tPA-磁性动力纳米棒对小鼠大脑中动脉梗塞的血流重建作用及机制研究

Stroke is the leading cause of death and disability in China. Systemic thrombolysis with intravenous tissue plasminogen activator (tPA) remains the only proven treatment to improve clinical outcome of patients with acute ischemic stroke. But because of the limitation in treatment window, only small population of stroke patients can benefit from tPA. To improve tPA-induced thrombolysis and recanalization rate, we have developed magnetic nanorods, which are capable of performing rotational and directional motion in an external magnetic field. In vitro, The nanorods-conjugated tPA can target the clot in specific locations and establish a high tPA concentration environment locally, such that the total dose of tPA needed would be lower and the time window of tPA treatment would be significantly prolonged. And furthermore, successful attempts on restoring the blood flow of obstructed femoral artery and common carotid artery in mice clot models have been achieved as well. In order to apply tPA-nanorods in ischemic stroke treatment clinically, we will use laser Doppler blood flow imaging, scanning electron microscopy technology, histological and neurobehavioral tests to test our specific aims, including: 1) to determine the tPA-nanorods mediated thrombolysis for restoring blood flow in mice middle cerebral artery clot model; 2) to identify the relevant treatment window, best dose, suitable injection routes，optimal magnetic field settings and the motor functional outcomes； 3） to explore the underlying mechanisms of tPA-nanorods mediated thrombolysis.

脑卒中是我国目前致死和致残的首位原因，除组织纤溶酶原激活物（tPA）静脉溶栓外，缺乏其他有效治疗手段。由于治疗时间窗的限制，tPA只适用于部分缺血性脑卒中患者。我们前期构建了能在外部磁场作用下旋转及定向运动的磁性动力纳米棒（tPA-Nanorods），通过体外实验发现tPA-nanorods运载系统不仅能将tPA靶向运送到血栓部位，降低tPA使用剂量，延长治疗时间窗；而且在小鼠股动脉及颈总动脉血栓模型上也已成功实现血流重建。为了将tPA-nanorods应用于临床，本课题拟通过激光多普勒血流成像、扫描电镜、组织学和神经行为学检测等技术，进一步研究：1）建立小鼠大脑中动脉血栓模型，探索tPA-nanorods对大脑中动脉梗塞的血流重建作用；2）探索tPA-nanorods溶栓的治疗时间窗、最佳药物剂量、最佳给药途径、最佳磁场条件和神经功能恢复情况；3）探索tPA-nanorods作用机制。

缺血性脑卒中除tPA外，目前仍缺乏有效治疗手段，治疗关键在于如何尽早进行血管再通和血流重建。在临床上，组织型纤溶酶原激活剂（tPA）静脉溶栓是当前唯一普遍认可的治疗手段，但受限于严格的治疗时间窗；而血管内机械取栓仅限于大型脑卒中医疗中心。项目组前期构建了能在外部磁场驱动下进行定向及旋转运动的tPA-磁性动力纳米棒（tPA-Nanorods，tPA-NRs）靶向溶栓复合体，并在体外实验、小鼠颅外动脉（股动脉、颈总动脉）血栓模型上成功实现血流重建。为进一步临床转化和应用，本项目围绕tPA-NRs对小鼠颅内动脉血栓的溶栓安全性、可行性、溶栓条件及机制开展研究，发现：1）采用热液合成制备Fe3O4磁性动力纳米棒（Fe3O4 NRs），长度L=1.3±0.2 μm，直径D=0.5±0.1 μm，应用戊二醛作为交联剂能将tPA分子共价结合到NRs上，所形成tPA-NRs复合体的tPA结合率为12.9%，tPA活性保留约70％；2）tPA-NRs具有良好的生物安全性，研究证实tPA-NRs无明显细胞毒性、肝肾毒性，不影响机体凝血功能，并能以原型经尿液中排出体外；3）应用10%FeCl3成功诱导小鼠远端大脑中动脉血栓模型，证实tPA-NRs能在外部磁场引导及驱动下靶向抵达颅内血栓局部，充分开通血管，高效恢复血流，缩小脑梗死范围，显著降低tPA相对使用剂量；4）tPA-NRs颅内溶栓时，项目组认为动脉途径优于静脉途径，tPA-NMs动脉溶栓的最佳剂量为1 mg/kg；5）tPA-NRs在外置旋转磁场驱动下同时发挥机械性及酶解性溶栓作用，两者相互协同，共同实现血流重建。