非病毒性基因治疗递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k的构建及性能研究

摘要

Objective To construct (mPEG5k-PCL1.2k)1.4-g-PEI10k, a copolymer designed as delivery vector for non-viral gene therapy, and explore its cytotoxicity and efficacy in delivery of plasmid DNA (pDNA).Methods The copolymer, mPEG5k-PCL1.2k-OH,was prepared by ring-opening polymerization and then followed by a conversion of hydroxyl teminal (-OH) into N-hydroxysuccinimide (NHS) to prepare mPEG5k-PCL1.2k-NHS.One of the branches, PEI10k, was then reacted with mPEG5k-PCL1.2k-NHS to synthesize a ternary copolymer, (mPEG5k-PCL1.2k)l.4-g-PEI10k.The structure of (mPEG5k-PCL1.2k)1.4-g-PEI10k was characterized with Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC).The cytotoxicity of (mPEG5k-PCL1.2k )1.4 -g-PEI10k was compared with that of PEI10k by MTT assay.The (mPEG5k-PCL1.2k)1.4-g-PEI10k/pDNA complexes were prepared by complex coacervation.In the in vitro transfection experiment, the expression of reporter gene and the changes thereof as influenced by the time needed for transfection or pH value of culture medium were observed.Results The ternary copolymer, (mPEG5k-PCL1.2k)1.4-g-PEI10k, was successfully synthesized, which could condense the green fluorescence protein (GFP) reporter plasmid (pEGFP)to form the compound (mPEG5k-PCL1.2k )1.4-g-PEI10k/pEGFP, and transfect cells to express the target genes effectively.The optimum expression of GFP was observed under weight ratio of vector to plasmid at 10:1.As for the cytotoxidty of (mPEG5k-PCL1.2k )1.4-g-PEI10k,a lower cytotoxicity was found at concentration less than 62.5μg/ml when compared with PEI10k.The transfection efficacy was found to be influenced by both the time needed for transfection, and it was found that 48 hours was better than 24 hours, and as for pH value of culture medium, 6.8 was better than 7.2.Conclusion The delivery vector (mPEG5k-PCL1.2k)1.4-g-PEI10k, with lower cytotoxicity, can deliver pDNA into cells, and express the target proteins.%目的 构建非病毒性基因治疗递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k,探讨其细胞毒性及对质粒基因(pDNA)的递送性能.方法 通过开环聚合反应制备共聚物mPEG5k-PCL1.2k-OH,将其羟基末端化学转化为N-羟基琥珀酰亚胺(-NHS)生成mPEG5k-PCL1.2k-NHS,随后同枝化PEI10k进行反应生成三元共聚物(mPEG5K-PCL1.2k)1.4-g-PEI10k.应用傅立叶变换红外光谱(FTIR)、核磁共振(NMR)和凝胶渗透色谱对(mPEG5k-PCL1.2k)1.4-g-PEI10k进行结构表征分析;通过MTT分析,比较(mPEG5k-PCL1.2k)1.4-g-PEI10K与PEI10k的细胞毒性;通过复凝聚法制备(mPEG5k-PCL1.2k)1.4-g-PEI10K/pDNA复合物;通过细胞转染实验,观察不同质量比的复合物转染细胞后报告基因的表达效果,考察转染时间和培养基pH值对报告基因表达的影响.结果 成功合成了递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k.浓度小于62.5g/ml时,(mPEG5k-PCL1.2k)1.4-g-PEI10k的细胞毒性显著小于PEI10k.(mPEG5k-PCL1.2k)1.4-g-PEI10k载体能够压缩绿色荧光蛋白质粒报告因(pEGFP)形成(mPEG5k-PCL1.2k)1.4-g-PEI10k/pEGFP复合物,并有效转染细胞获得目的 基因表达;当载体与质粒的质量比为10∶1时,绿色荧光蛋白的表达效果最好.转染时间和培养基pH值均可影响(mPEG5k-PCL1.2k)1.4-g-PEI10k/pEGFP的转染效率,转染效率在48h优于24h和72h,pH 6.8培养基优于pH 7.2培养基.结论 递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k细胞毒性较低,能够递送pDNA进入细胞并表达目的 蛋白.