Localized Nitric Oxide Release from a Ciprofloxacin Hybrid Conjugate Enables Potent Disruption of Gram-Positive and...

Biofilm-associated infections on medical devices contribute to treatment failure and antibiotic resistance by impeding drug penetration and sustaining persistent bacterial communities. Ciprofloxacin (CFX), a fluoroquinolone, is active against Gram-negative bacteria but has limited activity against Gram-positive bacteria and biofilm infections. However, nitric oxide (NO) is a widely used broad-spectrum antimicrobial agent with biofilm dispersion capabilities. To enhance CFX's therapeutic potential, a hybrid compound, SNAP_CFX, was synthesized by covalently conjugating S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, with CFX. This hybrid facilitates localized and sustained NO release, thereby enhancing the antimicrobial action of CFX. Antibacterial efficacy was assessed using the minimum inhibitory concentration (MIC) and biofilm reduction assays against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). SNAP_CFX exhibited greater stability than SNAP alone, prolonging NO release over 30 days. The MIC studies demonstrated a 200-fold improvement in SNAP_CFX's efficacy against S. aureus compared to CFX alone. At low micromolar bacterial concentrations (5 μM for S. aureus, 20 μM for P. aeruginosa), SNAP_CFX significantly disrupted preformed biofilms. SNAP_CFX reduced S. aureus biomass by ∼69%, biofilm cell viability by ∼88%, and planktonic bacteria by ∼99.5%. In P. aeruginosa, the hybrid achieved ∼51% biomass reduction, ∼98% reduction in viable biofilm cells, and near-complete eradication of planktonic populations. These outcomes far exceeded those of SNAP or CFX alone. Confocal microscopy confirmed widespread bacterial death and biofilm disintegration following SNAP_CFX treatment, in contrast to the partial effects seen with the individual agents. Importantly, cytotoxicity assays confirmed that SNAP_CFX exhibited improved biocompatibility relative to CFX. These results highlight SNAP_CFX as a promising dual-action therapeutic agent, combining antibiotic potency with NO-mediated biofilm disruption, paving the way for advanced treatments against antibiotic-resistant biofilm infections.