Light‐Inducible Bispecific DNAzymes Reveal Mitochondrial Fe ²⁺ /Fe ³⁺ Dynamics During Ferroptosis

Abstract

ABSTRACT Mitochondrial iron redox homeostasis plays critical roles in cellular function and disease progression; however, spatiotemporal measurement of Fe 2+ and Fe 3+ dynamics in mitochondria remains challenging, largely due to limited analytical tools. Here we report a light‐inducible bispecific DNAzyme sensor (LiBD) that enables simultaneous and spatiotemporally controlled imaging of mitochondrial labile Fe 2+ and Fe 3+ in living cells and in vivo. LiBD integrated two orthogonal iron‐specific DNAzymes into a single DNA scaffold, incorporating photocleavable (PC) linkers, rendering DNAzymes inactive until light‐triggered activation. Coupled with mitochondria‐targeted nanocarriers, LiBD demonstrated high spatial and temporal resolution for monitoring Fe 2+ and Fe 3+ level changes in mitochondria. We revealed remarkable mitochondrial Fe 2+ accumulation in tumor cells during drug‐induced ferroptosis, whereas chemotherapy‐resistant tumor cells exhibited substantially decreased Fe 2+ in mitochondria, both accompanied by slight increases in Fe 3+ . Moreover, an NIR‐activatable LiBD demonstrated in vivo visualization of mitochondrial Fe 2+ and Fe 3+ level changes during ferroptosis in tumor‐bearing mice. Collectively, this work presents LiBD as a powerful platform for investigating mitochondrial iron biology with spatiotemporal precision and provides insights into iron‐dependent mechanisms of ferroptosis and chemoresistance.