肽修饰羧基化多壁碳纳米管基因载体的构建与体外评价

Construction and in vitro evaluation of peptide-modified carboxylated multiwalled carbon nanotube gene vector

目的:制备一种肽修饰羧基化多壁碳纳米管(peptide modified carboxylated multiwalled carbon nanotubes,MHR)基因载体,考察其对HEK293细胞的转染效率及细胞毒性。方法:将羧化的多壁碳纳米管(MWCNTs)与精氨酸(arginine,R)和组氨酸(histidine,H)组成的短肽(HR)按照一定的质量比反应,通过酰胺键连接得到MHR。采用1 H-NMR、红外光谱以及热重分析对其结构进行鉴定。取纯化后的MHR,用激光粒度测定仪测定粒径和zeta电位,凝胶电泳法测定载体MHR对pEGFP质粒的包裹能力。用MHR/pEGFP纳米复合物与HEK293细胞共同培养,考察细胞摄取情况及相关基因转染情况,并测定MHR和MWCNTs-COOH对DU145和RAW264.7细胞的细胞毒性。结果:通过结构鉴定确定MHR合成成功。在氮磷比(N/P)=20时,HEK293细胞对MHR/pEGFP的摄取及转染效率高于其他N/P值时,其中N/P=20时,RAW264.7细胞对MHR/pGL3复合物的摄取率约为单体pGL3的2.4倍,差异具有统计学意义(P<0.05)。细胞毒性实验表明,MHR作用于DU145和RAW264.7细胞24和48h后,当MHR浓度达640μg/ml时,DU145和RAW264.7细胞的活性仍然>80%;而MWCNTs-COOH浓度达320μg/ml时,DU145和RAW264.7细胞活性明显降低;当浓度达640μg/ml时,细胞活性低于20%。结论:MHR有望成为一种高效、低毒的基因载体。

Objective：To prepare peptide modified carboxylated multiwalled carbon nanotubes（MHR）gene vector,and to evaluate its transfection efficiency and cytotoxicity in HEK293 cells.Methods：MHR was obtained by linking the carboxylated multiwalled carbon nanotubes（MWCNTs）with peptide（HR）consisting of arginine（R）and histidine（H）through amide group in a certain ratio of mass.The construction of MHR was characterized by 1 H-NMR,infrared spectrum and thermogravimetric analysis.The particle size and zeta potential of purified MHR were determined by laser particle size analyzer.The encapsulation ability of MHR to pEGFP was determined by gel electrophoresis.MHR/pEGFP nanoparticles were co-cultured with HEK293 cells and its cellular uptake and transfection efficiency were investigated.The cytotoxicity of MHR and MWCNTs-COOH to DU145 and RAW264.7 cells was also evaluated.Results：MHR synthesis was confirmed by its structural identification.The cellular uptake and transfection efficiency of HEK 293 cells to MHR/pEGFP at a N/P ratio of 20 was higher than that at other N/P ratios,and MHR/pGL3 compound uptake rate by RAW264.7 cells at a N/P ratio of 20 was about 2.4 folds higher than that of pGL3,and there was statistical significance when comparisons were made between the two（P〈0.05）.Cell toxicity test showed that the viability of DU145 and RAW264.7 cells treated by MHR for 24 hor 48 hwas still more than 80%,even though the concentration of MHR reached 640μg/ml.However,the viability of the cells decreased markedly when the concentration of MWCNTs-COOH decreased to 320μg/ml,and when the concentration of MWCNTs-COOH was 640μg/ml,the viability of the cell was lower than 20%.Conclusion：MHR seems to be a promising efficient gene vector with low cytotoxicity.