Dynamic in vivo imaging of microplastic-induced hepatic injury via enzyme-activated far-red fluorescent probe

Microplastic pollution, particularly from polystyrene (PS), poses increasing risks to human health due to its persistence in the environment, tendency to bioaccumulate, and ability to induce oxidative stress and hepatic injury. However, tools for dynamic imaging that elucidate the mechanistic link between microplastic exposure and liver toxicity remain lacking. Herein, we report a far-red fluorescent probe (emission maximum at 674 nm), DDAO-CT, derived from the hydroxyl-substituted red-emitting fluorophore DDAO (9,9-dimethylacridin-2(9H)-one derivative), designed for in vivo visualization of chymotrypsin activity, a key enzyme marker known to be upregulated during microplastic-induced liver damage. DDAO-CT is constructed by conjugating a 4-bromobutyryl recognition group to the red-emissive fluorophore DDAO, which quenches fluorescence by disrupting intramolecular charge transfer (ICT). Upon enzymatic hydrolysis by chymotrypsin, the ICT pathway is restored, resulting in a 14-fold enhancement in fluorescence at 674 nm with a detection limit of 3.5 ng/mL. The probe had high selectivity, low cytotoxicity, and excellent responsiveness to enzyme activity both in vitro and in PS-exposed hepatocytes. Notably, in vivo imaging in mouse models revealed dose-dependent fluorescence signals in the liver, which correlated closely with histopathological damage and elevated serum markers of liver injury. These results show that chymotrypsin activation is a downstream event of PS bioaccumulation, establishing DDAO-CT as an effective tool for visualizing pollutant-induced hepatic dysfunction. This study presents a novel chemical biology platform for noninvasive assessment of environmental hepatotoxins and offers mechanistic insights into microplastic-induced liver injury at the enzymatic level.