Activation-Triggered Lysosome-Retaining Visible/NIR-II Integrated Fluorescent Probe Enables Tracking of Lysosomal Changes during Ferroptosis in Cells and In Vivo

Lysosomes are the key organelles in regulating cellular homeostasis, posing a significant role from basic enzyme trafficking to cell death. Existing fluorescent probes for imaging of lysosomes have limitations, such as tumor-associated false positives due to pH-dependent retention and single-channel emission properties restricting their use to either cellular or in vivo imaging. Real-time and high-fidelity visualization of lysosomal changes during ferroptosis both at cells and the tumor level presents a big challenge. To overcome these challenges, we developed ACy-SPz-Np, a glutathione (GSH)-activatable, lipophilic, visible/NIR-II dual-channel emission probe. It integrates ACy (an anionic cyanine with NIR-II emission) and Np (a naphthalimide derivative with visible emission) via a GSH-responsive linker. Leveraging the high intracellular GSH concentration, upon GSH activation, hydrophilic and proton-combinable products ACy-Pz and Np-GS are released, enabling long-term lysosomal retention (over 24 h in cells and up to 15 days in tumor-bearing mice). Benefiting from the lysosome retention effect, we further achieved visible/NIR-II dual-channel longitudinal monitoring of lysosomal changes during erastin-induced ferroptosis, from cellular-level confocal to in vivo NIR-II imaging, revealing significant lysosomal destruction. This cross-scale functionality of ACy-SPz-Np greatly bridges cellular dynamics to organismal outcomes, addressing a long-standing gap in lysosomal research tools.