tPA nanoconjugate for stroke therapy

中风是死亡、长期残疾和社会经济成本的主要原因， 迫切需要更有效的治疗方法。组织纤溶酶原激活物（tPA）的静脉内施用是 唯一一种FDA批准的恢复脑血流的疗法但由于风险增加， 出血超过中风后4.5小时，很少有中风患者（<5%）受益于t-PA。此外，t-PA破坏了 血脑屏障完整性（BBB）和神经毒性，加重再灌注损伤。活性氧 (ROS)在缺血后不久和再灌注期间以及之后产生的，被认为是主要的 缺血/缺血再灌注损伤的介质。为了支持这一观点，在我们之前的研究中，我们表明， 先用t-PA治疗，然后再用抗氧化酶，超氧化物歧化酶 (SOD)和过氧化氢酶（CAT）封装在纳米颗粒（纳米SOD/CAT），两者都通过颈动脉给药 在血栓栓塞性大鼠中风模型中，缺血后3小时的动脉， 单独使用tPA。这种连续治疗中和了升高的活性氧水平，抑制了血管渗漏/预防了 水肿形成，减少炎症，并保护神经元细胞免于凋亡。最重要的是，我们 发现顺序治疗刺激神经元和循环祖细胞迁移到 梗死脑，而tPA单独抑制该运动。基于这些有希望的结果，我们设计了一种新的双作用tPA纳米缀合物- tPA缀合纳米SOD/CAT -实现神经和血管保护再灌注损伤，同时保留tPA的溶栓作用。我们的初步数据显示tPA纳米缀合物a）具有比单独tPA明显更好的溶栓效果（在tPA剂量的1/4时，缀合物产生相同程度的血栓溶解），B）不引起tPA相关的神经毒性，和c）当通过静脉内给药时在中风后6小时（尾静脉）注射，有效地减少梗塞体积，随着时间的推移，神经恢复得到改善，生存率提高（~85%），明显高于单独给予tPA的大鼠（~20%）。我们假设，我们的tPA纳米缀合物，由于其对氧化应激的持续神经保护作用和比单独的t-PA显著更好的溶栓作用，可以克服单独的tPA的局限性， 最小化再灌注损伤并实现神经/功能恢复，即使治疗延迟。欧 目的是研究和开发我们的tPA纳米缀合物作为中风的安全有效的治疗方法。具体 目的1：分析tPA纳米偶联物的神经保护/溶栓作用。目标2： 评估神经恢复和运动功能随时间的变化程度。目的3：确认脑修复 缺血/再灌注损伤的机制。有效的治疗可以使更多的中风患者受益 由于治疗窗口延长和风险降低， 出血性并发症这样的治疗可以显着减少中风后残疾的程度。

Stroke is a leading cause of death, long-term disability, and socioeconomic costs, highlighting the urgent need for more effective treatments. Intravenous administration of tissue plasminogen activator (tPA) is the only FDA-approved therapy to re-establish cerebral blood flow. But because of increased risk of hemorrhage beyond 4.5 hr post stroke, few stroke patients (<5%) benefit from t-PA. Further, t-PA disrupts the blood-brain barrier integrity (BBB) and is neurotoxic, aggravates reperfusion injury. Reactive oxygen species (ROS), generated soon after ischemia and during reperfusion and thereafter, are considered the main mediators of ischemia/ischemia reperfusion injury. To support this notion, in our previous studies, we showed that sequential treatment with t-PA first, followed by delivery of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) encapsulated in nanoparticles (nano-SOD/CAT), both administered via carotid artery 3 hr post ischemia in a thromboembolic rat stroke model, significantly reduced reperfusion injury than did tPA alone. This sequential treatment neutralized elevated ROS levels, inhibited vascular leakage/prevented edema formation, reduced inflammation, and protected neuronal cells from apoptosis. Most important, we found out that the sequential treatment stimulated migration of neuronal and circulating progenitor cells into the infarcted brain, whereas tPA alone inhibited that movement. Based on these promising results, we designed a novel dual-action tPA nanoconjugate – tPA conjugated to nano-SOD/CAT – to achieve neuro and vascular protection from reperfusion injury while retaining the thrombolytic effects of tPA. Our preliminary data show that the tPA nanoconjugate a) has markedly better thrombolytic effects than tPA alone (at 1/4 of the dose of tPA, the conjugate yielded the same degree of thrombolysis), b) causes no tPA-associated neurotoxicity, and c) when given via intravenous (tail vein) injection at 6 hr post stroke, effectively reduces infarct volume, resulting in improved neurological recovery over time and increased survival (~85%), significantly more so than in rats given tPA alone (~20%). We hypothesize that our tPA nanoconjugate, with its sustained neuroprotective effects from oxidative stress and significantly better thrombolytic effects than t-PA alone, could overcome the limitations of tPA alone in minimizing reperfusion injury and achieving neurological/functional recovery, even if treatment is delayed. Ou goal is to investigate and develop our tPA nanoconjugate as a safe and effective treatment for stroke. Specific aims are: AIM 1: To analyze the neuroprotective/thrombolytic effects of the tPA nanoconjugate. AIM 2: To evaluate the extent of neurological recovery and motor functions over time. AIM 3: To confirm the brain repair mechanisms following ischemic/reperfusion injury. An effective treatment could benefit more stroke patients than can be treated with tPA alone because of the extended window of treatment and reduced risk of hemorrhagic complications. Such a therapy could significantly diminish the extent of post-stroke disability.