基于超支化聚酰胺胺可生物降解阳离子基因递送系统的构建与体外评价
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摘要
背景:与其他聚阳离子相比,超支化聚酰胺胺的细胞毒性小、生物相容性好、溶血活性低、易于表面修饰,在基因递送中是一类具有广阔应用前景的阳离子聚合物。但目前应用于基因转染的超支化聚酰胺胺大多不可降解,易在体内聚集引起细胞毒性。目的:合成还原降解超支化聚酰胺胺(redox-degradable hyperbranched polyamidoamine,DHPAA),考察其作为基因递送载体的安全性和有效性。方法:以N,N'-双(丙烯酰)胱胺(BAC)和1-(2-氨乙基)哌嗪(AEPZ)为功能性单体,通过迈克尔加成一锅法合成DHPAA,采用核磁共振氢谱、凝胶渗透色谱和酸碱滴定法对其结构和性能进行表征。采用自组装法制备DHPAA/DNA复合物,使二者质量比分别为1∶1、5∶1、10∶1、15∶1、20∶1;采用动态光散射和透射电镜测定复合物的粒径、形貌和Zeta电势;采用琼脂糖凝胶阻滞电泳、Picogreen荧光分析和体外释放DNA实验检测DHPAA对DNA的固缩能力及DHPAA的还原降解性。以人肾上皮细胞系HEK293、人乳腺癌细胞系MCF-7、间充质干细胞和子宫颈癌细胞系Hela为细胞模型,采用MTT法检测聚合物DHPAA的细胞毒性;以间充质干细胞和子宫颈癌细胞系Hela为细胞模型,采用MTT法检测DHPAA/DNA复合物的细胞毒性。以绿色荧光蛋白报告基因为研究对象(绿色荧光蛋白报告基因与DHPAA的质量比分别为1∶1、5∶1、10∶1、15∶1、20∶1),使用流式细胞仪与共聚焦激光扫描显微镜考察DHPAA在间充质干细胞中的体外转染效率。结果与结论:(1)合成的聚合物DHPAA结构符合分子设计,在pH=3-10范围内具有良好的缓冲能力;聚合物DHPAA对DNA分子具有很好的固缩能力,形成的复合物在生理条件下很稳定,在还原性介质中能够快速释放出DNA;当DHPAA/DNA质量比由1∶1增加到10∶1时,复合物的包封率逐渐增加,此后质量比继续增加包封率不再增加;(2)在1-200 mg/L范围内,聚合物DHPAA对HEK293、MCF-7、间充质干细胞和Hela细胞的存活率无影响;(3)当质量比由1∶1增加到10∶1时,25 mg/L DHPAA/DNA复合物对间充质干细胞和Hela细胞的存活率无影响;(4)当质量比由1∶1增加到10∶1时,DHPAA/绿色荧光蛋白报告基因复合物的转染效率逐渐增加,当质量比继续增加时转染效率增加缓慢;(5)结果表明,还原降解DHPAA生物相容性好,能够高效地将基因递送到细胞内并高效表达基因。综合考虑DHPAA的DNA负载效率和复合物的转染效果,选择10∶1为最佳聚合物/DNA质量比。
BACKGROUND: Compared with other polycations, hyperbranched polyamidoamine(HPAA) is a kind of cationic polymer with broad application prospect in gene delivery due to small cytotoxicity, good biocompatibility, low hemolytic activity and easy surface modification. However, HPAAs used in gene transfection are mostly non-degradable and easy to accumulate in vivo and cause cytotoxicity. OBJECTIVE: To investigate the safety and efficiency of redox-degradable hyperbranched polyamidoamine(DHPAA) as the gene delivery carrier. METHODS: Using N,N'-bis(acryloyl)cystamine and 1-(2-aminoethyl)piperazine as functional monomers, DHPAA was synthesized with "one-pot" Michael addition polymerization, and its structure and performance were characterized by H-nuclear magnetic resonance, gel permeation chromatography and acid-base titration. The DHPAA/DNA complexes were prepared by self-assembly method with the mass ratios of 1:1, 5:1, 10:1, 15:1 and 20:1, respectively. The particle size, morphology and Zeta potential of DHPAA/DNA complexes were determined by dynamic light scattering and transmission electron microscopy. The capacity of DHPAA to compact DNA and reduction degradation of DHPAA were respectively investigated by using agarose gel retardation assay, Picogreen fluorescence analysis and in vitro release DNA test. The cytotoxicity of polymer DHPAA was detected by MTT method using human renal epithelial cell line HEK293, human breast cancer cell line MCF-7, mesenchymal stem cells and cervical cancer cell line Hela as cell models. The cytotoxicity of DHPAA/DNA complexes were evaluated by MTT method using mesenchymal stem cells and Hela of cervical cancer cells as cell models. Using green fluorescent protein reporter gene as research object, the in vitro transfection efficiency of DHPAA was evaluated in mesenchymal stem cells by flow cytometry and confocal laser scanning microscopy with the DHPAA/green fluorescent protein reporter gene mass ratios of 1:1, 5:1, 10:1, 15:1 and 20:1, respectively. RESULTS AND CONCLUSION:(1) The synthetic polymer DHPAA had the right structure consistent with the molecular design, low cytotoxicity and good buffer ability in the range of pH=3-10. The polymer DHPAA was able to condense DNA into the complexes with good stability under physiological conditions and to rapidly release DNA in the reducing medium. When the DHPAA/DNA mass ratio increased from 1:1 to 10:1, the encapsulation rate of complexes gradually increased, and then remained unchanged with the mass ratio more than 10:1.