Abstract

The glutathione peroxidase 4 (GPX4) inhibitor RSL3 exhibits limited selectivity, unfavorable pharmacokinetics, and dose-dependent toxicity, which restrict its therapeutic application. Meanwhile, the efficacy of photodynamic therapy (PDT) is compromised by tumor hypoxia and off-target phototoxicity. In this study, we synthesized a novel GPX4 inhibitor, RSL3-ClAc, via a Pictet-Spengler reaction followed by chloroacetylation. RSL3-ClAc shows enhanced GPX4 binding affinity and inhibitory activity, resulting in improved ferroptosis induction in tumor cells. RSL3-ClAc was further co-assembled with an NTR-activatable photosensitizer, C2-NO 2 , through nanoprecipitation to construct a carrier-free, hypoxia-responsive nanoplatform (RC/C2-NO 2 @PEG). The nanoparticles exhibit good colloidal stability and preferential tumor accumulation through the enhanced permeability and retention (EPR) effect. Under hypoxic conditions, nitroreductase-mediated activation of C2-NO 2 produces near-infrared fluorescence for tumor imaging and generates singlet oxygen ( 1 O 2 ) to enable localized PDT. Importantly, PDT-induced depletion of intracellular NAD(P)H suppresses the FSP1-CoQ 10 antioxidant pathway, thereby overcoming ferroptosis resistance. By integrating hypoxia-responsive imaging, GPX4 inhibition, and ferroptosis-sensitized PDT, this nanoplatform achieves potent antitumor efficacy with favorable biosafety. Overall, this work provides a rational and translatable strategy for precision cancer therapy.