T7/SHp介导nNOS-PSD-95解偶联剂ZL006双级脑靶向递药系统的构建及抗缺血性脑卒中研究

Ischemic stroke is a common and frequently occurring disease with high disability rate. Hitherto, there is no effective drug of stroke treatment in clinic. An nNOS-PSD-95 uncoupler named ZL006 independently innovated in our research centre is able to significantly reduce the release of pathological NO through the inhibition of NMDAR-PSD-95-nNOS pathway over-activation, which reduces the infarct volume of brain. However, it is difficult for ZL006 to penetrate through the blood-brain barrier (BBB) because of it's strong hydrophilicity, reducing the distribution of ZL006 in brain tissue. Therefore, in order to enhance the brain targeting of ZL006, the transferrin receptor (TfR) specific ligand T7 peptide and specific stoke homing peptide (SHp) were used to doubly modify ZL006 nanoliposomes. The dual targeting ZL006 liposome delivery system built by T7/SHp modification could penetrate through BBB by T7-mediated transcytosis and further concentrate in the ischemic lesion by the specific homing ability of SHp, which enhanced the anti-ischemic stroke effect of ZL006 and reduced it's toxicity for the normal brain tissue. The two-stage targeting mechanisms of the dual targeting drug delivery system for stroke treatment will be investigated by in vitro and in vivo studies, which will provide a new dual targeting strategy and study experiences on ischemic stroke therapy research. There are no relevant reports of this study at home and abroad.

缺血性脑卒中是常见病、多发病、致残率高，迄今临床上少见疗效确切的治疗药物。我中心自主创新的nNOS-PSD-95解偶联剂ZL006能够显著抑制NMDAR-PSD-95-nNOS通路过度激活而释放的病理性NO，缩小脑梗死容积。然而，ZL006亲水性较强，难以透过血脑屏障（BBB），脑组织分布不理想。因此，为增强ZL006的脑靶向性，本项目采用BBB表面TfR特异性配体T7肽和脑缺血特异性归巢肽(SHp)双重修饰ZL006纳米脂质体，构建T7/SHp共修饰的双靶向ZL006脂质体递药系统。该系统在T7介导下透过BBB后，再在SHp特异性归巢作用下进一步浓集于缺血病灶，实现ZL006的脑内双重靶向递药。从而提高解偶联剂ZL006治疗效果并降低副作用，通过细胞水平及体内外实验，探讨该系统的双级靶向机制，为缺血性脑卒中靶向治疗研究提供一种全新的双重靶向策略和研究路径。本研究内容未见国内外相关报道。

脑卒中是一种具有较高发病率和死亡率的急性脑血管疾病，其危险因素复杂多样，涉及生物、环境、社会以及心理等多学科领域。此外，由于脑组织血脑屏障(BBB)等结构精细复杂，治疗药物透过BBB能力有限，其生物利用度差；同时，由于脑组织结构十分精细，任何外源性物质(包括治疗性药物等)透过BBB进入脑组织都将对脑的正常生理功能构成极大威胁。因此，理想的治疗脑卒中疾病的脑靶向递药系统须将治疗性药物靶向递送入脑后，在脑内二次靶向富集于病灶部位，增加脑内病灶部位的药物浓度，提高疗效，并降低对脑正常生理功能的影响。.本课题以BBB靶向肽(T7)和缺血归巢肽(SHp)为靶向功能头基、小分子药物nNOS-PSD-95解偶联剂(ZL006)为模型药物、磷脂/胆固醇为载体材料，经mPEG-DSPE2000修饰增加其长循环作用，构建多肽修饰的双靶向递药系统治疗脑卒中。本文主要包括以下三部分：.1. 靶向材料合成及双靶向修饰脂质体递药系统的构建；.2. T7&SHp修饰双靶向脂质体递药系统的体内外靶向性评价；.3. T7&SHp修饰双靶向脂质体递药系统的药效学研究及体内初步安全性评价。.首先，通过马来酰亚胺与巯基的反应将T7肽和SHp制备为靶向材料，通过1H-NMR进行表征；采用乙醇注入法制备出多肽修饰的双靶向PEG化脂质体(T7&SHp-P-LPs)。脂质体粒径为96.2±1.3nm，外观圆整，分布均匀，淡蓝色乳光明显。多肽的修饰后对脂质体的外观、粒径、电位、包封率、载药量和泄漏率等性质均无明显影响。.其次，通过体外BCECs单细胞层转运研究发现，T7&SHp-P-LPs具有良好BBB靶向性，更容易透过血脑屏障；通过体外谷氨酸刺激高分化PC-12细胞的摄取情况研究发现，T7&SHp-P-LPs能更好地作用于受损神经细胞部位，同时对正常的细胞无明显影响；通过离体大鼠脑组织显微观察和大鼠脑组织分布观察两个实验，结果均表明T7&SHp-P-LPs能够靶向至MCAO大鼠的缺血病灶部位，降低载体在脑部其他区域分布。.最后，大鼠体内药效学研究包含MCAO大鼠神经功能缺损评分实验、大脑梗死灶面积实验及检测MCAO大鼠缺血部位细胞凋亡实验。结果表明，载有ZL006的双靶向脂质体(T7&SHp-P-LPs/ZL006)能够有效减轻MCAO大鼠的神经功能损伤，明显减少脑缺血梗死面积，表现出较好的神经功能