新型生物正交核医学-光学双模态靶向探针体系的构建及成像研究

Nuclear imaging is playing a crucial role in early precise diagnosis of cancer. However, identification of tumor margins remains difficult during resection operation. Combination of diagnosis and intraoperative image-guidance by dual-modal imaging fused with optical imaging will probably solve this problem. Despite high specificity and affinity of antibody-based dual-modal imaging probes, radionuclides with longer half-life is usually needed to match the long circulation time of the labeled antibodies, resulting in more radiation dose and more waiting time before resection operation. This research intends to realize pre-targeted dual-modal imaging of tumors through highly efficient tetrazine bioorthogonal reaction. Radionuclides with shorter half-life will be used to label a novel fluorogenic scaffold containing 1,2,4,5-tetrazine, antibodies will be modified with dienophiles. These two parts of entities will compose the intended universal pre-targeted probe system, and will be used for dual-modal imaging study in cancer models including breast cancer. Moreover, the fluorogenic scaffold displayed promising two-photon fluorescence properties, which will be tested for its potential application in dual-modal imaging and intraoperative image-guidance. The main purpose of this research is to significantly reduce the radiation dose of antibody-based dual-modal imaging, further improve the signal-to-noise ratio by pre-targeting strategy and fluorogenic properties, resulting in general improvement of dual-modal imaging and promotion of basic research on the integration of diagnosis and treatment.

核医学成像在肿瘤的早期精准诊断中发挥着越来越重要的作用，但在手术切除过程中对肿瘤边界的判断仍然困难，与光学成像融合的双模态成像可弥补这一不足，将术前诊断与术中导航有机结合。基于抗体的双模态探针具有高特异性和亲和力，但为匹配抗体较长的体内循环时间则需使用较长半衰期的放射性核素，增加了辐射剂量和术前等待时间。本研究拟使用高效的四嗪生物正交反应，以实现肿瘤的预靶向双模态成像。利用短半衰期核素标记一类包含四嗪结构的新型荧光生成骨架，搭配亲二烯体修饰的抗体，构建通用靶向探针体系，并对乳腺癌等肿瘤模型进行双模态成像研究。此外，所使用的荧光生成骨架具有双光子荧光性质，将探索双光子成像在双模态成像和术中导航中的潜在应用。本研究主要目的在于显著降低抗体类双模态成像的辐射剂量，借助预靶向策略和荧光生成性质进一步提升成像的信噪比，以期总体改进双模态成像效果，推动诊疗一体化的基础研究。

本研究首先对四嗪荧光生成骨架尝试进行修饰以进行放射性标记，但前期实验结果显示该骨架对18F标记的兼容性较差，后转为尝试金属螯合剂的偶联，以便于68Ga等放射性金属核素的标记；所利用的四嗪荧光生成骨架与亲二烯体TCO的反应速率快，但存在后续缓慢的氧化过程，显著影响荧光开启，我们对该氧化过程进行了详细研究并尝试对其进行优化和调控。此外利用一类新型四嗪生物正交反应，实现了对多种经典荧光基团的有效淬灭，与异腈发生生物正交断键反应后荧光开启，可用于活细胞成像；又通过核酸模板化学在低浓度条件下实现了显著加速的荧光增强，对miR-10b的检测具有很好的特异性、化学选择性和稳定性；此外还开发了一组全新的相互正交的生物正交断键反应，并首次报道了两种生物正交断键反应及荧光增强在活细胞内的同时进行。

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