Unexpected dual-functional cyanine fluorophores: NIR multifunctional fluorescent probes for simultaneous monitoring of mitochondrial cysteine /viscosity during kidney ferroptosis in vivo

Cysteine (Cys) level and mitochondrial viscosity are important factors controlling cellular lipid peroxide metabolism and are closely related to ferroptosis. However, simultaneous tracking of these two key target analytes in the context of ferroptosis has not been realized. In this study, we developed a series of novel NIR dual-functional cyanine fluorophores AR-Cy based on a modified Vilsmeier-Haack reaction for simultaneous visualizing of cysteine level and mitochondrial viscosity during kidney ferroptosis in vivo. Intriguingly, this modified Vilsmeier-Haack reaction provides additional electron-withdrawing molecular rotor for improving the sensing performance to Cys and mitochondrial viscosity at two separated fluorescence channels (λem = 650 nm, Cys; λem = 710 nm, viscosity). Notably, by dual-color fluorescent imaging with this excellent probe AR-Cy-3, we found that ferroptosis process (erastin, sorafenib and sulfasalazine as ferroptosis inducers) was accompanied by a slight decrease in Cys channel and a significant increase in mitochondrial viscosity channel for the first time. More importantly, we further revealed that Cys level gradually decreased and mitochondrial viscosity rapidly increased in mouse kidney ferroptosis model in vivo. Therefore, this work provides not only the first dual-functional probe for studying the dynamic changes between the Cys level and mitochondrial viscosity in kidney ferroptosis but also useful cyanine fluorophores for designing powerful probes of relevant analytes to other diseases.