αIIbβ3受体靶向性荧光磁性纳米探针的制备及其在动脉血栓多模态成像中的应用研究

Integrin αIIbβ3 is an important receptor on the membrane of platelets and plays a key role in the process of thrombosis. It is suggested to be an efficient target for molecular imaging of thrombosis. In this study, we will develop a novel αIIbβ3 targeting multimodal system (P1Cm-SPIO-Cy5.5), which could be used to image arterial thrombosis with both magnetic resonance imaging (MRI) and optical imaging, and describe the characterization of the nanoprobe. The thrombogenicity of P1Cm-SPIO-Cy5.5 is explored to deduce the safety of the probe as drug carrier. The in vitro specific binding ability of the probe on platelets is investigated by the methods of confocal laser-scanning microscopy and prussian blue staining. The rats of arterial thrombosis models are established. The animals are injected with nanoprobe (P1Cm-SPIO-Cy5.5) and then imaged by both MRI and near-infrared fluorescence imaging (NIRFI) to test the multimodal imaging capability in vivo. Immunohistologic staining and transmission electron microscope (TEM) of the thrombosis are performed to detect the targeting ability of probe for integrin αIIbβ3. It may provide a new idea for early diagnosis and targeted therapy of thrombotic diseases.

整合素αIIbβ3是血小板膜的主要受体，在血栓形成过程中起着关键作用，可作为血栓分子靶向成像的有效靶点。本研究拟利用靶向肽P1Cm偶联近红外荧光染料Cy5.5标记的顺磁性氧化铁颗粒（SPIO），构建以整合素αIIbβ3受体为靶点的多功能荧光磁性纳米探针 P1Cm-SPIO-Cy5.5用于动脉血栓的多模态成像，并对其进行表征；通过体外实验探讨探针的致栓性，评价其安全性；采用激光共聚焦显微镜和普鲁士蓝染色对活化血小板进行体外结合研究，评价探针对活化血小板的靶向性；建立大鼠颈总动脉血栓模型，用靶向探针进行在体磁共振成像及近红外荧光成像的多模态成像，免疫组化检测和透射电镜观察探针对整合素αIIbβ3的靶向结合能力，探索此探针实现对血栓的靶向磁共振和近红外荧光多模态分子成像的可行性，从而为血栓性疾病的早期诊断和靶向治疗提供新的思路。

血栓性疾病是严重危害人类生命健康的一组循环系统疾病，心肌梗死及脑梗死主要由动脉血栓形成所引起，早期诊断对于预防急性冠状动脉事件或缺血性脑卒中等疾病具有重要意义。先进的影像技术能早期检测动脉血栓，为心脑血管事件的预测和预防提供了光明的前景。GPIIb/IIIa复合物（整合素αIIbβ3）在活化的血小板膜表面高表达，在早期血栓形成过程中起关键作用。该项目以整合素αIIbβ3受体作为成像靶点，制备新型αIIbβ3配体（P1Cm靶向肽）修饰的双功能荧光磁性纳米探针，建立小鼠颈总动脉血栓模型，评价探针对血栓的靶向成像能力。结果显示：我们构建的多模态纳米探针以超顺磁氧化铁颗粒（SPIO）为基础，连接荧光分子Cy5.5及靶向αIIbβ3的P1Cm肽，在体外能与活化血小板特异性结合，且对凝血功能无明显影响，无明显细胞毒性；在体内MRI及近红外荧光成像实验中，探针可活体靶向小鼠颈动脉血栓，生物安全性良好。通过本课题，我们建立了活体颈动脉血栓检测多模态体系，采用MRI及近红外荧光成像方式对血栓进行活体成像，为血栓性疾病的早期诊断和防治提供了理论和实验基础。