Platelet-inspired Delivery System for Targeted Thrombolytic Therapy

 描述：在闭塞性血管疾病中，血栓的溶解和血流的快速恢复是预防组织发病率和死亡率的关键。为此，目前临床上使用血管内全身给药如tPA的溶栓策略存在着血浆诱导的药物失活、药物的血浆半衰期短、药物快速洗脱、靶点药物可获得性降低以及全身出血副作用等问题。这些问题可以通过从可注射给药载体中控制释放药物来解决，这些药物可以在血流动力学下选择性地积聚在血栓部位。为此，载体必须(I)主动结合到血栓部位，(Ii)在流动中保持不变，以及(Iii)允许局部药物释放以进行部位选择性作用。为了设计这样的载体，天然的血小板提供了一个很好的范例，因为血小板具有先天的能力，可以朝向血管壁边缘，通过特定的配体-受体相互作用主动与凝块结合，并释放细胞质内容物来调节凝块的活动。盘状、直径约2微米、模数约25~50kPa的血小板可促进其向血管壁的边集，而其在血栓部位的活性结合则分别通过与血小板表面整合素GPIIb-IIIa和P-选择素的配体纤维蛋白原(Fg)和PSGL-1的结合而实现。受天然血小板的这些物理-机械和生物交互作用线索的启发，我们建议设计一种具有(I)类似血小板的形状、大小和模数的载体，以促进向血管壁更多的边缘形成，以及(Ii)用GPIIb-IIIa和P-选择素结合的多肽配体进行异多价表面装饰，以在血流动力学下实现血栓选择性锚定和保持。我们的中心假设是，从这种锚定在血栓部位的血小板激发的载体上控制释放溶栓药物将增强局部特异性治疗效果，同时将全身药物分布和副作用降至最低。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，用活性的血小板靶向配体对其表面进行异价修饰，并评估其体外和体内的血栓锚定能力(Aim1)。同时，我们将对tPA(Mtpa)进行阳离子修饰，以促进其负载到白蛋白颗粒中，因为白蛋白I在生理pH下带负电荷，并在体外表征mtpa从这些颗粒中的负载和释放(目标2)。在通过这两个协同但独立的目标优化了血小板激发的血栓锚定机制和mtpa的加载/释放动力学之后，我们将把它们整合起来，创造出负载mtpa的血小板激发载体，并将在体外和体内对其靶向溶栓效果进行评估(目标3)。我们的主要创新 是一种输送载体的设计，它结合了关键的物理-机械和生物交互参数，灵感来自于增强血栓锚定能力的血小板。目前的应用将测试这项技术的定向溶栓治疗。这项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.