磁控释一氧化氮靶向纳米诊疗体系的构建及一氧化氮/光热联合治疗三阴乳腺癌作用机制研究

Nitric oxide (NO) is a green and safe active molecule, which has attracted great attention in cancer therapy. However, gaseous NO faces drawbacks of hard for storage, burst release and easy clearance in biological environment, which largely impeded its translation in clinical medicine. In the present project, we will design and synthesize multifunctional nanomaterials with targeting ability and magnetic field remote controlled NO release properties. The resultant nanomaterials can precisely deliver NO to the tumor tissue and control NO release on demand, which is supposed to have significant inhibitory effects on triple negative breast cancer. On this purpose, we will first fabricate brush-like block polymer coated magnetic nanoparticles. Then β-galactosidase (abbreviated for E) and NO prodrug (abbreviated for S) are loaded into the brush-like block polymer layer of the magnetic nanoparticles, followed by functionalization with AE105 peptide which can specifically target triple negative breast cancer cell, obtaining targeting nanosystem E and S, respectively. After intravenously injected, both nanosystem E and S can deliver directly to the tumor tissue with the aid of target AE105 peptide. When an external magnetic field is applied, nanosystem E and S located in the tumor site will interact strongly with each other to compress the protective polymer layer on nanoparticles' surface. Subseqently, enzyme catalytic reaction between β-galactosidase and NO prodrug is triggered to release NO. Moreover, the prepared theranostic nanomaterials can be used for magnetic resonance and photoacoustic dual-model imaging of triple negative breast cancer, and exert NO/photothermal synergistic anticancer effects. Finally, the mechanism regarding the synergistic NO/photothermal anticancer activity will be elucidated. The findings in this project will provide a novel strategy for cancer diagnosis and therapy with high efficiency.

一氧化氮（NO）作为绿色安全的气体疗法活性分子，近年来在肿瘤治疗领域受到广泛关注。然而，在生物环境中，NO存在难储存、易释放和易被清除等缺陷，极大限制了其在临床医学的转化应用。本课题拟结合酶催化和磁控释作用，设计合成具有磁控释NO性能的靶向纳米材料，实现NO在肿瘤病灶的精准递送和可控释放，用于三阴乳腺癌的高效诊疗。本课题拟以刷状聚合物修饰的磁性纳米材料为载体，将β-半乳糖苷酶（E）和酶响应的NO前药（S）分别载入磁性纳米材料中，并利用靶向多肽AE105对其进行修饰，获得靶向纳米体系E和S。在纳米体系E和S通过靶向作用到达肿瘤组织后，通过外加磁场的作用，促使纳米体系E和S发生充分接触融合，并触发酶催化NO前药反应释放NO气体。同时，本课题研究磁控释靶向纳米体系对三阴乳腺癌的磁共振/光声双模态成像诊断及NO/光热联合治疗效果，并阐明其具体的联合抗肿瘤作用机制，为肿瘤的精准诊疗提供科学依据。

该项目主要针对NO气体存在难储存、易释放和易被清除等缺陷，通过利用磁控释手段，设计合成具有磁控释NO性能的靶向纳米材料，实现NO在肿瘤病灶的精准递送和按需可控释放。基于纳米载体兼具优异的磁性及其在近红外区域的光学性能，在外加磁场和近红外激光的双重作用下，实现对三阴乳腺癌的NO/光热联合高效治疗，并阐明其NO/光热联合抗肿瘤作用机制。同时，研究纳米体系对三阴乳腺癌的磁共振/光声双模态高灵敏成像诊断性能，从而为肿瘤的精准诊疗提供新思路及科学依据。在项目执行过程中，项目负责人设计合成了具有超顺磁特性的有机/无机杂化材料，并将其作为载体材料实现了对NO气体的高效负载和磁控释放。研究表明，NO可以显著提升三阴乳腺癌细胞对光热效应的敏感性，进一步增强光热抗肿瘤效果。机制研究表明，光热/NO联合作用一方面通过破坏细胞线粒体，导致细胞色素C的释放引起TNBC细胞的凋亡；另一方面，通过下调Bcl-2蛋白表达，上调Bax蛋白表达，从而引起凋亡相关蛋白Caspase3和Caspase9的表达，最终导致TNBC细胞的凋亡。此外，进一步的研究发现，NO在协同增效光动力治疗、烷基自由基治疗、酶动力治疗等疗法的疗效方面也具有显著的效果。(1) NO具有协同增效烷基自由基抑制乳腺癌作用，NO/烷基自由基的协同抗肿瘤作用主要通过介导线粒体的凋亡途径来实现：一方面，释放的NO/烷基自由基联合作用于线粒体产生严重的氧化应激，造成线粒体膜的严重破坏，最终导致细胞凋亡。另一方面，NO/烷基自由基协同作用直接引起Bax的上调，以及Bcl-2蛋白的下调，随后触发凋亡相关蛋白Caspase-3和Caspase-9的激增，导致细胞凋亡。(2) NO具有协同增效酶动力抗肿瘤作用，NO协同增效EDT的抗肿瘤作用很可能通过破坏溶酶体的功能，阻断线粒体自噬体与溶酶体的融合来抑制线粒体的自噬作用，进而抑制相关蛋白的表达，最终触发线粒体凋亡相关的通路来实现。(3) NO在协同增效光热抗菌和修复方面效果显著。该项目启动至今，发表Advanced Functional Materials, Chemical Engineering Journal, Chemistry of Materials等高水平SCI论文8篇，以第一发明人获授权发明专利6件，申请发明专利3件。