P-gp单抗-USPIO载药纳米颗粒对难治性癫痫的MR分子显像及靶向治疗的研究

The pathogenesis of refractory epilepsy is complicated, and mostly appears drug resistance to conventional anti-epileptic drugs. Preliminary studies of our group confirmed that P-gp over-expression was higher in refractory epileptic rats，as compared with normal and non-refractory epileptic rat. P-gp was distributed on micro-vascular endothelial cells only in brain，leading to the possibility that P-gp can play as a target molecule for targeted drug delivery against refractory epileptic patients and conjugate a stable nano-particle with P-gpMab on its surface, antiepileptic drugs (AEDs) and superparamagnetic iron oxide ( USPIO) in the core(MAb-P-gp-USPIO-AEDs nano-particles,PUNP) by active targeting on the basis of the unique surface effct of nanoparticles. The structure using P-gp monoclonal antibody on the specific recognition of P-gp, reversible closed role and superparamagnetism, could early identification of targeting lesions, specific blocking mechanisms of resistance, magnetic resonance molecular imaging and targeted therapy in vivo. The study of refractory epilepsy is investigated from different levels in the BBB model cell model of epilepsy, and drug-resistant epilepsy model in rats by HPLC, atomic force microscopy, transmission electron microscopy, immune electron microscopy, patch clamp technique, magnetic resonance imaging and other methods. The purpose is to provide experimental and theoretical bases for early identification of the epileptic targeting lesions, efficacy monitoring, screening new drugs and new nano-oriented targeted drug delivery systems.

难治性癫痫的发病机制复杂、治疗效果不佳，对常规抗癫痫药物多有耐药性，本课题组前期研究证实其与病灶区脑毛细血管内皮细胞特异性高表达P-gp相关，针对这一主要耐药机制，结合磁共振分子影像、免疫以及纳米技术，制备一种基于P-gp靶向特异性识别的新型磁性微纳米载药分子探针MAb-P-gp-USPIO-AEDs nano-particles（PUNP）。该结构充分利用P-gp单抗对P-gp的特异性识别以及USPIO的超顺磁性，实现病灶靶向早期识别、特异阻断耐药机制、磁共振活体分子显像以及靶向治疗的疗效监测。在BBB模型、癫痫细胞模型及耐药癫痫大鼠模型上通过HPLC、原子力显微镜，透射电镜、免疫电镜、膜片钳技术、磁共振等方法从不同层次进行深入探讨，以期为临床上难治性癫痫耐药机制的早期识别、疗效监测、筛选新药及新型导向性纳米载药系统等研究提供实验基础和理论依据。

P-糖蛋白（P-glycoprotein, P-gp）过量表达可以降低抗癫痫药物浓度从而导致多药耐药，是难治性癫痫重要的生物学基础之一。然而目前临床上缺乏对P-gp活体检测手段，本研究评估了基于纳米颗粒的癫痫灶P-gp多模态靶向分子显像的可行性。利用USPIO 作为载体，连接对P-gp有特异性亲和力短肽Pepstatin A，以及IR783荧光探针，构建多模态靶向纳米颗粒PANP，并在体外实验验证PANP对P-gp的靶向结合能力。体内实验中，构建大鼠海伦酸癫痫模型，尾静脉注射PANP后，成功实现P-gp的多模态分子显像（MRI及活体光学显像）。靶向纳米探针PANP研制，有望对癫痫过表达P-gp进行多模态无创显像，早期诊断难治性癫痫，进一步了解P-gp过表达分子机制，并为难治性癫痫的治疗提供新的思路。富集P-gp单抗、内集AED拉莫三嗪(Lamotrigine, LTG)的新型聚合物胶束纳米载体P123/LTG，通过特异性抗体的空间位阻效应直接封闭、阻断P-gp的过度表达，具有较好的血脑屏障通透性，经分子影像技术确认，它能使AED拉莫三嗪顺利到达耐药性癫痫患者的致痫灶并抑制P-gp过表达从而实现靶向治疗的目标。