A reversible fluorescent probe based on On-Off-On switching cycle to track the dynamic signaling of ferroptosis

Ferroptosis, a distinct modality of necrotic cell death driven by excessive lipid peroxidation, has garnered significant attentions. However, the visualization of ferroptosis remains challenging due to the lack of specific probes, which severely restricts the study of ferroptosis execution mechanisms and the crosstalk between associated signaling pathways. Herein, a sulfonate-functionalized cyanine derivative (Cy7) was synthesized and firstly found based on On-Off-On switching cycle: Cy7 specifically chelated Fe 3+ to form Cy7-Fe 3+ complex by intramolecular charge transfer (ICT), with fluorescence restored via endogenous GSH. Leveraging this behavior, iron-free and iron-doped Cy7-nanomodels were then established to track the dynamic signaling of ferroptosis. Notably, high concentrations of iron-doped Cy7-nanomodels could be degraded within the acidic tumor microenvironment, releasing Fe 3+ and Cy7 fluorophore. Both in vitro and in vivo fluorescence imaging results showed that the iron-doped Cy7-nanomodels exhibited a characteristic ‌On-Off-On‌ fluorescence transition during cellular internalization and subsequent biological interactions: initial fluorescence quenching was through the chelation of locally high concentration of Fe 3+ with Cy7, followed by GSH-mediated reduction restoring fluorescence and generating the ferroptosis-promoting Fe 2+ . The fluorescence switching from On to Off reflected pre-ferroptotic cellular status, whereas the Off-to-On resurgence marked active ferroptotic signaling. This ‌On-Off-On‌ fluorescence signature provided direct evidence of tumor cells undergoing ferroptosis. Accordingly, our findings presented a novel fluorescent-based approach for visualizing the dynamic signaling of ferroptosis.