Matrix Metalloproteinase-9-Activated Dually-Quenched Fluorescence Nanoprobe for Sensitive Imaging of Rheumatoid Arthritis

Abstract Fluorescence probes that enable sensitive and real-time in vivo imaging of matrix metalloproteinase-9 (MMP-9) hold high promise for early clinical diagnosis of intractable rheumatoid arthritis (RA), but they remain scarcely developed. Herein, we developed the dually quenched fluorescence nanoprobe P-IR780-NP for sensitive in vivo imaging of RA. To prepare P-IR780-NP, a molecular precursor Cys(StBu)-Pro-Leu-Gly-Leu-Lys(IR780)-CBT (P-IR780) was synthesized. Under reductive conditions, P-IR780 undergoes a 2-cyanobenzothiazole-cysteine (CBT-Cys) click reaction to form a cyclic dimer (P-IR780-Dimer), which subsequently assembles into P-IR780-NP nanoparticles. The sequential cyclization and assembly processes cause intramolecular and intermolecular quenching of the conjugated IR780 fluorophore, respectively, thereby collectively minimizing background fluorescence. In vitro studies confirmed that MMP-9 specifically cleaved P-IR780-Dimer, triggering nanoparticle disassembly and consequent 8.6-fold fluorescence enhancement. In MMP-9-overexpressing macrophages, P-IR780-NP produced 1.8-fold stronger fluorescence signals compared to the MMP-9-inhibited control group. Notably, in vivo imaging of RA mice revealed a 3.7-fold higher fluorescence intensity in affected joints relative to healthy controls, demonstrating the enhanced sensitivity of P-IR780-NP for in vivo RA imaging. We anticipate that P-IR780-NP could be applied for early clinical diagnosis of RA in the near future.