中空碳纳米球靶向P-gp依赖耐药性癫痫的T1/T2双模式示踪与拮抗研究

Previous studies have demonstrated that N-methyl-D-aspartate (NMDA) signaling in brain capillaries contributes to the transcriptional activation of P-glycoprotein(P-gp). Regional over-expression of P-gp in blood–brain barrier(BBB) is considered as a major hurdle in the treatment of pharmacoresistant epilepsy. Previously, we developed a nanoagent by conjugating magnetic nanoparticles with pepstatin A(PA), a targeting peptide with specific affinity for P-gp. The nanoagent was readily and selectively accumulated within cerebral epileptogenic regions, which were detectable by magnetic resonance (MR) T2-weighted imaging. However, application of magnetic nanoparticles has several disadvantages, in particular, the negative contrast effect resulting in magnetic susceptibility artifacts. Besides, the low efficacies of brain targeting capacity and drug loading of magnetic nanoparticles limited their application as theranostics in the treatment of pharmacoresistant epilepsy. Therefore, improvements of the above-mentioned nanocarrier system are warranted to solve those drawbacks. T1/T2 dual-mode MR contrast agents have gained much attention because of their ability to improve accuracy by providing two pieces of complementary information simultaneously. Encouragingly, iron- and gadolinium-doped mesoporous invaginated hollow carbon nanospheres(HCNSs)were designed and synthesized successfully as the potential T1/T2-weighted dual-modal MR contrast agent in our study. The HCNSs as a novel platform have higher surface areas and larger pore volumes, which could be harnessed for drug loading as well as chemical modification. Evidence exists that SC-51089, an EP1 receptor antagonist, efficaciously prevented the seizure-associated induction of endothelial P-gp expression by blocking the NMDA-dependent signaling pathway in epileptogenic focus. Thus, the resultant HCNSs are expected to effectively diagnose and treat of epilepsy through the functionalization of targeting PA and loading of SC-51089. Compared with the previous studies, our nanosystem could target P-gp in pharmacoresistant epilepsy for T1/T2 dual mode precision imaging, simultaneously deliver SC-51089 to reverse the drug resistance of epilepsy by inhibiting the NMDA signaling pathway to reduce P-gp overexpression. The results of our project may provide research basis for development of novel nano theranostics to early diagnose and timely intervene drug resistant epilepsy.

P-糖蛋白（P-gp）参与癫痫（EP）耐药，致痫灶P-gp过表达与N-甲基-D-门冬氨酸（NMDA）信号途径激活密切相关。前期研究主要采用磁性纳米造影剂，通过修饰特异性结合P-gp的短肽胃酶抑素A（PA），对P-gp进行靶向T2成像，存在负性增强易产生磁敏感伪影及未能同步递药干预的不足。因此，本项目拟将PA修饰于具有T1/T2双模式成像功能的铁、钆掺杂中空碳纳米球（HCNSs），同时利用HCNSs介孔特性，高效负载NMDA信号通路拮抗剂SC-51089，构建可调控EP耐药微环境的T1/T2双模式靶向纳米系统。与前期研究相比，本纳米系统在实现P-gp靶向T1/T2双模式精准成像的同时可向致痫灶递送SC-51089，继而通过拮抗NMDA信号途径减少P-gp过表达，达到抑制EP耐药的目的，以期解决耐药性EP的早期诊断与及时干预的问题。本项目研究也有望为发展EP耐药新的诊疗方法提供实验依据和参考。

世界上约有5000万人罹患癫痫。尽管有多种抗癫痫药物问世，但同时对多种抗癫痫药物耐药的耐药性癫痫一直占所有癫痫患者的三分之一左右。P-糖蛋白（P-glycoprotein, P-gp）过量表达可以阻止抗癫痫药物由血脑屏障入脑，是导致癫痫多药耐药的重要生物学基础之一，然而目前临床上缺乏对P-gp的活体检测手段。我们的前期研究发现利用磁性纳米造影剂（SPION），通过修饰特异性结合P-gp的短肽胃酶抑素A（Pepstatin A, PA），对P-gp进行靶向T2成像。研究表明，SPION-PA可用于血脑屏障上过表达的P-gp的分子成像，有效并特异性地揭示癫痫发作引起的区域性变化存在，但T2负性增强容易产生磁敏感伪影，同时也未能同步递药逆转过表达P-gp。因此，我们首先制备了生物安全性良好的铁、钆掺杂中空碳纳米球（HCNSs），其具有良好的T1/T2双模式成像功能，将PA修饰在纳米球表面（pHCNSs-PA），同时利用HCNSs介孔特性，高效负载SC-51089。研究发现SC-51089作为EP1受体拮抗剂可通过阻断NMDA依赖性信号通路有效阻止癫痫发作时P-gp过表达。在大鼠海人酸癫痫模型，通过尾静脉注射纳米探针pHCNSs@ SC-51089-PA，实现了对过表达P-gp的T1/T2双模式磁共振成像，同时靶向递送的SC-51089可以通过拮抗NMDA信号途径减少P-gp过表达。本研究以HCNSs为载体的靶向纳米探针研制将有望实现对癫痫导致过表达P-gp进行多模态无创显像，有助于进一步了解P-gp过表达分子机制，早期预测多药耐药性，为完善癫痫治疗提供客观依据；同时拮抗剂应用将有望于在实现靶向给药将在降低抗癫痫活性的同时，进一步降低药物剂量和抗耐药性。这种方法不仅有助于提高对耐药性癫痫的诊断能力，治疗水平以及早期预防P-gp过度表达，为探索耐药性癫痫新的诊疗方法提供实验依据和参考。