关键过渡金属离子的活体荧光成像探针设计与应用研究

Comprehensive understanding of the physiological roles of essential transition metal cation demands still the in situ homeostasis informations of the transition metal cations especially in mammalian model such as mice. However, the in vivo fluorescence imaging for transition metal cations in mammalian model is still challenging. The individual and tissue differences lead to the deviations in photon attenuation and local microenvironment and sensor concentration, which interfere with the detected fluorescence intensity and concealed the signals for transition metal cations. On the other hand, there is no suitable sensor of high tissue penetration and fine selectivity for specific transition metal cation. In this proposal, the ratiometric NIR fluorescent sensors for essential transition metal cations will be constructed from the organic NIR fluorophore. With these sensors, the dual channel ratiometric in vivo imaging, instead of the normal intensity-based turn-on imaging, will be developed to realize the in vivo imaging for transition metal cations in mammalian model i.e. mice. The strategy of integration of organic NIR fluorophore with the chelator for specific transition metal cations should be helpful to form the ratiometric NIR fluorescent sensor of high tissue penetration and fine selectivity for the related transition metal cation. Moreover, the dual channel ratiometric imaging should reduce the interference found in the in vivo turn-on imaging due to the internal calibration effect of ratiometric sensing. This study will promote not only the development of in situ tracking technique for transition metal cation but also the clarification of the related physiology and pathology, which are helpful to develop the imaging-based techniques for diagnosis and therapy.

全面理解关键过渡金属离子的生理功能需要其活体尤其是模式哺乳动物中的实时原位信息，而相应的过渡金属离子的小鼠活体荧光成像仍面临两方面挑战：一是个体和组织导致的微环境、探针分布、荧光衰减的差异及活体位置扰动会严重干扰荧光信号强度，过渡金属离子快速变化引起的荧光强度变化易被掩盖；另一方面缺乏组织穿透能力强、过渡金属离子选择性高的荧光探针。本项目拟通过构建过渡金属离子的小分子近红外比例计量型荧光探针，以双通道比例成像模式代替简单的强度增强成像，实现模式动物尤其是模式哺乳动物小鼠中的过渡金属离子的活体成像。有机近红外荧光团偶联金属离子选择性螯合团构建的比例探针不仅提高了成像深度，也保证了对过渡金属离子的选择性。双通道比例成像利用了比例识别的内校正效应，有助于克服荧光增强型活体成像中的干扰因素。研究将不仅推动过渡金属离子活体检测技术的发展，也将促进阐明相关的生理功能和病理机制，推动相应诊疗技术的发展。

活体模式动物及组织中过渡金属离子的发光成像跟踪对理解和认识关键过渡金属离子的生理和病理功能至关重要。然而由于探针发光信号易受个体/组织差异、探针动态分布等干扰，兼之发光信号组织穿透深度的局限，相关成像还难以有效实现。针对这些问题，本项目通过BODIPY荧光团ICT扩展的NIR荧光团构建策略分别构建了过渡金属离子的NIR增强和比例有探针；通过双光子荧光团结合双荧光团的FRET效应构建了双光子比例响应探针。据此获得了可实现活体和组织Cu2+/Cu+、Zn2+成像的近红外比例探针3种、双光子荧光探针2种、近红外增强探针2种，均可实现目标离子的可逆响应。针对Fe3+的猝灭效应，克服常见反应性增强响应铁离子探针不可逆的不足，发展了基于新型铁离子螯合团的Fe3+可逆性增强和比例荧光探针各1种，为进一步发展可逆铁离子NIR比例荧光探针奠定了基础。据此项目分别实现了基于双光子比例探针的心脏组织中Cu+的比例荧光成像，还发现了内质网应激中内质网的Cu+增大现象。利用铜离子的NIR探针不仅实现了的小鼠活体Cu2+增强成像，还开展了有效的活体及分离器官的Cu2+比例成像跟踪，为FDA批准的Menkes病新药组氨酸铜的代谢提供了重要信息。利用引入双光子荧光的FRET探针不仅实现了斑马鱼Zn2+的3D活体比例荧光成像还实现了小鼠脑部组织Zn2+的3D双光子比例荧光成像。利用锌离子NIR比例探针对小鼠活体、分离器官同样实现了Zn2+比例成像跟踪，为全营养补锌后的器官代谢途径研究提供了重要信息。项目在完成原定目标外，还根据最新活体成像技术的发展，还开发了Zn2+的光声比例成像探针，进一步提升了活体小鼠的Zn2+成像深度，为关键过渡金属离子的成像跟踪提供了新选择。项目实施以来发表了包括Angew. Chem. Int. Ed., J. Am. Chem. Soc., ACS Nano, Inorg. Chem., Dyes &Pigments等在内的标注资助的SCI论文13篇，申请请国家发明专利7项。毕业硕士生4人、博士生7人。项目积极参与国际交流，并建立了稳定的国际合作， 为后续高分辨成像研究奠定了基础。