程序化癌细胞和线粒体双重靶向纳米载体用于提高青蒿素类药物的抗肿瘤性能

Up to today, several hundreds of nanoparticle-based formulations have been investigated in different stages of clinical trial, and some of them have been approved and successfully applied in clinical cancer therapy. Although the improvement of patient safety and decrease of toxic side effects have been realized in these nanoformulations, many of these strategies fail to overcome various biological barriers in cancer treatment. Both increasing chemotherapeutics concentration and improving anticancer efficacy of chemotherapeutics in cancer cells are important in the current development of novel cancer nanomedicines. Active cellular targeting and subcellular targeting, especially mitochondrial targeting, are two well-known strategies in improving anticancer efficacies of nanomedicines. However, simple combination of cellular targeting ligand and mitochondrial targeting ligand, such as TPP with highly positive charge, in one nanocarrier, cannot often lead to success in synergetic enhancement of anticancer ability in vivo. In this project, on the basis of our previous work in the construction of redox-triggered activation of nanocarriers for mitochondria-targeting cancer chemotherapy, we develop programmed cellular and mitochondrial dual-targeting nanomedicine for precise cancer chemotherapy. The triple functions, including passive targeting, active targeting and mitochondrial targeting are integrated in one nanocarrier, can play a role successively. There is no interfere each other. Artemisinin and its derivatives, drugs with moderate anticancer ability, will be then encapsulated in the dual-targeting nanocarrier and used in cancer therapy. It is expected that the anticancer efficacies of programmed dual-targeting artemisinin nanoformulation will be greatly improved. We will also systematically evaluate the anticancer mechanism, possible resistance of artemisinin nanoformulation in cancer therapy, and further explore the mechanism of resistance and the strategy for overcoming the resistance.

临床上批准的纳米化疗剂尽管在减少毒副作用、改善病人生活质量等方面取得显著进步，但是通常不能克服生物体内的多重屏障，仅通过被动靶向增加药物在肿瘤组织中的富集，只能有限提高体内抗肿瘤效果。构建主动靶向纳米载体增加肿瘤细胞内药物浓度、设计肿瘤细胞内亚细胞靶向特别是线粒体靶向纳米载体是提高药效的两种重要策略。简单地将两种靶向基团集成到一个纳米载体上，往往达不到提高疗效的目的。本项申请在前期构建肿瘤细胞活化线粒体靶向纳米载体的基础上，提出程序化肿瘤细胞和线粒体双重靶向纳米载体的新策略，整合被动靶向、细胞靶向和线粒体靶向三种功能，依次发挥作用，互不干扰，预期可获得更好的体内疗效。在此基础上研发负载青蒿素及其衍生物的纳米药物，系统研究双靶向青蒿素纳米药物的体内外抗肿瘤性能、作用机制。探索双靶向青蒿素纳米药物可能存在的耐药性、耐药机制和克服耐药性的方法。开发具有自主知识产权和应用前景的青蒿素纳米新剂型。

为了发展新的癌症治疗方法，本项目分别在肿瘤组织和肿瘤细胞内活化的线粒体靶向纳米药物的制备，提高抗肿瘤效果、克服肿瘤耐药和增强肿瘤免疫治疗等方面进行了系统的研究。主要包括：（1）构建胞内可活化的线粒体靶向纳米载体显著提高青蒿素对多种癌症细胞的毒性。发现癌细胞经线粒体靶向青蒿素（ART）处理后，在线粒体内持续产生有毒的ROS，损伤线粒体，诱导癌细胞死亡。（2）虽然线粒体靶向青蒿素单药具有良好的体内外抗肿瘤效果，然而，ART在达到一定浓度后，细胞毒性几乎不再增强。为逆转癌细胞对ART治疗产生的抵抗，构建了线粒体靶向ART/GPX4抑制剂((1S,3R)-RSL3)组合纳米药物。发现线粒体靶向共输送RSL3/ART对癌细胞毒性表现协同作用。RSL3在线粒体中与GPX4特异性结合使其失活，导致MCF-7细胞发生铁死亡，也降低了细胞通过GPX4对自由基或ROS的抵抗。在体内外均表现出优异的抗肿瘤效果。（3）考虑到青蒿素激活的线粒体自噬不利于青蒿素发挥抗癌作用，使用莲心碱阻断线粒体自噬下游通路，抑制自噬体与溶酶体的融合抑制线粒体自噬，导致自噬积累。通过抑制线粒体自噬提高线粒体靶向青蒿素抗肿瘤效果。莲心碱增加了MCF-7细胞对青蒿素的药物敏感性。(4) 发展肿瘤酸度活化的线粒体靶向纳米载体克服肿瘤耐药性。利用聚乙烯亚胺和2,3-二甲基马来酸酐合成了肿瘤酸度触发电荷反转的阴离子聚合物，通过静电相互作用吸附在正电性载阿霉素纳米粒子表面。在弱酸性的肿瘤部位发生酰胺键水解，转变为正电荷的PEI，与TPP之间失去了静电相互作用而脱离，从而暴露出内层正电荷，增加了耐药细胞MCF-7/ADR的摄取。进入细胞后，一方面避免耐药细胞对DOX的泵出，另一方面又能够将DOX靶向输送到线粒体中，在细胞水平和整体动物水平均表现出克服MCF-7/ADR耐药的能力。（5） 利用肿瘤酸度响应电荷反转载阿霉素纳米粒子调控免疫下调微环境，提高癌症免疫治疗效果。我们发现肿瘤酸度响应电荷反转载药纳米粒子到达肿瘤组织后，一方面导致癌细胞发生免疫原性细胞死亡；另一方面下调肿瘤微环境中的一些免疫抑制细胞（M2巨噬细胞，MDSCs，Tregs细胞等），降低肿瘤组织中的免疫抑制细胞群数量，还能将部分M2巨噬细胞再极化为M1巨噬细胞，改善肿瘤微环境的免疫抑制状态，并联合PDL1抗体获得良好的的抗肿瘤效果。

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