Architecture-driven design of near-infrared probes for bioimaging of senescence in biological systems

Cellular senescence contributes to aging, tissue remodeling, pathologies including cancer and fibrosis, motivating the development of sensitive and selective tools to detect senescent cells in complex biological settings. Here we report SenNIR, a lysosome-targeted near-infrared (NIR) fluorescent and colorimetric probe engineered for selective imaging of senescence. SenNIR integrates: a senescence-associated β -galactosidase (SA- β -gal)-responsive trigger, a lysosomal localization element, and a dicyanomethylene-4H-pyran fluorophore. Upon SA- β -gal activation, the lead probe, SenNIR-1 ( meta-ether linkage), displays a NIR fluorescence turn-on response (~17-fold enhancement), enabling sensitive detection of senescent cells. To interrogate how molecular architecture governs function, we evaluated structural analogues altering the connectivity and tethering topology of the modules. Strikingly, these systematic variations revealed that linker identity and the substitution position of the lysosomal targeting unit strongly regulate the probe’s photophysical response. The effect arises from architecture-dependent control of excited-state energetics, specifically the relative ordering of charge-transfer versus locally excited states, which is reorganized upon enzymatic unmasking to produce an efficient fluorescence turn-on. Transitioning to an ortho -amide linkage (SenNIR-2) maximized the electronic coupling, yielding a >100-fold NIR turn-on, whereas an ortho-ether linkage (SenNIR-3) produced a ~9-fold enhancement but at deeper NIR wavelengths. All SenNIR probes showed no detectable cytotoxicity in normal human skin fibroblasts and robustly differentiated proliferating from prematurely senescent cells. Probe performance and SA- β -gal specificity were further validated in ex vivo models. Collectively, these results demonstrate that not only the choice of functional modules but also their mode of assembly is decisive for diagnostic performance, providing design rules for next-generation senescence probes.