血小板膜蛋白-脂质体纳米载体靶向递送雷帕霉素治疗动脉粥样硬化的实验研究

Atherosclerosis is the leading cause of cardiovascular diseases worldwide, and the reversal and stabilization of atherosclerotic plaque remains a challenge. Rapamycin (Rap) and its rapalogs have potent atheroprotective effects. However, systemic administration of Rap is impossible due to the severe side effects, and the stent/ballon-based targeting approach is limited by the local distribution, short-term release and invasive delivery of drug. It is highly desirable to establish a new targeting delivery system with extensive coverage of plaques and repeatable administration. Platelets play an important role in the initiation and development of atherosclerosis, and we confirmed that platelets distributed not only on the surface of plaque, but also within plaques. The platelet membrane proteins endows platelets with the inherent adhesive ability to atherosclerotic plaques. Therefore, it is reasonable to speculate that platelets can serve as an ideal targeting carrier of anti-atherosclerotic drugs. We recently demonstrated platelet membrane-coated nanoparticles could highly accumulated in atherosclerotic plaques and reduced atherosclerotic area. However, platelet membrane-coated nanoparticle had a very short circulating half-life, and low utilization efficiency of platelet membrane proteins. In this project, we will integrate the platelet membrane proteins to liposome (PL), then construct a novel platelet-simulation carrier with long circulation time. The novel delivery system, using the platelet membrane proteins as the targeting moieties, liposome nanoparticles as the carrier, will bring Rap to atherosclerotic plaques based on the inherent adhesive ability of platelets to atherosclerotic plaques. We will evaluate the binding efficiency and specificity of PL with constituents of atherosclerotic plaque both in vitro and in vivo and then assess the anti-atherosclerosis effect of PL encapsulating Rap (Rap-PL) in the atherosclerotic model of apolipoprotein E-deficient (ApoE-/-) mice. Our study offers a brand-new approach for targeting Rap to the atherosclerotic plaques, and lays the foundation for clinical translation.

动脉粥样硬化是心血管疾病的主要病因，斑块逆转和稳定依然是治疗的难点和热点。雷帕霉素具有强大的抗粥样硬化作用，但大剂量系统给药副作用明显，介入给药存在局部性、短期性、有创性等缺陷。建立斑块广覆盖、可反复缓释给药的新型靶向递药系统意义重大。血小板对动脉粥样斑块具有血小板膜蛋白介导的天然靶向性，广泛分布于斑块表面和内部，可作为比较理想的药物靶向载体。最近我们发现血小板膜包被的纳米粒可靶向富集到斑块部位，但存在半衰期短、靶向效率低及生物膜蛋白利用效率低等局限性。本项目拟将血小板膜蛋白与长循环脂质体融合，构建具有长循环特性的新型模拟血小板纳米载体，以ApoE-/-小鼠动脉粥样硬化为疾病模型，以雷帕霉素为模型药物，通过模仿血小板对斑块的天然趋向行为，将雷帕霉素靶向递送至斑块局部，提高稳定、逆转斑块的疗效。本项目旨在挖掘动脉粥样硬化靶向治疗新方法，为临床转化奠定基础。

动脉粥样硬化（Atherosclerosis，AS）是一种发生在动脉壁的纤维脂肪病变，且AS是心血管疾病的主要病理基础。雷帕霉素及其衍生物是一种mTOR的新型免疫抑制剂，对于AS的治疗具有抗炎、抗血管内皮增生等多重治疗效果。然而雷帕霉素治疗斑块具有剂量依赖性，低浓度的雷帕霉素不足以有效治疗，高浓度的雷帕霉素具有全身性的副作用，因此需要维持斑块局部的有效浓度，靶向递药则显得尤为重要。血小板参与了AS的发生发展全过程，与斑块局部的成分具有多重交互作用。基于此，我们提出了一种新的雷帕霉素仿生递送策略，将血小板膜和脂质体挤压融合，制备血小板仿生的脂质体，同时包被雷帕霉素，模拟血小板靶向斑块的行为，实现靶向递送雷帕霉素到斑块局部的目的。.①Rap-P-Lipo的制备、表征和稳定性：提取血小板膜并整合到脂质体表面（R-Lipo），测量粒径电位，P-Lipo的粒径分布主要在90 nm左右，DLS测得P-Lipo的PDI为0.17±0.04，Zeta电位为-38.63±3.40 mV，且粒径大小稳定；②P-Lipo体外靶向性的研究：DiD标记脂质体，体外比较P-Lipo和Lipo对胶原和vWF的结合能力，以及被泡沫细胞的摄取情况，发现P-Lipo结合胶原、vWF和巨噬细胞的能力强于Lipo；③P-Lipo体内靶向性：建立ApoE-/-高脂饲养模型，通过离体器官成像、荧光显微镜、荧光分光光度法观察P-Lipo对AS的主动脉及斑块的靶向能力和分布特点，证实了P-Lipo对于主动脉斑块的靶向性；④Rap-P-Lipo治疗AS：建立ApoE-/-高脂饲养模型，尾静脉注射Rap-P-Lipo，病理染色和荧光染色评价主动脉斑块面积和稳定性，检测斑块内巨噬细胞的数量，证实了Rap-P-Lipo的治疗可以减少AS的斑块大小，减少巨噬细胞含量；⑤P-Lipo的安全性：Rap-P-Lipo给药四周后，检测炎症因子水平，免疫球蛋白水平和器官毒性：P-Lipo具有生物安全性。.本项目首次提出以血小板膜-脂质体融合体为主动靶向载体，靶向递送Rap治疗AS。递药系统的革新意义可能不亚于抗AS新药的研发，血小板膜-脂质体融合体是独立于球囊/支架的新型靶向给药载体，为AS 的靶向治疗提供了新的思路。