(2) In the range of 1-200 mg/L, the polymer DHPAA had no effect on the cell viability of HEK293, MCF-7, mesenchymal stem cells and Hela cells.(3) When the DHPAA/DNA mass ratio increased from 1:1 to 10:1, the complex with the concentration of 25 mg/L had no effect on the cell viability of mesenchymal stem cells and Hela cells.(4) The transfection efficiency of DHPAA/green fluorescent protein reporter gene complexes increased gradually when the mass ratio increased from 1:1 to 10:1 and increased slowly when the mass ratio continued to increase. To conclude, DHPAA, with good biocompatibility, cannot only efficiently deliver the gene into cells but also correctly express the gene. Integrally considering the efficiency of loading DNA of DHPAA and the transfection effect of the complexes, 10:1 is determined to be the optimal polymer/DNA mass ratio.
BACKGROUND: Compared with other polycations, hyperbranched polyamidoamine(HPAA) is a kind of cationic polymer with broad application prospect in gene delivery due to small cytotoxicity, good biocompatibility, low hemolytic activity and easy surface modification. However, HPAAs used in gene transfection are mostly non-degradable and easy to accumulate in vivo and cause cytotoxicity. OBJECTIVE: To investigate the safety and efficiency of redox-degradable hyperbranched polyamidoamine(DHPAA) as the gene delivery carrier. METHODS: Using N,N'-bis(acryloyl)cystamine and 1-(2-aminoethyl)piperazine as functional monomers, DHPAA was synthesized with "one-pot" Michael addition polymerization, and its structure and performance were characterized by H-nuclear magnetic resonance, gel permeation chromatography and acid-base titration. The DHPAA/DNA complexes were prepared by self-assembly method with the mass ratios of 1:1, 5:1, 10:1, 15:1 and 20:1, respectively. The particle size, morphology and Zeta potential of DHPAA/DNA complexes were determined by dynamic light scattering and transmission electron microscopy. The capacity of DHPAA to compact DNA and reduction degradation of DHPAA were respectively investigated by using agarose gel retardation assay, Picogreen fluorescence analysis and in vitro release DNA test. The cytotoxicity of polymer DHPAA was detected by MTT method using human renal epithelial cell line HEK293, human breast cancer cell line MCF-7, mesenchymal stem cells and cervical cancer cell line Hela as cell models. The cytotoxicity of DHPAA/DNA complexes were evaluated by MTT method using mesenchymal stem cells and Hela of cervical cancer cells as cell models. Using green fluorescent protein reporter gene as research object, the in vitro transfection efficiency of DHPAA was evaluated in mesenchymal stem cells by flow cytometry and confocal laser scanning microscopy with the DHPAA/green fluorescent protein reporter gene mass ratios of 1:1, 5:1, 10:1, 15:1 and 20:1, respectively. RESULTS AND CONCLUSION:(1) The synthetic polymer DHPAA had the right structure consistent with the molecular design, low cytotoxicity and good buffer ability in the range of pH=3-10. The polymer DHPAA was able to condense DNA into the complexes with good stability under physiological conditions and to rapidly release DNA in the reducing medium. When the DHPAA/DNA mass ratio increased from 1:1 to 10:1, the encapsulation rate of complexes gradually increased, and then remained unchanged with the mass ratio more than 10:1.