A lysosome-targeted fluorescent probe for ratiometric imaging of in vitro and in vivo

Peroxynitrite (ONOO^-) dysregulation is closely linked to the pathogenesis of various inflammatory diseases, making accurate real-time monitoring of ONOO^- levels in specific subcellular compartments critical for elucidating its molecular mechanisms and enabling early disease diagnosis. Herein, we report Ro-Np, a novel lysosome-targeted ratiometric fluorescent probe based on Förster Resonance Energy Transfer (FRET) for specific ONOO^- detection. This probe employs 5-carboxytetramethylrhodamine (Ro) and naphthalimide (Np) as fluorophores, integrates a pyrrolidine group for precise lysosome targeting, and utilizes a phenylhydrazine moiety as the ONOO^--responsive site. Biochemical evaluations demonstrate that Ro-Np exhibits rapid ONOO^- detection (within 10 min), high sensitivity (limit of detection = 80 nM), excellent selectivity against other reactive species and biological interferents, and strong anti-interference capability. In the presence of high concentrations of biological thiols (Glutathione (GSH), Cysteine (Cys), Homocysteine (Hcy)), which partially reduce fluorescence intensity but do not disrupt its core ratiometric feature. Cellular colocalization experiments confirm its specific lysosome-targeting property with a high Pearson's correlation coefficient of 0.85. Importantly, Ro-Np successfully detects both endogenous ONOO^- (induced by Lipopolysaccharide (LPS)/Interferon-γ (IFN-γ)/Phorbol-12-myristate-13-acetate (PMA)) and exogenous ONOO^- (induced by 3-morpholino-sydnonimine (SIN-1)) in BV-2 microglial cells, with reversible fluorescence changes upon ONOO^- scavenging by minocycline. Furthermore, the probe shows reliable imaging performance in live mouse models, where distinct green-to-red fluorescence shifts are observed in ONOO^--treated tissues. Additionally, two-photon imaging in a lipopolysaccharide (LPS)-induced neuroinflammatory mouse model revealed enhanced ONOO^- levels in brain tissues, demonstrating the probe's capability for high signal-to-noise and high-resolution visualization of ONOO^- in deep tissues. These findings validate Ro-Np as a promising chemical tool for lysosome-targeted ONOO^- detection in biological systems, with potential for investigating lysosome-related ONOO^- functions and supporting inflammatory disease research. © 2025 Elsevier Ltd. All rights reserved.