肽修饰的多功能仿生纳米颗粒诱导选择性自噬降解肿瘤细胞中突变p53蛋白的研究

The gene encoding p53 (TP53) was characterized as a one of the most important tumor suppressor. More than 50% of all human cancers involve a mutant p53 gene. In fact, mutant p53 protein was often high expressed in cancer cells and difficult to degrade, not only abrogates p53 tumor-suppressive functions, but in some instances can also endow mutant proteins with novel activities to promote the development of cancer (Gain of function). Selective autophagy is a biological degradative process through which cellular components are recognized by autophagy receptors and delivered into autophagosome to degradation. Based on this biological principles of selective autophagy and the rare earth surface coating peptide RE-1 we have previously discovered and published on Nature Materials, we have designed and synthesized a multifunctional biomimetic nanoparticles. For this nanoparticle, RGD peptide, the mutant p53 protein binding peptide and the LC3 protein binding peptide were self-assembled onto the surface of gadolinium oxide nanoparticles with RE-1 as a linker, respectively, so that the nanopaticles will have multifunctions of tumor cells targeting, endocytosis, autophagy induction and simulating selectivity autophagy receptor protein to deliver mutant p53 protein into the autophagosome degradation. In our preliminary work, we found a good autophagic dependent degradation effect on the mutant p53 protein by biomimetic nanoparticles modified with functional peptides. In this project, we will further optimize the structure and function of the above biomimetic nanoparticles and assess its ability to degrade a variety of different mutant p53. In addition, we will also elucidate the molecular mechanism underlying the selective autophagy and clearance of mutant p53 protein by biomimetic nanoparticles, and verify the potential of tumor treatment through mutant p53 clearance in both cell and tumor-bearing mouse model. Successful implementation of this project may have implications and provide a new strategy for developing nanomaterial-based therapeutics targeting mutant p53 for tumor precise treatment.

p53是最重要的抑癌基因之一，超过50%人类肿瘤携带TP53基因突变。突变p53在癌细胞中高表达难以降解，不仅丧失了抑癌能力还产生促进癌症的新功能。选择性自噬是通过特异性受体蛋白将待降解蛋白带入自噬途径选择性降解的生物学过程。基于这一原理及我们发现的稀土表面涂层肽RE-1(Nature Materials)，设计合成了一种多功能仿生纳米颗粒，以RE-1为Linker将RGD、突变p53结合肽、LC3结合肽自组装到纳米氧化轧表面，从而将肿瘤靶向入胞、诱导自噬、模拟受体蛋白将突变p53带到自噬体中降解这几种功能合为一体，初步结果显示对突变p53的自噬依赖性降解效应。本项目将对该颗粒进行结构和功能优化，评估其对多种不同突变p53的降解能力，阐明其诱导选择性自噬的分子机制，在细胞及荷瘤小鼠中验证其通过清除突变p53实现肿瘤治疗的可行性。有望为发展以突变p53为靶点的新型肿瘤精准治疗药物提供新策略。

本项目自启动以来，紧密围绕课题任务书的要求以及结合目前国内外的研究进展，开展了以锌基纳米材料调控突变p53稳定性为主要目标的相关研究。在此项目的资助下，课题任务按照计划执行并顺利完成，发表SCI论文6篇，申请国家发明专利2项，PCT 1项，授权一项。培养博士研究生1名，硕士研究生5名。在本项目的资助下，我们主要围绕降解肿瘤细胞中的突变p53蛋白为核心以及针对项目书的研究目标和拟解决的关键科学问题，我们按照研究计划较好地完成了预期研究成果。首先我们采用具有独特生物活性的工程化纳米材料策略来调控突变型p53的稳定性。通过优化锌和铁的比例，研发出一种锌铁比例为1比2的锌掺杂氧化铁纳米颗粒，能够在肿瘤细胞内酸性溶酶体中释放锌离子，介导蛋白酶体依赖的突变型p53蛋白降解作用，逆转突变p53的获得性功能，诱导肿瘤细胞的死亡。研究结果显示，锌铁纳米粒能显著提高突变p53肿瘤细胞对化疗药物的敏感性，并在人卵巢癌以及病人来源的乳腺癌等突变p53肿瘤模型验证了抗肿瘤活性，为实现突变p53肿瘤的诊疗一体提供新思路新方法。此外，我们发现并验证具有响应pH变化释放锌离子的多孔的ZIF-8纳米材料具备广谱的降解突变p53的能力，并对其降解机制进行了研究。证明了锌离子升高后引发的突变p53发生谷胱甘肽化修饰是其通过泛素-蛋白酶体途径降解的关键步骤。该研究一方面利用多肽对ZIF-8纳米材料进行改造，提高了ZIF-8纳米材料在肿瘤微环境的稳定性，增强了材料的肿瘤靶向性，显著改进了ZIF-8材料降解突变p53的效应，在增强ZIF-8材料治疗肿瘤效果的同时大大提高了其稳定性和生物安全性。为以化疗药物联合“突变p53靶点”的药物装载、改造和靶向输运提供了借鉴和依据。另一方面该研究证明了锌离子降解突变p53的广谱性并对其降解通路进行了研究，为将来基于降解突变p53治疗肿瘤的理论研究和药物研发提供了支撑和依据。