Covalent Anchoring of Enzyme-Activatable Fluorescent Signals for in Situ Imaging and Longitudinal Staging of Cellular Senescence in Pulmonary Fibrosis.

Authors

Feiyi Chu, Bin Feng, Huanfeng Tian, Jin Zhou, Chengmin Li, Yingli Zhu, Jing Zhou, Xueping Feng, Jie Dong, Fei Chen, Wenbin Zeng

Published in

Small (Weinheim an der Bergstrasse, Germany). Pages e75215. Aug 11, 2026. Epub Aug 11, 2026.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease caused by the pathological accumulation of senescent cells. In this fibrotic microenvironment, senescence-associated β-galactosidase (SA-β-Gal) serves as a key biomarker. However, existing SA-β-Gal molecular probes suffer from signal diffusion and rapid clearance due to the extracellular leakage, compromising imaging fidelity. To address this issue, an enzyme-activated covalent labelling strategy is proposed, and as a design paradigm developing TCFM-Gal, a imaging tool integrating β-galactosidic fluorophore with an ortho-difluoromethyl leaving group. Upon SA-β-Gal-mediated hydrolysis, TCFM-Gal generates a quinone methide intermediate that covalently anchors to surrounding nucleophiles, confining fluorescence within lysosomes. In senescent cells, TCFM-Gal achieves extended lysosome-retained imaging, maintaining over 85% signal retention within 24 h. In the IPF model, TCFM-Gal enables longitudinal tracking of senescent cells and maintains a high signal intensity after 24 h. Rapid circulatory washout and high interstitial fluid pressure make conventional probes ineffective in the lungs. TCFM-Gal overcomes this not just by sensing, but by anchoring the signals in situ. Moreover, TCFM-Gal facilitates staging assessments of fibrosis progression by quantifying the senescence burden across disease phases. This study establishes an enzyme-activated covalent labeling paradigm for high-fidelity senescence imaging, advancing the spatiotemporal mapping of senescence in lung diseases.

PMID:
42581603
Bibliographic data and abstract were imported from PubMed on 12 Aug 2026.

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