Dual-Channel NIR-II/Red Probe for Simultaneously Visualizing Mitochondrial Viscosity and NAD(P)H Dynamics Reveals Cuproptosis Mechanisms

Cuproptosis, a copper-dependent mitochondrial cell death pathway, holds therapeutic potential for cancers reliant on mitochondrial respiration. While NAD(P)H, a core electron donor in mitochondrial metabolism, regulates this process, the dynamics of NAD(P)H levels and key microenvironments such as viscosity during cuproptosis remain unresolved, impeding mechanistic insight. Here, we developed Cy-N, a unimolecular fluorescent probe enabling simultaneous visualization of mitochondrial viscosity and NAD(P)H levels via distinct red (633 nm) and NIR-II (944 nm) emission channels. Utilizing Cy-N in vitro and in vivo within a hepatocellular carcinoma model, we uncovered that cuproptosis progression triggers a significant increase in mitochondrial viscosity coupled with NAD(P)H depletion. Mechanistically, lipoylated DLAT oligomerization drives viscosity elevation, while TCA cycle disruption, specifically downregulation of isocitrate dehydrogenase 3 (IDH3), impairs NAD(P)H synthesis. Our study establishes Cy-N as a powerful dual-parameter imaging tool for monitoring cuproptosis progression and delineates the roles of DLAT oligomerization and IDH3 suppression, advancing both mechanistic understanding and therapeutic targeting of copper-dependent cell death.