tPA nanoconjugate for stroke therapy

中风是导致死亡、长期残疾和社会经济损失的主要原因，突出了 迫切需要更有效的治疗方法。静脉注射组织型纤溶酶原激活剂(TPA) FDA批准的唯一一种重建脑血流的治疗方法。但由于风险增加， 卒中后出血超过4.5小时后，很少有中风患者(&lt；5%)受益于t-PA。此外，t-PA还破坏了 血脑屏障完整性(BBB)和神经毒性，加重再灌注损伤。活性氧物种 (ROS)在缺血后不久、再灌流期间和之后产生，被认为是主要的 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，我们展示了 即先用t-PA顺序治疗，然后递送抗氧化酶、超氧化物歧化酶 (超氧化物歧化)和过氧化氢酶(CAT)包裹纳米粒(纳米-SOD/CAT)，两者都通过颈动脉给药 血栓栓塞型大鼠卒中模型中动脉缺血3小时后再灌注损伤显著减少 只有TPA。这种顺序治疗中和了升高的ROS水平，抑制了血管渗漏/预防了 水肿形成，炎症减轻，并保护神经细胞免于凋亡。最重要的是，我们 研究发现，序贯治疗刺激神经元和循环中的祖细胞迁移到 脑梗塞，而tPA单独抑制这种运动。基于这些有希望的结果，我们设计了一种新型的双作用tPA纳米结合物-tPA与纳米-SOD/CAT偶联-在保留tPA的溶栓作用的同时，实现了对神经和血管的保护作用。我们的初步数据显示，tPA纳米结合物a)明显比tPA单独使用有更好的溶栓效果(在tPA剂量的1/4时，结合物产生相同程度的溶栓)，b)没有引起tPA相关的神经毒性，c)当在中风后6小时通过静脉(尾静脉)注射时，有效地减少梗塞体积，随着时间的推移，神经功能恢复改善和存活率增加(~85%)，显著高于单独使用tPA的大鼠(~20%)。我们推测，我们的tPA纳米结合物，由于其持续的氧化应激神经保护作用和显著优于t-PA单独的溶栓作用，可以克服tPA单独治疗的局限性 最大限度地减少再灌注损伤，实现神经/功能恢复，即使治疗被推迟。我们 目标是研究和开发我们的tPA纳米结合物作为一种安全有效的中风治疗方法。特定的 目的：目的1：分析tPA纳米结合物的神经保护/溶栓作用。目标2：实现 随着时间的推移，评估神经恢复和运动功能的程度。目标3：确认大脑修复 缺血/再灌注损伤后的机制。有效的治疗可能使更多的中风患者受益 而不是单独用tPA治疗，因为治疗窗口延长了，降低了 出血性并发症。这种疗法可以显著降低中风后残疾的程度。

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.