( P188)1-PEI( N/P = 24) was significantly higher than that of PEI( N/P = 6). Conclusion The P188 modifed PEI can serve as a effective non-viral gene carriers to transfect Hela cells.
引文
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[12]梁爽,韩雷强,张娜.结构修饰的基于聚乙烯亚胺的载体在基因递送方面的研究进展[J].药物生物技术,2017,24(4):334-340.
[2] XIANG D,SHIGDAR S,QIAO G,et al.Aptamer-mediated cancer gene therapy[J]. Curr Gene Ther,2015,15(2):109-119.
[3] DE MENDOZA C,BARREIRO P,BENITEZ L,et al. Gene therapy for HIV infection[J].Expert Opin Biol Ther,2015,15(3):319-327.
[4] TOUZOT F,HACEIN-BEY-ABINA S,FISCHER A,et al.Gene therapy for inherited immunodeficiency[J]. Expert Opin Biol Ther,2014,14(6):789-798.
[5] IBRAHEEM D,ELAISSARI A,FESSI H.Gene therapy and DNA delivery systems[J]. Int J Pharm,2014,459(1/2):70-83.
[6] RAMAMOORTH M,NARVEKAR A.Non viral vectors in gene therapy an overview[J]. J Clin Diagn Res,2015,9(1):1-6.
[7]颜丹萍,张洪,马频.聚乙烯亚胺促进腺病毒体外基因表达[J].医药导报,2017,36(8):862-864.
[8]史疆,尹东锋.泊洛沙姆407修饰对聚乙烯亚胺的毒性和转染性质的影响[J].中国药师,2012,15(1):26-29.
[9] QUAZI T,SHUBHRA H,JUDIT T,et al.Poloxamers for surface modification of hydrophobic drug carriers and their effects on drug delivery[J]. Polymer Rev,2014,54(1):112-138.
[10] VILALTA A,MAHAJAN R K,HARTIKKA J,et al.Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine:evaluation of plasmid DNA biodistribution/persistence and integration[J]. Human Gene Therapy,2005,16(10):1143-1149.
[11] WEI F,XIN W,DING B,et al.Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine:preparation,characterization,intracellular trafficking,and cellular distribution[J]. Int J Nanomed,2012,45(9):1127-1138.
[12]梁爽,韩雷强,张娜.结构修饰的基于聚乙烯亚胺的载体在基因递送方面的研究进展[J].药物生物技术,2017,24(4):334-340.