Department of Chemical and Materials and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan.
Department of Physiology and Pharmacology and Healthy Aging Research Center, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan.
Int J Mol Sci. 2020 Apr 13;21(8):2690. doi: 10.3390/ijms21082690.
Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single -SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area.
重组组织型纤溶酶原激活剂（rtPA）是唯一一种获得美国食品药品监督管理局（FDA）批准用于治疗缺血性脑卒中的溶栓药物。然而，由于溶栓药物的半衰期较短，需要高剂量静脉输注以维持有效的药物浓度，而一个重要的限制是出血风险。我们设想 rtPA 递药的双重靶向策略将是可行的，可以最大限度地减少治疗所需的 rtPA 剂量。为此，将 rtPA 和纤维蛋白亲和肽共同固定在聚乳酸-共-羟基乙酸（PLGA）磁性纳米颗粒（PMNP）上，制备肽/rtPA 缀合 PMNPs（pPMNP-rtPA）。在制备过程中，首先用亲和素对 PMNP 进行表面修饰，亲和素可以与生物素相互作用。然后，将 PMNP-亲和素与生物素-聚乙二醇-rtPA（或生物素-聚乙二醇-肽）结合，这是通过在点击化学反应中使用马来酰亚胺和 rtPA（或肽）中的单个 -SH 基团之前，将 rtPA（或肽）与生物素-聚乙二醇-马来酰亚胺结合而制备的。理化性质表征表明成功制备了保留 rtPA 纤维蛋白溶解活性的磁性纳米颗粒，而体外细胞毒性和溶血试验中的生物反应研究强调了所有磁性纳米颗粒的高生物相容性。还证实了磁导向和纤维蛋白结合作用，这导致在与血块静态或动态孵育后，与游离 rtPA 相比，使用 PMNP-rtPA 或 pPMNP-rtPA 时体外的溶栓率更高。在流动系统中的压力依赖性血栓溶解模型中，与相同药物剂量的游离 rtPA 相比，双重靶向 pPMNP-rtPA 可将再灌注的血栓溶解时间减少 40%。在大鼠栓塞模型中的体内靶向溶栓实验中，pPMNP-rtPA 的用量为游离 rtPA 的 20%，以恢复通过向后肢区域注射血栓形成的血管血栓中的髂血流。
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