Molecular Engineering of 2′, 7′‐Dichlorofluorescein to Unlock Efficient Superoxide Anion NIR‐II Fluorescent Imaging and Tumor Photothermal Therapy

Abstract Although classical fluorescent dyes feature advantages of high quantum yield, tunable “OFF‐ON” fluorescence, and modifiable chemical structures, etc., their bio‐applications in deep tissue remains challenging due to their excessively short emission wavelength (that may lead to superficial tissue penetration depth). Therefore, there is a pressing need for pushing the wavelength of classical dyes from visible region to NIR‐II window. As a representative classical dye, the 2′,7′‐Dichlorofluorescein (DCF), a derivative of Fluorescein, is selected and rationally engineered to develop a novel NIR‐II platform, CR‐OH, which exhibits a substantial red‐shift in the wavelength from the visible region to the NIR‐II region. This achievement is attributed to molecular modification strategies that include extending π‐conjugation, enhancing molecular rigidity, and incorporating strong electron‐withdrawing groups. Furthermore, based on this developed NIR‐II platform, a NIR‐II fluorescence probe and a photothermal nanoagent are successfully constructed to unlock its bio‐application in the NIR‐II fluorescence imaging of endogenous O₂^·– fluctuations in a CIRI model for the first time, as well as effective photothermal therapy for 4T1 tumors with a high photothermal conversion efficiency (44.0%). Significantly, this work overcomes the wavelength limitation of classical dyes, effectively unlocking their applications for the diagnosis and treatment of early disease in the NIR‐II window.