携带cRGD肽的靶向纳米粒超声造影剂的制备以及体外寻靶实验研究  

Experimental study on preparation and targeting research in vitro of targeted nanoparticle ultrasound contrast agent carrying cRGD

作　　者：宣吉晴[1] 陈瑜莉 敖梦[1,2] 王志刚[1,2] 郑元义[1,2,3] 汪朝霞[4] 李奥[5] XUAN Jiqing;CHEN Yuli;AO Meng;WANG Zhigang;ZHENG Yuanyi;WANG Zhaoxia;LI Ao(Chongqing Key Laboratory of Ultrasound Molecular Imaging,the Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China;Department of Ultrasound,the Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China;Department of Ultrasound,Shanghai Jiao Tong University Affiliated Shanghai Sixth People’s Hospital,Shanghai 200233,China;Department of Ultrasound,the Children’s Hospital of Chongqing Medical University,Chongqing 400016,China;Department of Ultrasound,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]重庆医科大学附属第二医院超声分子影像学重庆市重点医学实验室,重庆400010 [2]重庆医科大学附属第二医院超声科,重庆400010 [3]上海交通大学附属第六人民医院超声科,上海200233 [4]重庆医科大学附属儿童医院超声科,重庆400016 [5]南京医科大学第一附属医院超声科,江苏南京210029

出　　处：《中国医学影像技术》2017年第6期810-815,共6页Chinese Journal of Medical Imaging Technology

基　　金：国家自然科学基金青年基金(81501482;81401427);国家自然科学基金(81227801);国家杰出青年科学基金(81425014)

摘　　要：目的制备携带精-甘-天冬氨酸(cRGD)肽的聚乳酸/羟基乙酸(PLGA)纳米粒超声造影剂,观察其在体外对大鼠肝星状细胞(HSC)的靶向能力。方法以PLGA-COOH和全氟新溴烷(PFOB)为原料,采用双步乳化法制备PLGAPFOB纳米粒,采用光学显微镜、扫描电镜以及透射电镜观察其表面以及内部结构;采用马尔文粒径分析仪测量其粒径大小、分布以及表面电位;通过碳二亚胺法连接cRGD肽制备靶向纳米粒超声造影剂(cRGD-NPs),用激光共聚焦显微镜以及流式细胞仪检测PLGA-PFOB纳米粒造影剂与cRGD肽的连接情况;在大鼠HSC中验证该造影剂的靶向性能。结果所得样品为乳白色混悬液,纳米粒大小均匀,粒径分布较窄,平均粒径(255.3±66.8)nm,多分散指数为0.025,Zeta电位为(-16.4±5.1)mV;扫描电镜示纳米粒表面光滑规则、透射电镜示PLGA外壳里包裹液氮氟碳PFOB;共聚焦显微镜观察到连接FITC-cRGD显示为绿色,与显示红色的纳米粒共同融合成橙色,靶向纳米粒超声造影剂大量向大鼠HSC聚集并被其吞噬,红色荧光强度明显多于普通非靶向造影剂。结论成功制备的携带cRGD肽的纳米粒超声造影剂,在体外实验中对大鼠HSC有较强的特异性亲和力。Objective To prepare a poly （lactic-co-glycolic acid） （PLGA） nanoparticles ultrasound contrast agent carry ing cRGD, and to investigate its targeting ability to the rat hepatic stellate cells （HSC） in vitro. Methods PLGA-PFOB nanoparticles were prepared by a double emulsion solvent evaporation process with a modified polymeric shell （PLGA- COOH） encapsulating perflurooctyl bromide （PFOB）. The morphological and structural characteristics of the nanoparticles were observed by an optical microscope, scanning electron microscopy and transmission electron microscopy. The size, dis- tribution and Zeta potential were observed using Malvern particle size analyzer. The cRGD was conjugated onto the nanop- articles by carbodiimide technique. The combination of cRGD with nanoparticles （cRGD-NPs） and the targeting function of cRGD-NPs in vitro were proved by laser scanning confocal microscopy （LSCM）. Results The samples were milk white in deionized water, with a mean diameter of （255.3±66.8）nm and a polydispersity index of 0. 025. Zeta potential was （-16.4 ±5.1）inV. Scanning electron microscopy images reveal that majority of capsules were spherical with smooth surface. Transmission electron microscope resented spherical particle with a core-shell structure. By observing LSCM, Nile red- dyed NPs emitted red fluorescence, FITC-labeled eRGD emitted green fluorescence, and overlay fluorescence image emitted bright homogeneous yellow fluorescence. It was found that greater and stronger red fluorescence representing cRGD-NPs could be observed in the cytoplasm of HSC while fewer NPs remained within HSCs in the non-targeted group. Conlusion The PLGA encapsulated PFOB nanopartieles carrying cRGD （cRGD-NPs） can be prepared successfully. The cRGD-NPs contrast agent has strongly specific affinity on the HSC.

关 键 词：靶向 纳米粒 超声检查 造影剂 

分 类 号：R445.1[医药卫生—影像医学与核医学]