Antiaromaticity-Based General Molecular Design of Activatable Near-Infrared Photoacoustic Probes with High Acoustic Loudness

Abstract

Photoacoustic (PA) imaging offers high spatial resolution and deep tissue penetration, and the development of activatable PA probes has greatly expanded its potential for detecting disease-associated molecular biomarkers. Optimization of the probe performance has long been a central challenge. Recently, the proposed acoustic loudness factor (ALF = ε × k_nr) established that strong absorption and ultrafast nonradiative decay are key for maximizing PA output, yet a general molecular design strategy under this framework remains elusive. Here we report PhAF, an antiaromatic fluorene dye platform in which ground-state antiaromaticity and excited-state aromaticity reversal under Baird’s rule induce strong vibronic coupling and naturally high k_nr. PhAF exhibits maximal absorption at 950 nm, an ultrashort 3.37 ps lifetime, and a high ALF of 7.25 × 10¹⁵ M^–1 cm^–1 s^–1, while maintaining excellent stability in aqueous and biological media. Masking the aniline donor as an amide yielded nitroreductase-responsive PhAF-N and peroxynitrite-responsive PhAF-R, both showing robust in vivo imaging of tumor hypoxia and drug-induced liver injury. This work establishes a molecular foundation for designing high-performance activatable PA probes for future theranostic applications.