共轭聚合物拉曼探针及其在高分辨多色拉曼成像中的应用

As a superb tool to visualize and study the spatial-temporal distribution of chemicals, Raman imaging has attracted great attention in biomedicine. Development of Raman-active materials with enhanced and distinctive Raman vibrations in the cell-silent Raman window (1800-2800 cm-1) is of great importance to Raman imaging of living cells with high spacial resolutions. Poly(deca-4,6-diyunedioic acid), or PDDA, is a water-soluble conjugated polymer, the carboxyl groups on the side chains on which are chemically reactive and can be modified to different functional groups. Strikingly, the Raman spectrum of PDDA shows a strong and narrow characteristic peak of alkyne stretching at 2100 cm-1. The Raman intensity is 1000 times stronger than alkyne-containing small molecules, due to synergistic enhancement by the perfectly planar backbone of PDDA. For our proposed work, we will study how molecular weight and chemical environment will affect the Raman frequency and intensity of alkynes in PDDA, and develop methods to modulate signal-to-noise ratio and multicolor properties of PDDA as Raman probes. We will also prepare a series of PDDA derivatives, through the side chain modifications, with enhanced cell penetrating and subcellular organelle-targeting capabilities as bioorthogonal Raman reporters. These Raman reporters will consequently be integrated as multicolor Raman probes through various construction strategies, including self-assembly, nano encapsulation, etc. In addition, we will apply the prepared Raman probes in different Raman imaging techniques, such as spontaneous Raman scattering, surface-enhanced Raman scattering, and stimulated Raman scattering, to achieve high-resolution multiplex Raman imaging of living cells at subcellular level.

发展能在细胞拉曼沉默区（1800-2800cm-1）有高强度特征峰的拉曼探针，对实现活细胞的高分辨多色拉曼成像至关重要。聚4,6-二亚炔癸二酸（PDDA）是我们前期合成的一种水溶性好且侧链可修饰的共轭聚合物，由于PDDA主链共平面结构的协同增强效应，PDDA在2100cm-1左右有超高强度的C≡C拉曼特征峰(可达炔类小分子探针强度的1000倍)。本项目将在此基础上，通过对PDDA侧链的化学修饰制备一系列具有细胞器靶向性、环境响应性等功能的PDDA衍生物作为拉曼信号报告分子，并将这些报告分子构建为拉曼成像探针；研究拉曼特征峰强度和频移随不同PDDA分子量、侧链修饰基团结构、修饰度以及组装/解组装行为等的变化关系，并探索如何通过结构与环境变化调控此类拉曼探针的信噪比和多色性；将所制得拉曼探针应用于活细胞成像，获得亚细胞水平的高分辨多色拉曼成像，为推动拉曼成像在生物医学等领域的应用提供有力支持。

如何发展高效安全的拉曼成像探针是促进拉曼成像在生物医学中应用与临床转化的主要挑战。本项目设计制备了一系列多种功能性官能基团，与聚4, 6-二亚炔癸二酸（PDDA）反应，获得了一系列拉曼信号报告分子。并考察了取代基团与取代度等因素对拉曼峰峰强与频移的影响，以及PDDA中C≡C拉曼峰峰强和频移随溶剂、水溶液pH值、分子量等的变化关系。本项目通过对PDDA主链中C≡C基团的精准13C同位素掺杂，实现了拉曼峰峰强与频移的连续调控。在此基础上，将所制得的拉曼成像分子探针应用于细胞拉曼成像，实现了亚细胞水平的高效活细胞细胞器靶向拉曼成像。本项目还基于主客体模板聚合法构建了侧链为单个碘原子的聚1，4-二碘代二炔（PIDA），PIDA在保留了聚二炔材料高拉曼强度特性的同时，还兼具超强的X射线吸收，进而可通过PIDA进行高效CT-拉曼双模态成像，将PIDA应用于肿瘤手术辅助，实现了术前规划与术中导航。此外，针对PDDA在生物医学应用中所面临的安全性问题，本项目系统研究了PDDA的可控降解性，PDDA在活性氧条件下可完全氧化降解至小分子丁二酸。PDDA还可与天然普鲁兰糖交联，得到PPG载药水凝胶，将光敏剂与肿瘤免疫治疗抗体aCD47载入PPG，应用于肿瘤术后，可实现光动力治疗和免疫治疗的高效协同，有效防止了肿瘤的复发与转移。本项目还制备了PDDA与普鲁兰糖的纳米凝胶，用于药物在小鼠原位脑胶质瘤的深部富集。这些凝胶的降解和药物释放可通过PDDA的拉曼信号进行示踪，促进了拉曼可视化诊疗体系的构建与发展。在本项目的支持下，申请人在国内外高水平期刊等发表论文11篇，授权3项中国发明专利，申请一项PCT国际专利，培养博士后3名，博士生2名，硕士生5名。