Glutathione (GSH) is a crucial indicator for mapping the redox state of living organisms and has involved in numerous physiological and pathological processes. Real-time visualization of the GSH dynamics is significant for disease diagnosis and therapeutic evaluation. The second near-infrared (NIR-II, 1000–1700 nm) window fluorescence imaging demonstrates promise for detecting biological species in vivo, however, GSH-activatable NIR-II imaging probes remain relatively limited and encounter issues regarding detection sensitivity. In this study, we integrated molecular engineering approaches with structure-reactivity relationships to develop NIR-II fluorescent probes designed to elucidate diseases-related GSH variations. Two proof-of-concept probes (XD-1-GSH, XD-2-GSH) have fabricated based on an improved hemicyanine dye platform. Upon evaluating the structure-reactivity relationship, we identified the ideal probe, XD-1-GSH, which exhibits linearity with GSH concentration ranged from 4 to 10 mM and shows the NIR-II signal “on” at 905 nm after GSH activation. Utilizing the NIR-II fluorescence imaging, XD-1-GSH facilitates the determination and visualization of GSH dynamics in both diabetes-induced liver injury and hepatocellular carcinoma with high specificity and accuracy. Notably, XD-1-GSH demonstrates excellent performance in diagnosing clinical breast cancer specimens. We expect our propose fluorescence platform can be generalized to design imaging probes for various biomarkers through flexible incorporation of the recognition sites.

Scheme 1. (a) Molecular engineering approaches with structure-reactivity relationships to develop NIR-II fluorescent probes XD-1-GSH&XD-2-GSH for detection of GSH. (b) Schematic diagram showing the construction of primary liver tumor model and its imaging. (c) Schematic diagram showing the imaging of cancer tissues in human specimens.

Fig. 1. (a) Normalized absorption and fluorescence emission spectra of 10 μM HDS and XD-1 in PBS buffer. (b) Absorption and (c) fluorescence spectra of XD-1-GSH (10 μM) upon treatment with various concentrations of GSH (0–15 mmol). (d) NIR-II FL intensity at 905 nm of XD-1-GSH in PBS buffer containing different levels of GSH. (e) Linear relationship of fluorescence intensity (d) and GSH concentrations. (f) NIR-II FL intensity at 905 nm of XD-1-GSH in PBS buffer upon treatment with various biological species. Inset: fluorescent photographs taken by NIR-II imaging system. The data are expressed as the mean ± s.d. (n = 3). NIR-II spectra: 808 nm excitation, collection 870–1200 nm.

Fig. 3. (a) Schematic illustration of the establishment of a primary liver tumor model and imaging. (b) Bioluminescent image of the 4T1-Luc primary liver tumor, verifying the successful construction of the model. (c) Time-dependent NIR-II fluorescent images of mice bearing 4T1-Luc primary tumors (n = 3) following intravenous injection of 100 μL of 1 mM XD-1-GSH. (d) Signal-to-background ratio (SBR) of liver tumors as indicated in (c) (marked as a, b, c, and d) after 6 h post-injection of XD-1-GSH. (e) Representative ex vivo NIR-II imaging and bioluminescent images of isolated liver tissue. (f) Normalized fluorescence intensity in regions of interest (ROI-A and ROI-B) of the liver as shown in (e). (g) Hematoxylin and eosin (H&E) staining of liver tissues from ROI-A and ROI-B. The data are presented as the mean ± SD (**p < 0.01). NIR-II imaging of the liver region was conducted using an in vivo imaging system with an excitation wavelength of 808 nm (100 mW/cm²), an exposure time of 500 ms, and a 900 nm long-pass filter.

Fig. 4. Investigation of the feasibility of XD-1-GSH in clinical cancer samples application. (a) Schematic diagram illustrating the detection of cancer tissues in human specimens. (b) Longitudinal ex vivo NIR-II images of cancer tissue from patients after in-situ spraying of (100 nM) XD-1-GSH. (c) Time-depdendent fluorescnce intensity changes in the ROI-a and ROI-b of cancer tisuse shown in (b).(d) Signal-to-background ratio (SBR) of the tumor regions a and b after 60 min incubation of XD-1-GSH. (e) H&E staining of tumor sample from ROI-a. Ex = 808 nm (100 mW/cm²), an exposure time of 300 ms, and a 900 nm long-pass filter.
4. Conclusion
In conclusion, by integrating the molecular engineering approaches and structure-reactivity relationships, we developed GSH-activatable NIR-II fluorescent probes for in vivo imaging of GSH dynamics and its associated dis-ease diagnostic applications. Two proof-of-concept probes for GSH, namely XD-1-GSH and XD-2-GSH, were fabricated based on an improved HDs platform. Evaluation of their photophysical properties revealed that XD-1-GSH shows the ideal combination of high specificity, NIR-II emission, and suitable response sensitivity after GSH-mediated activation, permitting it a promising candidate for the imaging of GSH fluctuations. Leveraging the ad-vantages of NIR-II fluorescence imaging, XD-1-GSH was effectively utilized to detect GSH in cases of diabetes-induced liver injury and hepatocellular carcinoma model, thereby facilitating diagnosis of the related diseases. More importantly, XD-1-GSH demonstrated its utility in dis-criminating between clinical cancer samples with high imaging contrast. We believe that our molecular platform can be generalized to develop imaging agents for different biological species through flexible modification of the response sites.