肿瘤微环境双重响应的多肽类智能诊疗探针模块化设计、合成及其应用研究

For nano cancer diagnosis and therapy, new strategies need to be developed in terms of design of the theranostic probe and intelligent imaging. The proposed research targets at the effectiveness of cancer diagnosis and therapy by emphasizing on bio-safety and intelligent probe design based on our preliminary experimental results. Our approach combines multifunctional peptide, Au3+, Gd3+, and Doxorubicin for developing highly efficient theranostic probes. Specific functional sequence of peptide will be identified and selected for assembling of the systems that are capable of cell targeting, imaging, therapy, and response to tumor environmental changes. The goal of the research is to establish an entirely new theranostic system with high bio-safety, imaging sensitivity, and targeting membrane penetration, based on which, the continuous, dynamic, multimodal, non-invasive tumor imaging-therapeutic system can be realized. With such a system, the theranostic probe can be stimulated by enzym in micro-environment when circulating through the tumor lesions. In this way, the targeting and therapeutic modules will disassemble from the probe leading to rapid reduction in molecular weight. This sequential reaction will facilitate permeation of targeting and therapeutic components into the tumor. Under acidolysis, Doxorubicin dissociates into the original compound for the most effective treatment of tumor. This project proposes a new strategy in developing a multi-modal imaging probe with efficient therapeutic means. By modulating the system with a novel design, all components, including peptide, Au3+, Gd3+, and Doxorubicin, can be assembled in an intelligent fashion for precision delivery and release of drugs without any side effects. The development of this theranostic system will provide a new theoretical and experimental base for accurate cancer diagnosis, monitoring, and effective treatment.

设计、制备智能显像和治疗一体化探针被视为肿瘤诊疗的新策略。本项目围绕提高肿瘤诊疗效果这一中心目标，抓住分子探针智能设计和生物安全性等重点，基于前期研究基础，拟采用生物相容性好的功能多肽、Au3+、Gd3+和阿霉素等合成高效智能荧光/MRI诊疗探针。设计和筛选特定功能序列的多肽模块化可控合成显像、靶向&治疗和肿瘤微环境响应组件。构建全新模式的生物安全、显像灵敏和靶向穿膜性强的新型智能诊疗探针；实现对肿瘤非侵入性的连续动态双模态显像。并在此基础上，血液长循环的诊疗探针经肿瘤微环境酶刺激，靶向&治疗组件将脱离，分子量骤减，有助于靶向&治疗组件瘤内渗透，且在瘤内酸解作用下，阿霉素前药快速分解成原药以发挥抗肿瘤活性。本项目利用模块化多肽和双重生物响应等策略，发展肿瘤双模态灵敏显像和药物高效智能递送精准治疗，降低全身副反应，为恶性肿瘤分子水平的动态精确诊断、治疗及实时监测提供新的理论和实验依据。

敏感、特异和安全的双（多）模态分子探针已成为当今肿瘤早期影像学诊断的核心问题与迫切需要。可利用探针的特异性和高灵敏性，实现肿瘤病灶的定向富集，从而更加精确地进行早期诊断，进一步利用肿瘤部位的微环境响应性实现药物的智能投递，达到诊疗一体化的目的。尽管双模态纳米探针研究已经取得了一定的进展，但其诊疗效果还有待提高；且合成的原材料毒性较大，合成方法繁琐，医用性较差。针对这些问题，本项目重点研究了双/多模态探针的合成方法，并着实提升了所合成探针的肿瘤成像和治疗效果。本项目首先设计和制备了模块化多肽，利用各模块氨基酸序列，实现了探针指定位置的仿生合成，获得了性能优异的荧光/MR双模态影像探针。数据显示该探针具有超小尺寸，具有深红色荧光，且弛豫率可达3倍于临床钆剂；肿瘤增强效果显著。接着，依据肿瘤微环境碱性磷酸酶（ALP）和半胱氨酸蛋白酶-3（Caspase-3）高表达特征，设计和制备了可酶切激活的肿瘤靶向多模块多肽诊疗分子，实现了在肿瘤组织中特异性增强显像和增效治疗的目的。成像模块可在肿瘤微环境中被点亮，治疗模块可在肿瘤组织中发挥长效作用。基于模块化多肽策略，相比传统化学合成方法，我们较容易获得了肿瘤微环境特异性响应的高质量诊疗探针，且由于所使用的原材料生物相容性好，所制备的探针医用性大大增强。另外，受多肽合成的启发，我们尝试完成了从多肽到蛋白仿生合成高质量分子探针的研究。数据显示蛋白丰富的氨基、羧基或巯基等活性残基的氨基酸，可与多种显像或治疗性金属阳离子、阴离子或其他小分子物质结合，再在还原性的芳香族氨基酸（如W和Y）作用下，进行原位生物模拟仿生合成。基于蛋白策略，我们实现了系列单/双/多模态影像探针或诊疗探针；提出了“蛋白仿生集成”概念。本项目通过一系列体外实验和肿瘤动物模型验证了该类探针的可靠性。在项目实施过程中发表SCI收录论文22篇；申报专利3项，其中已授权1项；主编英文专著一部；获得奖励2项；超额完成了预期目标。