| 郭惠庄,罗文峰,余盛龙,李莉,陈汉威.制备并初步评价KGDS靶向活化血小板纳米钆对比剂[J].中国医学影像技术,2021,37(3):360~364 |
| 制备并初步评价KGDS靶向活化血小板纳米钆对比剂 |
| Preparation and preliminary evaluation of activated platelets KGDS-targeted nano gadolinium contrast agent |
| 投稿时间：2020-04-28 修订日期：2020-11-30 |
| DOI：10.13929/j.issn.1003-3289.2021.03.010 |
| 中文关键词: 磁共振成像 对比剂 赖氨酸-甘氨酸-天冬氨酸-丝氨酸 活化血小板 |
| 英文关键词:magnetic resonance imaging contrast media KGDS activated platelet |
| 基金项目:广州市科学研究计划一般项目（201804010036）。 |
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| 中文摘要: |
| 目的 制备赖氨酸-甘氨酸-天冬氨酸-丝氨酸（KGDS）修饰的去铁铁蛋白靶向活化血小板纳米钆（Gd）对比剂（Gd-Afn-KGDS），并评价其理化性质及作为MR对比剂的能力。方法 利用去铁铁蛋白自组装特性，通过调节反应液pH值，将Gd螯合剂装载于去铁铁蛋白空腔内；在1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐（EDC）/N-羟基琥珀酰亚胺（NHS）催化下，将可特异性结合血小板膜糖蛋白GPⅡb/Ⅲa受体的KGDS多肽耦联到去铁铁蛋白表面，经纯化制备出Gd-Afn-KGDS。采用透射电镜、马尔文粒径分析仪观察纳米颗粒形态、大小及分布；采集T1WI测试纳米对比剂增强效果；以细胞计数试剂盒（CCK-8）检测Gd-Afn-KGDS的细胞毒性；采用荧光显微镜观察Gd-Afn-KGDS与活化血小板和体外血栓靶向结合的特异性。结果 ①成功构建了Gd-Afn-KGDS，在透射电镜下呈球形，结构完整，粒径10~15 nm，分布均一。水合纳米颗粒粒径（11.0±1.2）nm，具有超微纳米结构。Gd载药率约为17.07个/去铁铁蛋白。②MRI信号强度随Gd-Afn-KGDS溶液浓度增加而增强，对比剂T1弛豫时间随Gd-Afn-KGDS浓度增高而缩短，且与弛豫率存在线性关系。③Gd-Afn-KGDS对人脐静脉内皮细胞无细胞毒性。④Gd-Afn-KGDS能在体外靶向特异性结合活化血小板及血栓。结论 构建成功的Gd-Afn-KGDS呈球形结构，大小适宜，靶向特异性强，生物相容性好，并具备良好的MR对比增强效果，有望成为可用于诊断早期动脉血栓的高效特异性MR对比剂。 |
| 英文摘要: |
| Objective To prepare activated platelets targeted nano gadolinium contrast agent Lys-Gly-Asp-Ser (KGDS) peptide conjugated magnetic-apoferritin nanoparticles (Gd-Afn-KGDS), and to evaluate the physicochemical properties as well as MR imaging ability of Gd-Afn-KGDS. Methods Gd chelates were loaded into the apoferritin cage through adjusting pH of the reaction liquid by using the self-assembly characteristics of apoferritin, and then under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). KGDS polypeptide which could specifically bind to platelet glycoprotein GPⅡb/Ⅲa receptor were coupled to the surface of apoferritin, and then purified to prepare Gd-Afn-KGDS. Transmission electron microscope and Malvern particle size analyzer were used to observe the size, distribution and morphology of nanoparticles. T1W scanning was performed to test the enhancement effect of nano contrast agent. The cytotoxicity of Gd-Afn-KGDS was analyzed with cell counting kit (CCK-8). The in vitro targeting ability of Gd-Afn-KGDS to activated platelets and blood clots was investigated with fluorescence microscopy. Results ①Gd-Afn-KGDS was successfully constructed. Under the transmission electron microscope, it showed spherical structure, complete structure, and uniform particle size of 10-15 nm. The hydrated particle size of nanoparticles was (11.0±1.2) nm by Malvern particle size analyzer. Using inductively coupled plasma (ICP) detection, the drug loading rate of Gd was about 17.07/apoferritin. ②With the increase of Gd-Afn-KGDS concentration, MRI signals enhanced gradually. T1 relaxation time of Gd-Afn-KGDS contrast medium was shortened with the increase of Gd-Afn-KGDS concentration, which had a certain linear relationship with the relaxation rate. ③Gd-Afn-KGDS had no cytotoxicity on human umbilical vein endothelial cells. ④Gd-Afn-KGDS could specifically bind to activated platelets and thrombi in vitro. Conclusion Gd-Afn-KGDS was successfully constructed with spherical structure, suitable size, strong targeting specificity, good biocompatibility and good MR contrast enhancement effect, which was expected to be an effective and specific MR contrast agent for diagnosis of early arterial thrombosis. |
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