(2) In the range of 1-200 mg/L, the polymer DHPAA had no effect on the cell viability of HEK293, MCF-7, mesenchymal stem cells and Hela cells.(3) When the DHPAA/DNA mass ratio increased from 1:1 to 10:1, the complex with the concentration of 25 mg/L had no effect on the cell viability of mesenchymal stem cells and Hela cells.(4) The transfection efficiency of DHPAA/green fluorescent protein reporter gene complexes increased gradually when the mass ratio increased from 1:1 to 10:1 and increased slowly when the mass ratio continued to increase. To conclude, DHPAA, with good biocompatibility, cannot only efficiently deliver the gene into cells but also correctly express the gene. Integrally considering the efficiency of loading DNA of DHPAA and the transfection effect of the complexes, 10:1 is determined to be the optimal polymer/DNA mass ratio.
引文
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[2]李丽春,李剑平.基因转染间充质干细胞的临床治疗:特点与问题[J].中国组织工程研究,2014,18(14):2257-2262.
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[6]El Aneed A.An overview of current delivery systems in cancer gene therapy.J Control Release.2004;94(1):1-14.
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[8]Lehrman S.Virus treatment questioned after gene therapy death.Nature.1999;401:517-518.
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[12]黄玉,王斯滕,朱新远.生物可降解超支化聚合物的研究进展[J].高分子学报,2017,61(2):1-13.
[13]胡楚玲,顾芬芬,台宗光.可生物降解多肽基因载体的构建与体外评价[J].第二军医大学学报,2016,37(11):1353-1359.
[14]杨远,贾文祥,祁欣.可生物降解材料作为基因载体的研究[J].生物医学工程学杂志,2006,23(3):573-577.
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[16]Lin C,Engbersen JF.Effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection.J Control Release.2008;132(3):267-272.
[17]于世钧,赵洪霞,郭宏.超支化聚酰胺的合成与表征[J].应用化学,2004,21(6):625-628.
[18]Kloeckner J,Bruzzano S,Odris M,et al.Gene carriers based on hexanediol diacrylate linked oligoethylenimine:Effect of chemical structure of polymer on biological properties.Bioconjugate Chem.2006;17(5):1339-1345.
[19]吴珊,钟延强,邹豪.生物还原响应型聚合物用于基因传递系统的研究进展[J].中国医药工业杂志,2014,45(11):1078-1085.
[20]Ping Y,Wu D,Kumar JN,et al.Redox-Responsive Hyperbranched Poly(amido amine)s with Tertiary Amino Cores for Gene Delivery.Biomacromolecules.2013;14(6):2083-2094.
[21]Zhan C,Fu XB,Yao Y,et al.Stimuli-responsive hyperbranched poly(amidoamine)s integrated with thermal and pH sensitivity,reducible degradability and intrinsic photoluminescence.Rsc Adv.2017;7(10):5863-5871.
[22]Christensen LV,Chang CW,Yockman JW,et al.Reducible poly(amido ethylenediamine)for hypoxia-inducible VEGF delivery.J Controll Release.2007;118(2):254-261
[23]Tian W,Li X,Wang J.Supramolecular hyperbranched polymers.Chem Commun.2017;53(17):2531-2542.
[24]Tian W,Li XX,Wang J X.Supramolecular hyperbranched polymers.Chem Commun.2017;53(17):2531-2542.
[25]Federico M,Martin P,Johan FJ,et al.Effects of branched or linear architecture of bioreducible poly(amido amine)s on their in vitro gene delivery properties.J Control Release.2012;164:372-379.
