Platelet-inspired Delivery System for Targeted Thrombolytic Therapy

 产品说明：在闭塞性血管疾病中，溶解血栓和快速恢复血流对于预防组织发病率和死亡率至关重要。为此，目前临床上使用血管内全身给药溶栓药物如tPA的“血栓破坏”策略存在血浆诱导的药物失活、药物的血浆半衰期短、药物快速洗脱、靶部位药物可用性降低和全身出血副作用的问题。这些问题可以通过控制药物从可注射递送载体的释放来潜在地解决，所述可注射递送载体可以在血液动力学流动下选择性地积聚在凝块部位。为此，载体必须（i）主动结合到血栓部位，（ii）在流动中保持保留，以及（iii）允许局部药物释放以进行位点选择性作用。为了设计这样的媒介物，天然血小板提供了极好的范例，因为血小板具有向血管壁边缘化、通过特异性配体-受体相互作用主动结合凝块并释放细胞质内容物以调节凝块活性的先天能力。血小板的盘状形状、~2 µ直径大小和~25-50 kPa模量促进了血小板向血管壁的边缘化，而它们在血栓部位的主动结合是通过配体纤维蛋白原（Fg）和PSGL-1分别与血小板表面整合素GPIIb-IIIa和P-选择素结合介导的。受天然血小板的这些物理机械和生物相互作用线索的启发，我们提出设计具有（i）血小板模拟形状、大小和模量以促进朝向血管壁的增强的边缘化，和（ii）具有GPIIb-IIIa-和P-选择素结合肽配体的异多价表面装饰以使得能够在血液动力学流动下实现血栓选择性锚定和保留的载体。我们的中心假设是，溶栓药物从锚定在血栓部位的血小板激发的载体中的受控释放将增强位点特异性治疗功效，同时最小化全身药物分布和副作用。为了测试这一点，我们将制造基于白蛋白的血小板激发的盘状柔性载体，用活性血小板靶向配体对其表面进行异多价修饰，并在体外和体内评估其血栓锚定能力（Aim 1）。平行地，我们将阳离子修饰tPA（mtPA）以促进其加载到白蛋白颗粒中，因为白蛋白在生理pH下带负电荷，并表征mtPA从这些颗粒的体外加载和释放（目的2）。在通过这两个协同但独立的目标优化血小板激发的血栓锚定机制和mtPA加载/释放动力学之后，我们将整合它们以创建加载mtPA的血小板激发的载体，其将在体外和体内评估靶向溶栓功效（目标3）。我们的主要创新 是一种递送载体的设计，其结合了受血小板启发的关键物理机械和生物相互作用参数，以增强血栓锚定能力。目前的申请将测试该技术用于靶向溶栓治疗。该技术还有望成为血管疾病其他靶向治疗的平台。

 DESCRIPTION: In occlusive vascular diseases, dissolution of thrombus and rapid restoration of blood flow is critical in preventing tissue morbidity and mortality. To this end, current clinicl `clot-busting' strategies using intravascular systemic administration of thrombolytic drugs like tPA present issues of plasma-induced drug deactivation, short plasma half-life of drugs, rapid drug washout, reduced drug availability at target site, and systemic hemorrhagic side effects. These issues can be potentially resolved by controlled release of the drugs from injectable delivery vehicles that can selectively accumulate at the clot site under hemodynamic flow. For this, it is necessary for the vehicles to (i) actively bind to the clot site, (ii) stay retained uner flow, and (iii) allow localized drug release for site-selective action. For designing such a vehicl, natural platelets provide an excellent paradigm, since platelets have the innate capability to marginate towards the vascular wall, bind actively to clot via specific ligand-receptor interactions, and release cytoplasmic contents to modulate clot activities. The margination of platelets towards the vascular wall is facilitated by their discoid shape, ~2 µ diameter size and ~25-50 kPa modulus, while their active binding at the thrombus site is mediated via ligands fibrinogen (Fg) and PSGL-1 binding to platelet surface integrin GPIIb-IIIa and P-selectin respectively. Inspired by these physico-mechanical and biointeractive cues of natural platelets, we propose to engineer vehicles that will have (i) platelet-mimetic shape, size and modulus to facilitate enhanced margination towards the vascular wall, and (ii) heteromultivalent surface-decoration with GPIIb-IIIa- and P-selectin-binding peptide ligands to enable thrombus-selective anchorage and retention under hemodynamic flow. Our central hypothesis is that controlled release of thrombolytic drugs from such platelet-inspired vehicles anchoring at the thrombus site will enhance the site-specific therapeutic efficacy, while minimizing systemic drug distribution and side-effects. To test this, we will fabricate albumin-based platelet-inspired discoid flexible vehicles, modify their surface heteromultivalently with the active platelet-targeting ligands and evaluate their thrombus anchoring capability in vitro and in vivo (Aim1). In parallel, we will cationically modify tPA (mtPA) to facilitate its loading into the albumin particles since albumin i negatively charged at physiological pH, and characterize the loading and release of mtPA from these particles in vitro (Aim 2). After optimizing the platelet-inspired thrombus- anchoring mechanisms and the mtPA loading/release kinetics via these two synergistic yet independent aims, we will integrate them to create mtPA-loaded platelet-inspired vehicles which will be evaluated for targeted thrombolysis efficacy in vitro and in vivo (Aim 3). Our principal innovation is the design of a delivery vehicle that combines key physico-mechanical and biointeractive parameters inspired by platelets for enhanced thrombus-anchoring ability. The current application will test this technology for targeted thrombolytic therapy. The technology also holds the promise to become a platform for other targeted therapies in vascular diseases.