“协同靶向”策略构建两性寡肽脂质体介导三氧化二砷前药脑胶质瘤胞内递药及机理研究

Arsenic trioxide (As2O3), an active ingredient of Traditional Chinese Medicine White Arsenic, is a potential agent for anti-glioma therapy for long time. However, its clinical application is limited largely because of the poor tumor distribution, high toxicity and narrow therapeutic windows. Otherwise, although delivered by targeted drug delivery system, As2O3 is still difficult to be targeted accurately and precisely due to the multiple physiological and pathological barrier. In this study, we will construct a multifunctional glioma active targeting drug delivery system embedding the characters of “synergistic targeting” and drug release monitoring. The property of As2O3, the biological characteristics of gliomas and the tumor microenvironment will be considered comprehensively through the combination of As2O3 prodrug, Angiopep-2 and zwitterionic oligopeptide to structure the synergy-targeted liposomes. Our synergistic targeting strategy will effectively assistant As2O3 to overcome multiple barriers, actively target glioma and precisely release in the cytoplasm of cancer cells as well as quantitative monitor the drug release. In addition, we will build patient derived glioma xenografts model and further using in vitro blood-brain barrier and glioma model and gene knockout model to study the transport mechanisms and evaluate the drug delivery system in vivo and in vitro from the molecular, cellular and animal levels. Our project will provide initial strategy and methods for the precise targeting and drug release monitoring Traditional Chinese Medicine in brain glioma.

中药砒霜有效成分三氧化二砷（As2O3）对脑胶质瘤有良好疗效，但因其分布无特异性，毒性大、治疗窗窄等原因限制其临床应用。经文献及前期研究发现，靶向递药系统受多重机体屏障阻碍仍难以将As2O3准确递送至脑胶质瘤细胞内，因此本项目提出整体考虑As2O3理化性质、机体屏障及脑胶质瘤微环境特点的新型靶向策略，构建兼具“协同靶向”和释药监测的脑胶质瘤递药系统，使As2O3前药、两性寡肽修饰脂质体与Angiopep-2靶头在递药系统透血脑屏障，多级pH响应靶向脑胶质瘤细胞以及前药溶酶体逃逸全过程协同作用实现肿瘤细胞内定位释药与释药监测。本项目将构建患者来源脑胶质瘤移植瘤模型、体外血脑屏障-三维脑胶质瘤细胞模型与CRISPR/cas9技术构建转基因细胞模型，从动物-细胞-分子三个水平研究载体协同靶向、胞内释药及转运机理。本项目的实施将为实现中药的脑胶质瘤胞内递送和释药监测提供新策略与新方法。

脑胶质瘤是一种常见的原发性恶性肿瘤，化疗效果因为血脑屏障的低通透性而受到阻碍。三氧化二砷(ATO)是中药砒霜的有效成分，能抑制脑胶质瘤细胞的生长并诱导细胞凋亡，但其穿透血脑屏障的能力差，体内分布无特异性，毒性大，治疗窗窄，限制了在治疗脑胶质瘤方面的应用。本项目通过氢化大豆磷脂包载亚砷酸钠溶液，将高浓度锰离子（Mn2+）被动包裹脂质体内部，使亚砷酸根离子主动且快速地载入脂质体内部并与Mn2+结合，使砷元素“固定”在脂质体内，表面修饰Angiopep-2 和两性寡肽，构建了angiopep-2修饰砷锰复合物脂质体纳米粒（LP@MnAsx），表征LP@MnAsx及其相关中间产物的结果和特征，结果显示LP@MnAsx可明显减小药物漏释现象，且具有显著的缓慢释放和pH响应突释效应。药动学实验表明，LP@MnAsx显著减缓了药物的血浆清除，提高了药物在体内的循环时间。细胞毒性研究表明，空白脂质体具有较好的安全性，砷锰复合物脂质体低浓度时对脑胶质瘤细胞既有较强的选择杀伤作用。细胞摄取动力学与入胞机制研究表明，LP@MnAsx的摄取需要消耗能量；体外研究显示angiopep-2介导脂质体跨BBB，且增强C6细胞摄取。药效学研究表明，LP@MnAsx可以使砷元素聚集在肿瘤部位，从而改善砷元素在体内的分布，抑制肿瘤细胞增殖和诱导肿瘤细胞凋亡的同时降低对正常细胞的神经毒性，最终达到增效减毒的目的。综上本项目所构建砷基纳米递药系统能够改善ATO体内分布，延长病灶部位滞留时间，达到增效减毒的目的，为中药靶向脑胶质瘤递药系统的构建提供了新的思路与实验依据。课题研究期间本课题组成员共发表相关学术论文14篇，其中SCI论文10篇（Top期刊5篇），在国内一级期刊发表论文2篇；申请国内专利2项，授权发明专利2项；培养与联合培养研究生7名。已基本完成课题申请时设定的研究目标。