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 and1-(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 of1:1,5:1,10:1,15:1and20:1,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 of1:1,5:1,10:1,15:1and20:1,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 from1:1to10:1,the encapsulation rate of complexes gradually increased,and then remained unchanged with the mass ratio more than10:1.(2)In the range of1-200mg/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 from1:1to10:1,the complex with the concentration of25mg/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 from1:1to10:1and 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,10:1is determined to be the optimal polymer/DNA mass ratio.
作者
王杨
聂锦山
顾准
朱铠
Wang Yang;Nie Jinshan;Gu Zhun;Zhu Kai(Suzhou Chien-Shiung Institute of Technology, Taicang 215411, Jiangsu Province, China;Department of Gastroenterology, Taicang No. 1 People Hospital, the Affiliated Hospital of Soochow University, Taicang 215400, Jiangsu Province, China;Department of Cardiac Surgery, Zhongshan Hospital, Fudan University & Shanghai Institute of Cardiovascular Diseases,Shanghai 200032, China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第6期936-944,共9页
Chinese Journal of Tissue Engineering Research
基金
江苏省高校自然科学研究面上项目(16KJB430037)
项目负责人:王杨
苏州健雄职业技术学院"三级联动"科研基金项目(2017SJLD14)
项目负责人:顾准
太仓市基础研究计划项目(TC2016YYJC09)
项目负责人:顾准
江苏省高校"青蓝工程"优秀青年骨干教师项目
项目负责人:王杨~~
作者简介
通讯作者:王杨,女,1979年生,江苏省连云港市人,博士,讲师,主要从事功能高分子与生物大分子研究,苏州健雄职业技术学院,江苏省太仓市215411
