Rational design of a lysosome-targeted fluorescent probe for monitoring the generation of hydroxyl radicals in ferroptosis pathways

Ferroptosis is a newly discovered iron-dependent form of regulated cell death associated with high levels of hydroxyl radical (OH) production. Meanwhile, lysosome dysfunction has been shown to be one of the causes of ferroptosis. Although a variety of OH-responsive fluorescent probes have been developed for detecting intracellular OH in living cells, there are still only few lysosome-targeted probes to monitor the variation in lysosomal OH levels during ferroptosis. Herein, we report a novel OH-specific fluorescent probe HCy-Lyso, which is composed of the hydrocyanine and morpholine moiety. Upon treatment with OH, its hydrocyanine unit was converted to the corresponding cyanine group, thus leading to a large pi-conjugation extension of HCy-Lyso, accompanied by a significant fluorescence off-on response. Moreover, after reacting with OH in an acidic environment, the protonation product of HCy-Lyso exhibits a higher fluorescence enhancement, which is suitable for detecting lysosomal OH variation. HCy-Lyso has been utilized for imaging endogenous OH in living cells under phorbol myristate acetate (PMA) stimuli and monitoring the changes in lysosomal OH levels during ferroptosis. Thus, our study proposes a new strategy to design lysosome-targeted and OH-responsive fluorescent probes to investigate the relationship between lysosomes and ferroptosis. A novel hydrocyanine-based turn-on fluorescence probe HCy-Lyso, with excellent lysosome-targeting ability and OH-responsive efficiency, has been applied for monitoring the variation in lysosomal OH levels during ferroptosis.