双靶点磁性、荧光双模态分子探针的制备及对乳腺癌体外靶向结合的可行性  

Synthesis of a novel long-circulating dual-receptor targeting and dual-modal molecular probe： an in-vitro study

作　　者：杨蕊梦[1] 唐文洁[1] 韦敏燕[2] 赖胜圣[3] 徐向东[1] 梁思敏[2] 魏新华[1] 江新青[1] 

机构地区：[1]广州医科大学附属广州市第一人民医院放射科,510180 [2]广州医科大学药学院 [3]广东食品药品职业学院医疗器械学院

出　　处：《中华放射学杂志》2016年第1期41-46,共6页Chinese Journal of Radiology

基　　金：国家自然科学基金（81201087,81402881,81571665）;广东省自然科学基金（2014A030313647）;广东省自然科学基金博士启动项目（$2013040014348）;广东省医学科学技术研究基金（B2014343,B2014080）;广州市科技计划项目（1563000477）

摘　　要：目的探讨双靶点磁性、荧光双模态分子探针多肽RGD@BBN．脂质体（量子点）-超顺磁性氧化铁[RGD@BBN-lipo（QDs）-SPIO]的构建方法及对乳腺癌体外靶向结合的可行性。方法采用薄膜分散法合成聚乙二醇（PEG）修饰的长循环脂质体，在其脂质核心亲水层装载水溶性超顺磁性氧化铁（sPIO）纳米颗粒、脂质双分子疏水层装载量子点（QDs）、表面偶联RGD及BBN多肽，制备同时靶向整合素受体（仪，良）和胃泌素释放肽受体（GRPR）的双靶点磁性、荧光双模态分子探针RGD@BBN-lipo（QDs）．SPIO。观察其在不同生理溶液中的稳定性；采用电子显微镜及Zeta电位和粒度分析仪测量其粒径及Zeta电位；采用磁共振波谱仪元素分析法对其进行表征及估算RGD及BBN的偶联率；行MRIT2map扫描，计算双靶点磁性、荧光探针的T2弛豫率；采用细胞生长测试（MTS）法检测其体外细胞毒性；以乳腺癌细胞为测试模型，采用普鲁士蓝染色和荧光成像方法检测分子探针对乳腺癌的体外靶向结合特性。结果RGD@BBN-lipo（QDs）-SPIO在生理盐水、磷酸盐缓冲液、细胞培养基、胎牛血清中均具有较好的稳定性，静置3个月后无混浊、沉淀，明显优于未经修饰的SPIO组。电子显微镜下粒径为（118．2±3．9）nm；Zeta电位为（-24．78±1．68）mY；分子探针上RGD及BBN受体成功偶联，偶联率分别为33．05％、45．06％；弛豫率0．4981×10^6M^-1·s-10RGD@BBN—lipo（QDs）-SPIO即使在Fe浓度达到100μg／ml，细胞存活率仍保持在80％以上，具有较低细胞毒性。普鲁士蓝和荧光成像显示可以灵敏地靶向整合素仅，B3和GRPR任何一个受体高表达的乳腺癌细胞。结论双靶点磁性、荧光双模态分子探针多肽RGD@BBN-lipo（QDs）-SPIO具有优良的理化性质及稳定性，生物安全性好，T3弛豫率高，肿瘤靶向结合能力强。Objective To explore a novel long-circulating dual-receptor targeting and dual-modal molecular probe and investigate its physicochemical properties and targeting effect on breast cancer ceils in vitro. Methods Dual-receptor targeting and dual-modal molecular probe RGD@BBN-lipo（QDs）-SPIO was synthesized in the following steps： long-circulating liposome was prepared by film dispersion method; water-soluble superparamagnetic iron oxide （SPIO） nanoparticles and Quantum dots （QDs） were loaded in the hydrophilic and hydrophobic layer of liposome, respectively; RGD and BBN polypeptides were coupled on the former functional magnetic/fluorescent liposomes. Stability of the probe in different physiological solutions was investigated. Transmission electron microscopy （TEM） and particle size analyzer were used to measure nanoparticle sizes and the Zeta potential. Characterization of RGD and BBN was investigated through 1H-NMR and elemental analysis. The MRI T2 relaxivities （1/T2） of RGD@BBN-lipo（QDs）-SPIO was measured through T2 map scanning on 3.0 T MR system. HUV-EC-C cells were used for assessment of cells viability by MTS assay. Prussian blue staining and fluorescence imaging were carried out to determine the targeted breast cellular uptake of RGD@BBN-lipo（QDs）-SPIO nanoparticles. Results The targeting magnetic/fluorescent dual-model molecular probes appeared spherical or para-spherical, with a mean diameter of （118.2±3.9）nm,Zeta potential of （- 24.78±1.68） mV,MR T2 magnetic relaxation rate of 0.498 1×10^6 M^-1·s^-1.RGD and BBN polypeptides were successfully coupled on the former functionally magnetic/ fluorescent liposomes with the bind rates of 33.05 % and 45.06 %, respectively. There was low cytotoxity of the molecular probe on human umbilical vein endothelical cells （HUV-EC-C）by MTS study. Prussian blue staining and fluorescence imaging studies showed that the RGD@BBN-lipo（QDs）-SPIO nanoparticles could target any ctv133 or gastrin releasing peptide receptor overexpres

关 键 词：分子探针 磁共振成像 乳腺肿瘤 

分 类 号：R737.9[医药卫生—肿瘤] R445.2[医药卫生—临床医学]