An NIR-II anionic heptamethine cyanine probe enables real-time in vivo imaging of tumor-associated glutathione

Abstract

Sensitive and specific in vivo glutathione (GSH) detection is critical for the early diagnosis of cancer, yet current methods are restricted by poor tissue penetration and low specificity. We report T2SHNO2, an NIR-II anionic heptamethine cyanine probe, capable of selective and sensitive imaging of tumor-associated GSH. The probe is incorporated with 4-nitrobenzenethiol (NBT) as a GSH-responsive moiety that quenches fluorescence by photoinduced electron transfer (PET). Reaction with GSH triggers nucleophilic substitution, removing the NBT quencher and restoring NIR-II emission at 924 nm. In addition to being highly selective, T2SHNO2 is also highly stable at physiological pH. It has a low cytotoxicity and excellent biocompatibility, as confirmed by cytotoxicity assays. In vivo imaging of 4T1 tumor-bearing mice reveals strong tumor-specific fluorescence with high signal-to-background contrast. In a density functional theory analysis, the quenching mechanism and restoration of fluorescence are confirmed using PET. These observations render T2SHNO2 a sensitive, biocompatible, and stable probe for deep tissue imaging of tumor-related GSH, and this holds potential as an early cancer diagnostic and real-time therapeutic monitoring agent. © 2025 Elsevier Ltd