[26]杨文.荧光超支化聚合物的合成与应用[D].合肥:中国科技大学,2010.
[27]CaoL,Yang W,Wang C,et al.Synthesis and striking fluorescence properties of hyperbranched poly(amido amine).J Macromol Sci.2007;44(4):417-424.
[28]Nam HY,Hahn HJ,Nam K,et al.Evaluation of generations 2,3 and 4arginine modified PAMAM dendrimers for gene delivery.Int J Pharm.2008;363(1):199-205.
[29]Caminade AM,Yan D,Smith DK.Dendrimers and hyperbranched polymers.Chem Soc Rev.2015;44(12):3870-3873.
[30]Meng F,Hennink WE,Zhong Z.Reduction-sensitive polymers and bioconjugates for biomedical applications.Biomaterials.2009;30(12):2180-2198.
[31]Schafer FQ,Buettner GR.Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.Free Radic Biol Med.2001;30(11):1191-1212..
[32]唐忠科,熊兴泉,蔡雷.还原响应型药物载体的研究进展[J].药学学报,2011,46(9):1032-1038.
[33]李义,佘英奇,朱和康.二硫键交联的巯基化壳聚糖水凝胶对蛋白质还原响应的控制释放[J].精细化工,2017,34(1):28-33.
[34]Pichon C,Lecam E,Guérin B,et al.Poly[Lys-(AEDTP)]:a cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection.Bioconjugate Chem.2002;13(1):76-82.
[35]Ou M,Wang XL,Xu R,et al.Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity.Bioconjug Chem.2008;19(3):626-633.
[36]赵亮,苏畅,马洪林.阳离子聚合物基因载体的理论研究[J].中国组织工程研究与临床康复,2009,13(42):8329-8332.
[37]任先越,杨立群,梁玄,等.阳离子聚合物在非病毒基因转染中的研究进展[J].生物工程学报,2013,29(5):568-577.
[38]Ding M,Beck RJ,Keller CJ,et al.Cloning and analysis of MAGE-1-related genes.Biochem Biophys Res Commun.1994;202(1):549-555.
[39]黄倩,李川源.绿色荧光蛋白质粒DNA作为活细胞标记基因的研究[J].中华肿瘤杂志,2001,23(3):264.
[2]李丽春,李剑平.基因转染间充质干细胞的临床治疗:特点与问题[J].中国组织工程研究,2014,18(14):2257-2262.
[3]Wirth T,Parker N,Yl?-Herttuala S.History of gene therapy.Gene.2013;525(2):162-169.
[4]孟凡荣,陈琛万,海粟.慢病毒载体及其研究进展[J].中国肺癌杂志,2014,17(12):870-876.
[5]Wang WW,Li WZ,Ma N,et al.Non-Viral Gene Delivery Methods.Curr Pharm Biotechno.2013;14(1):46-60.
[6]El Aneed A.An overview of current delivery systems in cancer gene therapy.J Control Release.2004;94(1):1-14.
[7]Ferber D.Gene therapy:Safer and virus-free?Science.2001;294:1638-1642.
[8]Lehrman S.Virus treatment questioned after gene therapy death.Nature.1999;401:517-518.
[9]Luo D,Saltzman WM.Synthetic DNA delivery systems.Nat Biotechnol.2000;18:33-37.
[10]江献芳,周诺.脂质体在基因治疗中的应用研究及进展[J].中国组织工程研究,2012,16(8):1463-1466.
[11]张颖,周志平.阳离子聚合物基因载体:进展与展望[J].应用化工,2018,47(1):145-149.
[12]黄玉,王斯滕,朱新远.生物可降解超支化聚合物的研究进展[J].高分子学报,2017,61(2):1-13.
[13]胡楚玲,顾芬芬,台宗光.可生物降解多肽基因载体的构建与体外评价[J].第二军医大学学报,2016,37(11):1353-1359.
[14]杨远,贾文祥,祁欣.可生物降解材料作为基因载体的研究[J].生物医学工程学杂志,2006,23(3):573-577.
[15]Richardson SC,Pattrick NG,Man YK,et al.Poly(amidoamine)s as potential nonviral vectors:ability to form interpolyelectrolyte complexes and to mediate transfection in vitro.Biomacromolecules.2001;2(3):1023-1028.
