Hydroxyl Radical–Activatable NIR-II Ratiometric Fluorescence Probe for Visualization In Vivo Tracking of Oxidative Stress and Therapeutic Response in Arthritis

Oxidative stress plays a pivotal role in the onset and progression of arthritis, in which the abnormal elevation of hydroxyl radical (•OH) has been recognized as a key molecular hallmark of early inflammation. However, effective tools capable of simultaneously visualizing •OH dynamics and tracking therapeutic efficacy remain elusive. Herein, we developed a self-calibrated near-infrared II (NIR-II) ratiometric imaging probe responsive to •OH. The probe integrates downconversion nanoparticles (DCNP) as a stable internal reference (F1550nm,980Ex) and a •OH-triggered red-shiftable dye Hydro-830 (F930nm,808Ex), enabling ratiometric imaging through the signal ratio “F930nm,808Ex/F1550nm,980Ex.” The probe exhibits high sensitivity and specificity toward •OH in vitro and effectively captures the upregulation of oxidative stress at early inflammatory stages in vivo. In a longitudinal “treatment–imaging” study, the ratiometric signal gradually decreased following methotrexate (MTX) therapy, consistent with reductions in joint swelling, inflammatory cytokines, and histopathological lesions. This study establishes a self-calibrated NIR-II ratiometric imaging strategy for precise monitoring of oxidative stress, enabling early molecular diagnosis and dynamic therapeutic evaluation of arthritis, and offering a promising paradigm for imaging other ROS-related inflammatory disorders.