主动靶向乳腺癌的pH响应纳米粒子的构建及体外评价

Preparation and in vitro evaluation of active targeting pH-responsive nanoparticles for breast cancer

目的制备主动靶向乳腺癌的pH响应负载多烯紫杉醇(docetaxel,DTX)的纳米粒子,并考察其理化性质、载药和药物缓释特征及对人乳腺癌MCF-7细胞的靶向和杀伤效果。方法采用纳米沉淀法和基于聚多巴胺(polydopamine,PDA)的表面修饰方法制备主动靶向MCF-7细胞的载DTX的叶酸(folic acid,FA)和PDA修饰的胆酸-聚乳酸-羟基乙酸共聚物纳米粒子(DTX-loaded CA-PLGA@PDA-PEG-FA/NPs);采用透射电镜观察纳米粒子的形貌,纳米粒度仪分析纳米粒子的粒径和zeta电位,X射线光电子能谱仪(XPS)分析纳米粒子表面修饰情况;采用透析法和高效液相色谱法研究纳米粒子的载药率、包封率以及体外释放曲线;采用激光扫描共聚焦显微镜和流式细胞仪分析负载荧光探针的纳米粒子的体外细胞摄取;采用MTT法研究载药纳米粒子对MCF-7细胞的存活率的影响。结果本研究制备的DTX-loaded CA-PLGA@PDA-PEG-FA/NPs呈"核-壳"结构,水合粒径为(166.4±3.9)nm,zeta电位为(-11.7±3.8)mV,载药量为(9.67±0.45)%,包封率为(88.32±3.10)%,在pH 5.0的释放介质中药物释放较在pH7.4的释放介质中快,XPS分析结果显示PDA和叶酸在纳米粒子表面的修饰,MCF-7细胞摄取的主动靶向的纳米粒子多于未连接主动靶向配体的纳米粒子,载药主动靶向纳米粒子的细胞毒性明显优于DTX的临床制剂泰素帝○R。结论主动靶向乳腺癌的pH响应的载药纳米粒子表现出良好的主动靶向性和MCF-7细胞杀伤效果。

Objective To prepare docetaxel（DTX）-loaded active breast cancer-targeted pH-responsive nanoparticles and to determine its chemo-physical properties,drug loading and releasing characteristics,and targeting ability and cytotoxity against MCF-7cells.Methods The nanoparticles were synthesized by nanoprecipitation method and surface modification based on polydopamine（PDA）.The morphology,size and zeta potential,and surface modification of the nanoparticles were characterized by transmission electron microscopy（TEM）,dynamic light scattering（DLS）and X-ray photoelectron spectroscopy（XPS）,respectively.Drug loading content,encapsulation efficiency,and in vitro drug release profiles were measured by dialysis and high performance liquid chromatography（HPLC）.The in vitro cellular uptake was analyzed by confocal laser scanning microscope（CLSM）and flow cytometry（FCM）,and the the effect of drug-loaded nanoparticles on the viability of MCF-7cells was determined by MTT assays.Results The DTX-loaded nanoparticles,CA-PLGA@PDA-PEG-FA/NPs,exhibited a core-shell structure,with hydrodynamic size of（166.4±3.9）nm,zeta potential of（-11.7±3.8）mV,drug loading efficiency of（9.67±0.45）%,and encapsulation efficiency of（88.32±3.10）%.Furthermore,the drug release rate of the nanoparticles in pH 5.0release medium was faster than that in pH 7.4.XPS spectra showed that PDA and folic acid were modified on the surface of the nanoparticles.The active targeting nanoparticles ingested by MCP-7cells were more than the nanoparticles not linked to active targeting ligands,and the cytotoxicity of active-targeted nanoparticles was significantly superior than that of Taxotere○R（clinical preparation of DTX）.Conclusion The active breast cancer-targeted pH-responsive nanoparticles（DTX-loaded CA-PLGA@PDA-PEG-FA/NPs）exhibits promising targeting ability and efficient antitumor activity in vitro against MCF-7cells.