[16]Lin C,Engbersen JF.Effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection.J Control Release.2008;132(3):267-272.
[17]于世钧,赵洪霞,郭宏.超支化聚酰胺的合成与表征[J].应用化学,2004,21(6):625-628.
[18]Kloeckner J,Bruzzano S,Odris M,et al.Gene carriers based on hexanediol diacrylate linked oligoethylenimine:Effect of chemical structure of polymer on biological properties.Bioconjugate Chem.2006;17(5):1339-1345.
[19]吴珊,钟延强,邹豪.生物还原响应型聚合物用于基因传递系统的研究进展[J].中国医药工业杂志,2014,45(11):1078-1085.
[20]Ping Y,Wu D,Kumar JN,et al.Redox-Responsive Hyperbranched Poly(amido amine)s with Tertiary Amino Cores for Gene Delivery.Biomacromolecules.2013;14(6):2083-2094.
[21]Zhan C,Fu XB,Yao Y,et al.Stimuli-responsive hyperbranched poly(amidoamine)s integrated with thermal and pH sensitivity,reducible degradability and intrinsic photoluminescence.Rsc Adv.2017;7(10):5863-5871.
[22]Christensen LV,Chang CW,Yockman JW,et al.Reducible poly(amido ethylenediamine)for hypoxia-inducible VEGF delivery.J Controll Release.2007;118(2):254-261
[23]Tian W,Li X,Wang J.Supramolecular hyperbranched polymers.Chem Commun.2017;53(17):2531-2542.
[24]Tian W,Li XX,Wang J X.Supramolecular hyperbranched polymers.Chem Commun.2017;53(17):2531-2542.
[25]Federico M,Martin P,Johan FJ,et al.Effects of branched or linear architecture of bioreducible poly(amido amine)s on their in vitro gene delivery properties.J Control Release.2012;164:372-379.
[26]杨文.荧光超支化聚合物的合成与应用[D].合肥:中国科技大学,2010.
[27]CaoL,Yang W,Wang C,et al.Synthesis and striking fluorescence properties of hyperbranched poly(amido amine).J Macromol Sci.2007;44(4):417-424.
[28]Nam HY,Hahn HJ,Nam K,et al.Evaluation of generations 2,3 and 4arginine modified PAMAM dendrimers for gene delivery.Int J Pharm.2008;363(1):199-205.
[29]Caminade AM,Yan D,Smith DK.Dendrimers and hyperbranched polymers.Chem Soc Rev.2015;44(12):3870-3873.
[30]Meng F,Hennink WE,Zhong Z.Reduction-sensitive polymers and bioconjugates for biomedical applications.Biomaterials.2009;30(12):2180-2198.
[31]Schafer FQ,Buettner GR.Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.Free Radic Biol Med.2001;30(11):1191-1212..
[32]唐忠科,熊兴泉,蔡雷.还原响应型药物载体的研究进展[J].药学学报,2011,46(9):1032-1038.
[33]李义,佘英奇,朱和康.二硫键交联的巯基化壳聚糖水凝胶对蛋白质还原响应的控制释放[J].精细化工,2017,34(1):28-33.
[34]Pichon C,Lecam E,Guérin B,et al.Poly[Lys-(AEDTP)]:a cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection.Bioconjugate Chem.2002;13(1):76-82.
[35]Ou M,Wang XL,Xu R,et al.Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity.Bioconjug Chem.2008;19(3):626-633.
[36]赵亮,苏畅,马洪林.阳离子聚合物基因载体的理论研究[J].中国组织工程研究与临床康复,2009,13(42):8329-8332.
[37]任先越,杨立群,梁玄,等.阳离子聚合物在非病毒基因转染中的研究进展[J].生物工程学报,2013,29(5):568-577.
[38]Ding M,Beck RJ,Keller CJ,et al.Cloning and analysis of MAGE-1-related genes.Biochem Biophys Res Commun.1994;202(1):549-555.
[39]黄倩,李川源.绿色荧光蛋白质粒DNA作为活细胞标记基因的研究[J].中华肿瘤杂志,2001,23(3):264.