基于Thrombin/pH程序化响应释药的血小板膜仿生智能共递送载体及其抗缺血性脑卒中研究

Ischemic stroke is a serious brain diseases that threaten human beings. It is potential to treat ischemic stroke by combination thrombolytic agents tPA and neuroprotective agents ZL006 which was created innovatively in our research centre. However, the present shortcomings of the combination therapy include the short half time, weak targeting capacity to thrombus and narrow thrombolytic time window of tPA, as well as the poor penetration into brain of ZL006 and poor controllability of temporal and spatial selectivity in vivo. In this research project, we study the solving strategy from the point of drug delivery system based on our preliminary studies. Herein, we use the thrombin up-regulated in thrombus and low pH microenvironment in ischemic brain tissue as smart "switch" of drug release to develop a "core - shell" co-delivery carrier of ZL006 and tPA. The mutifunctional nanocarrier has a core of ZL006 loaded pH-sensitive polymer nanoparticles and a outer shell of platelet membrane that grafted by tPA via thrombin cleavage amino acid sequence conjugated with Tat peptides. The programmed and responsive platelet biomimetic delivery system will target to thrombus and release tPA by thrombin cleaved, which in situ exposed the Tat peptides outside of the carrier that mediates the remain carrier penetrate BBB into the brain. Then, the ZL006 will be released rapidly from the carrier disassembled in the acid microenvironment of ischemic brain tissue. The co-delivery system developed in this project will prolong the tPA's half time and enhance its thrombus targeting ability as well as improve the BBB penetration of ZL006 by "in-situ exposure" of Tat peptides, which satisfied the request of in vivo temporal and spatial selectivity of tPA/ZL006 release for stroke treatment. Our proposal will provide a novel co-delivery strategy for the drug delivery system study of ischemic stroke.

缺血性脑卒中是一种严重危害人类健康的脑部疾病，我中心自主创新的神经保护剂ZL006与溶栓剂tPA协同用药是极具潜力的治疗方案。然而，两药合用时存在tPA循环时间短，血栓靶向性弱，溶栓时间窗窄、ZL006脑组织渗透差及二者体内时空选择控制性差等问题。本项目在前期研究基础上，从共递送载体出发，利用血栓部位高表达Thrombin和缺血脑组织酸性pH为智能释药“开关”，以载ZL006的pH敏感聚合物纳米粒为核，可被Thrombin酶切释放并串联Tat肽的tPA接枝血小板膜为壳，构建程序化响应释药的“核-壳”型血小板膜仿生共递送载体。该载体先靶向脑血栓，在血栓处响应性释放tPA溶解血栓，随后“原位暴露”的Tat介导载体透过BBB进入缺血脑组织，pH响应下释放ZL006。达到了长循环、血栓靶向、脑靶向及两药体内时空选择性释药的tPA/ZL006共递送，开辟了全新的缺血性脑卒中药物共递送载体构建策略。

目前，临床上大约87%的缺血性脑卒中是由脑动脉血栓的形成所致。血栓造成局部脑组织区域血液供应障碍，从而引起的脑组织缺血缺氧性病变坏死。现阶段，临床上使用的tPA溶栓治疗，但往往会造成缺血再灌注，对患者脑组织造成进一步损伤。联合溶栓药物和神经保护剂可以实现在恢复血流供应的同时，挽救濒临死亡的受损神经元。目前，程序化靶向递送溶栓药物和神经保护剂到血栓和缺血半暗带的治疗策略面临着巨大挑战。受启发于血小板在血栓形成中发挥的重要作用，本项目设计了一种thrombin/pH程序化响应的血小板仿生纳米递药系统（简称为：tP-NP-rtPA/ZL006e）用于程序化病灶靶向递送溶栓药物rtPA和神经保护剂ZL006e，来治疗缺血性脑卒中。该载体系统可以整合血脑屏障的靶向通过性和血栓位点的特异亲和性，以分别提高神经保护剂ZL006e和溶栓剂rtPA的治疗效果。本项目研究主要包含以下三个部分：（一）材料合成及Thrombin/pH程序化响应释药的血小板膜仿生共递送智能载体（tP-NP-rtPA/ZL006e）的构建与表征；.（二）Thrombin/pH 程序化响应释药的血小板膜仿生共递送智能载体系统的体外生物性能研究；（三）Thrombin/pH 程序化响应释药的血小板膜仿生共递送智能载体系统的体内生物性能研究。.tP-NP-rtPA/ZL006e由载有神经保护剂ZL006e的葡聚糖衍生物纳米粒内核和修饰有可被凝血酶剪切的Tat-rtPA多肽复合物的血小板膜外壳构成。研究结果显示，该体系在血小板膜上激活的GPIbα等蛋白介导血栓靶向作用下，吸附聚集于血栓位点，随后原位释放出溶栓剂后暴露出Tat穿膜肽，接到载有神经保护剂ZL006的聚合物纳米粒从而透过血脑屏障，特异性递送ZL006e至缺血脑组织处。从一系列的体内外评价，我们发现tP-NP-tPA/ZL006e在MCAO大鼠模型中具有明显改善的抗缺血性脑卒中的治疗效果。