超声/光声介导RGD纳米分子探针可视化指导冠脉易损斑块精准诊疗一体化研究

Vulnerable plaques are an important mechanism of acute coronary syndrome. However, the means to identify vulnerable plaques are limited, and there are lack of accurate prevention and treatment methods of thrombosis induced by vulnerable plaques. Photoacoustic imaging technology can achieve multi-scale tracer and functional imaging from the cells to the organization, which provides strong support for identifying coronary vulnerable plaques, the liquid-gas phase transition effect by laser not only can improve the signal intensity of photoacoustic imaging and ultrasound contrast imaging, but also can be used to enhance the efficiency of ultrasound microbubble thrombolysis. Molecular probe RGD was introduced in ultrasound/photoacoustic contrast agent to specifically identify the receptor of activated platelet membrane glycoprotein GP IIb/IIIa in vulnerable plaques, inhibit platelet aggregation and prevent thrombosis. In addition, thrombolysis was performed by using the cavitation effect of ultrasonic microbubbles in the formed thrombus. That can play a dual role of accurate prevention and treatment. Contents of our study are as below: preparation and qualification of the RGD nano-agent is the first step. The parameter optimization of ultrasonic/photoacoustic irradiation of RGD nanobubbles in identifying vulnerable plaques, specific adsorption efficiency of RGD, and in vitro artificial thrombolysis. With ultrasonic and photoacoustic irradiation in vivo coronary vulnerable plaques micro-embolism, the therapeutic functions and safety of thrombus dissolute are to be evaluated. Based on this study, a novel integrated solution of accurate diagnosis and treatment of coronary vulnerable plaques is to be established with targeted, high efficiency and safety.

易损斑块破裂是急性冠脉综合征的重要机制，然而识别易损斑块的手段有限，且缺乏精准防治易损斑块诱发血栓的方法。光声成像可从细胞到组织多尺度示踪及功能成像，精确识别冠脉易损斑块，其产生的激光致液气相变效应，不仅可大幅提高成像信号强度及造影效果，还可增强超声微泡的溶栓效率。在超声/光声造影剂中引入分子探针RGD，可特异性识别易损斑块中的活化血小板膜糖蛋白GP IIb/IIIa受体，抑制血小板聚集，防止血栓形成；对已经形成的血栓，利用超声微泡的空化效应可进行有效溶栓，进而实现易损斑块精确识别、血栓精准防治的双重作用。本研究主要内容为：制备携RGD纳米分子探针相变微泡及鉴定；超声/光声辐照RGD纳米微泡识别易损斑块、RGD特异性吸附效率，体外人工血管溶栓等参数优化；检测冠脉易损斑块急性栓塞模型超声/光声靶向识别、溶栓治疗及安全性评估。藉此创建一种靶向、高效、安全的急性冠脉综合征精准诊疗一体化解决方案。

动脉粥样硬化易损斑块引起的心脑血管意外事件严重威胁人类健康，超声/光声介导的分子影像技术为动脉粥样硬化斑块无创监测及治疗、斑块破裂血栓防治等提供重要方法。本研究主要内容为：第一，制备特异性靶向动脉粥样硬化斑块并具有光声/超声双模态显像功能的RGD纳米分子探针cRGD-GNR-PFP-NPs，对其基本性能进行表征，体外评价其相变性能、双模态成像能力及细胞水平安全性；第二，构建小鼠动脉粥样硬化斑块模型，探索利用RGD纳米分子探针介导斑块光声/超声双模态分子成像评估斑块易损性的可行性。第三，为进一步提高纳米分子探针的靶向性，构建血小板膜仿生纳米分子探针PLT-RAP@NPs，探索其体外免疫逃逸能力、靶向及黏附能力、细胞毒性和血液相容性以及联合UTMD后药物释放情况；研究血小板膜仿生纳米药物体内长循环特点以及联合UTMD后在体内靶向斑块情况、延缓动脉粥样硬化进展、改善斑块稳定性情况。结果显示：成果制备RGD纳米分子探针平均粒径258.03±6.75 nm，电位−9.36±0.53 mV，透射电镜显示GNR被成功包载，在780~790 nm波长范围具有特征吸收峰，其表面高效偶联cRGD肽，连接率达92.79%，LIFU辐照激发下易发生相变，具有优良的体外光声/超声双模态成像能力。cRGD-GNR-PFP-NPs对斑块具有特异靶向性，体内循环6h后在斑块处富集最多；cRGD-GNR-PFP-NPs介导成像结果显示高脂组斑块具有更强的光声/超声/荧光信号，信号强度与斑块富集程度、整合素αvβ3表达及斑块易损性均具有很好的一致性，且生物安全性良好。成功制备血小板膜仿生纳米分子探针PLT-RAP@NPs，其表面保留血小板膜蛋白组成，为其生物学功能提供结构基础和保障，特异性靶向病变区域能力更强，提高局部药物浓度，抑制巨噬细胞和血管平滑肌增殖，从而实现减少斑块大小、改善斑块成分、降低脂质含量、减少坏死核形成、增加斑块稳定性等表现出较好的治疗效果。血小板膜仿生纳米药物结合UTMD具有优异的血液相容性和生物安全性，可用作动脉粥样硬化斑块的长期治疗，为心血管疾病提供了新的辅助诊疗手段，具有广泛的应用前景。