Profiling Inflammatory Pathogenesis and Therapy: Ratiometric Photoacoustic Imaging of Energy Metabolism Dynamics With Upconversion Nanoprobes

Abstract

NAD(P)H is a critical redox cofactor in cellular energy metabolism, yet real-time monitoring of its spatiotemporal dynamics in pathological contexts remains challenging due to the limited penetration depth and resolution of conventional imaging modalities. In this study, we developed a dual-modal ratiometric imaging nanoprobe that enables precise photoacoustic (PA) and upconversion luminescence imaging of this energy metabolism substrates. The nanoprobe conjugates an NAD(P)H-responsive chromophore and an internal reference dye onto NIR-excited upconversion nanoparticles, enabling self-calibrated quantification through ratiometric UCL and mPAT signals. This design minimizes artifacts from nanoprobe distribution and environmental variations, enhancing spatiotemporal accuracy. The utility of the nanoprobe is demonstrated in multiple disease models: it visualizes metabolic reprogramming during macrophage M1 polarization, tracks aberrant NAD(P)H accumulation in drug-induced and acute liver injury (ALI), and monitors metabolic responses to NAC and 2-DG interventions. Furthermore, in a calorie restriction (CR) combined immunotherapy model, the nanoprobe reveals that the TLR7/8 agonist R848 and glycolytic inhibitor 2-DG synergistically suppress tumor growth by reprogramming tumor-associated macrophage metabolism. This work provides a robust imaging platform for tracking energy metabolism dynamics, offering insights into the interplay between metabolic pathways and inflammatory diseases for developing targeted therapies.