EphA2受体特异性的纳米免疫毒素EphrinA1-NP/PE38靶向治疗胶质瘤及机制研究

Recent evidence has shown EphA2 as a novel molecular marker and target in glioblastoma multiforme. We have constructed an anti-EphA2 immunotoxin and demonstrated EphrinA1-PE38 have therapeutic efficacy in vitro and in a malignant glioma model. Howerver the failure of location and the nonspecific toxicity remained unresolved.To overcome the limitations, we hypothesize that PE38-loaded anti-EphA2 nanoparticles could target glioma cells. With this in mind, we plan to develope PE38-loaded nanoparticles conjugated with EphrinA1 fragments which bind specifically to EphA2 receptors. Employing PTMC as carriers, we intend to prepare PE38-loaded nanoparticles by double-emulsion evaporation method. The PE38-loaded anti-EphA2 targeted nanoparticles were fabricated by conjugation of EphrinA1 fragments onto the surface of nanoparticles according to carbodiimide method．The ex vivo and in vivo studies will confirmed the therapeutic efficacy and reveal the mechanism of targeted delivery system.This study focus on the novel nanotechnology applied in immunotoxin-based targeted therapy, providing a potentially useful strategy for cancer treatment.

EphA2受体作为胶质瘤新的分子标记物，是免疫毒素治疗胶质瘤的新靶点。我们前期成功构建了靶向EphA2受体的重组免疫毒素EphrinA1-PE38，对胶质瘤具有显著的杀伤作用，但是仍面临免疫毒素难以穿透到肿瘤局部并由此引起的非特异性毒性问题。载药纳米微粒具有一定的靶向性、可缓控释等优点，已成为抗肿瘤药物的良好载体。由此，我们提出载药纳米微粒介导免疫毒素靶向治疗胶质瘤的可能性。我们拟用聚三亚甲基碳酸酯（PTMC）为载体，通过复乳法制备PE38毒素纳米微球，再将导向性EphrinA1片段连接在纳米毒素表面，形成PTMC包裹的靶向纳米免疫毒素EphrinA1-NP/PE38，并研究其理化特征。通过静脉途径给药，观察其对胶质瘤细胞靶向杀伤作用，旨在获得该新型纳米免疫毒素靶向治疗胶质瘤的可靠依据及作用机制。该课题探索新型纳米免疫毒素治疗胶质瘤的新方法，将为免疫毒素靶向治疗肿瘤提供新的思路。

EphA2受体作为胶质瘤新的分子标记物，是治疗胶质瘤的新靶点。本课题拟在前期工作基础上制备靶向纳米免疫毒素EphrinA1-NP/PE38并研究其理化性质，通过体内外实验观察其对胶质瘤的杀伤作用。项目实行过程中，我们作了一些调整，以EphA2受体特异性结合肽SWL代替EphrinA1，以脂质体代替PTMC作为纳米载体，制备了S-NP/PE38。体外实验结果显示S-NP/PE38具备稳定的理化性质，对EphA2高表达的胶质瘤细胞具备特异性亲和性，能特异性靶向胶质瘤细胞并起到杀伤效果。体内实验结果显示其能穿透血脑屏障到达胶质瘤靶部位，并起到治疗胶质瘤的作用。在此基础上，我们以SWL和cRGD双肽修饰肝素载体制备了可同时靶向EphA2和αvβ3受体的可重新自组装纳米粒H-S-R NPs。通过表征检测发现H-S-R NPs在血液模拟环境中可达到适合穿透BBB的粒径并具备良好的稳定性；细胞摄取实验和流式实验结果显示其具备对EphA2和αvβ3过表达细胞的特异性靶向能力；CCK8实验和三维培养实验结果证明H-S-R NPs可体外抑制EphA2和αvβ3过表达细胞增殖和微管形成；通过小动物活体成像检测进行的体内分布实验证实其可在胶质瘤皮下瘤和原位瘤模型鼠中穿透BBB和BBTB到达胶质瘤靶部位；通过尾静脉给药注射H-S-R NPs证明其可在胶质瘤皮下瘤和原位瘤荷瘤鼠模型中达到抑制胶质瘤生长和延长荷瘤鼠生存期的作用；免疫组化检测Ki-67和CD34/PAS证实H-S-R NPs通过抑制胶质瘤细胞增殖和阻断胶质瘤血供发挥作用；对各治疗组的各器官进行HE染色未发现H-S-R NPs具备明显毒性。综上，我们在针对EphA2受体这个胶质瘤的新靶点治疗胶质瘤方面进行了有意义的探索。