植物源性双配体阿霉素纳米药物应用于胶质瘤治疗的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive form of malignant primary brain tumors, stands out as one of the world’s most devastating diseases. Limited choices in therapeutic drugs contribute to the modest treatment outcomes of patients suffering GBM. Poor efficiency of drug delivery to glioma significantly restricts the application of many potent therapeutic agents due to several physiologic barriers including blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). Therefore, it is important to explore drug delivery system to bypass BBB and BBTB to improve current GBM therapeutic situation. Plant-derived exosomes are nanocarrier with a large-scale producation, biocompatibility, brain delivery ability, and appilication potential. We previously conducted the isolation of exosomes derived from plant and developed nanoparticles with dual-ligands for inhibiting angiogenesis and vasculogenic mimicry of glioma. Dual peptides cRGD and SWL, which has high affinity with αvβ3 integrin and EphA2 overexpressed in giloma, enable to inhibit endothelium blood vessels and vasculogenic mimicry of glioma, respectively. In this study, plant-derived exosomes will be modified by cRGD and SWL thereby encapsulating doxorubicin (DOX) to fabricate dual-ligand DOX nanomedicine derived from plant. It is expected to bypass BBB and target glioma thereby suppressing glioma cells by assistance of dual-ligand. The isolation procedure of plant-derived exosomes and preparation method of nanomedicine are optimized. In vitro experiments of nanomedicine is conducted and in vivo experiments of nanomedicine is eveluated at intracranial glioma cells xenograft models using intravenous and intranasal injection. At last, we further investigate nanomedicine mechanism and the initial dosage form of nanomedicine. It is expected to obtain an effective, low cast and potential nanomedicine for inhibiting glioma. The strategy enables to design drug delivery system for other cancer.

胶质瘤临床死亡率极高，有效化疗药不多，血脑屏障严重限制传统化疗药的实际应用。因此，极有必要研究穿越血脑屏障又靶向胶质瘤的纳米载药系统。植物源性外泌体是一种易获得、有生物相容性、有脑部递药特性、极具应用前景的纳米载体。我们前期初步开展植物外泌体提取分离，并研制抑制胶质瘤血管生成和血管拟态的双配体纳米粒子。本项目拟利用受体在胶质瘤高表达、对胶质瘤血管生成和血管拟态有抑制作用的双配体（cRGD肽和SWL肽），修饰植物外泌体，再物理包裹阿霉素，构建能穿越血脑屏障、高度靶向胶质瘤的双配体阿霉素纳米药物，并通过双配体进一步增强药物对胶质瘤的杀伤作用。然后，优化载体提取工艺和纳米药物制备工艺，考察其体外生物活性，利用原位胶质瘤动物模型进行鼻内给药和静脉给药体内实验。最后，进一步探索药物作用机制及药物初始剂型，从而获得一种低成本、高效、有应用前景的抗胶质瘤纳米药物。该体系可普适化其他类型肿瘤的纳米药物开发

胶质瘤是最常见的中枢神经系统恶性肿瘤，化疗是常用的治疗手段。尽管一线化疗药物对胶质瘤细胞有较好杀伤作用，但血脑屏障/血脑肿瘤屏障（BBB/BBTB）的存在限制了它们在胶质瘤的应用。植物来源的细胞外囊泡（Extracellular vesicles, EVs）具有磷脂双分子层结构，能够穿越多种生理屏障深层次穿透组织的能力，且植物来源的EVs提取方便、产量高、成本低、生物相容性好，因此植物EVs作为药物载体，应用于胶质瘤药物开发展现出巨大的应用前景。尽管如此，细胞外囊泡载药效率低且没有靶向修饰，是其应用的瓶颈。相比较而言人工合成的纳米粒子能够实现表面靶向修饰、载药量高，可携带化疗药物用于肿瘤治疗，然而人工合成的纳米粒子穿越血脑屏障效率极低，限制了它们在胶质瘤的药物开发。基于此，我们应用西柚来源的EVs携带肝素为载体的阿霉素（DOX）酸敏纳米粒子（DNs），构建阿霉素纳米粒子与西柚细胞外囊泡的联合复合物（EV-DNs）开发其用于胶质瘤治疗的研究，其中DNs是以肝素为骨架，连接胶质瘤高表达受体的配体cRGD肽，并通过腙键连接阿霉素制备而成。.我们成功提取具有明显的脂质双层膜结构、纳米粒径的西柚EVs，研究表明其在模拟血清环境下有较好的稳定性，且对胶质瘤细胞几乎没有毒性。我们制备了三种不同配比的EV-DNs（EV-DN1、EV-DN2和EV-DN3），通过物化性能表征筛选出EV-DN2为最佳剂型，经检测表明EV-DN2载药效率高于传统载药方法至少4倍以上。研究发现经DNs修饰后的EV-DN2具有酸敏感特性，且具有抑制补体激活的特性。体外BBB模型实验表明，EV-DN2能够更高效地穿过BBB并被胶质瘤细胞的摄取；同时EV-DNs能够有效杀伤胶质瘤细胞。体内实验表明，相比较EVs，EV-DN2延长了体内的循环时间，并能穿过BBB/BBTB高效蓄积在原位胶质瘤部位。体内抑制实验表明，与DOX和DNs相比，EV-DN2显著减小原位胶质瘤荷瘤裸鼠瘤体体积，并明显延长其生存期。本课题我们通过新颖的设计方法成功构建了仿生复合物EV-DNs，该方法大大提高了EVs的载药效率，并能高效穿透BBB/BBTB靶向胶质瘤组织，从而有效发挥抗胶质瘤作用，具有较好的应用前景。