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合成cRGD-羧甲基壳聚糖-软脂酸(cRGD-CMCh-PA)载体,以紫杉醇(paclitaxel,PTX)为模型药物,薄膜分散法制备pH敏感性载紫杉醇-cRGD-羧甲基壳聚糖-软脂酸胶束(PTX-cRGD-CMCh-PA),采用FT-IR、~1H NMR对相关产物结构进行表征,并对胶束粒径、电位和形态进行测定;透析法考察载药胶束在pH 5.3和7.4的磷酸缓冲盐溶液(phosphate buffered saline,PBS)中体外释药行为。采用MTT法考察了细胞毒性,共聚焦显微镜及活细胞工作站动态观察胶束的细胞动态摄取过程;近红外小动物活体成像技术探索胶束的肿瘤靶向性。结果显示:合成的cRGD-CMCh-PA羧甲基化程度为45.0%,软脂酸接枝率为15.0%;载药胶束包封率和载药量分别为99.67%和28.5%,粒径为(162.9±1.5)nm。pH 7.4 PBS条件下PTX释放缓慢,释药遵循Higuchi方程;pH 5.3 PBS中2 h内出现突释现象,呈现pH敏感特性。PTX-CMCh-PA的IC50为2.077μg·mL~(-1),PTX-cRGD-CMCh-PA的IC50为0.876μg·mL~(-1);共聚焦显微镜和活细胞工作站均显示肿瘤细胞对PTX-cRGD-CMCh-PA摄取率更为显著,近红外小动物活体成像技术显示其对肿瘤具有更高的靶向性。本实验中合成的cRGD-CMCh-PA胶束具有pH敏感释药特点和良好的肿瘤靶向性,是一种具有开发潜力的新型药物载体。
The cRGD-carboxymethyl chitosan-cRGD-CMCh-PA carrier was synthesized, and paclitaxel (PTX) was used as model drug to prepare pH-sensitive paclitaxel- (PTX-cRGD-CMCh-PA). FT-IR and ~ 1H NMR were used to characterize the structure of the product. The micelles were characterized by particle size, potential and morphology. In vitro release behavior of micelles in phosphate buffered saline (PBS) at pH 5.3 and 7.4. MTT assay cytotoxicity, confocal microscopy and dynamic cell workstation dynamic observation of micellar cell dynamic uptake process; near infrared small animal live imaging technology to explore the micellar tumor targeting. The results showed that the degree of carboxymethylation of cRGD-CMCh-PA was 45.0% and the rate of grafting of palmitic acid was 15.0%. The entrapment efficiency and drug loading of drug-loaded micelles were 99.67% and 28.5% (162.9 ± 1.5) nm. The release of PTX was slow at pH 7.4 PBS. The release was followed by Higuchi’s equation. The pH was released within 2 h at pH 5.3 and showed pH-sensitive properties. The IC50 of PTX-CMCh-PA was 2.077μg · mL -1, and the IC50 of PTX-cRGD-CMCh-PA was 0.876μg · mL -1. Confocal microscopy and live cell workstation showed that the tumor cell pairs PTX-cRGD-CMCh-PA uptake rate is more significant, near-infrared small animal biopsy technology shows that it has a higher tumor targeting. The cRGD-CMCh-PA micelles synthesized in this study possesses the characteristics of pH-sensitive drug release and good tumor targeting. It is a novel drug carrier with potential for development.