New SubmissionGold Nanoparticle-Mediated Delivery of Methylene Blue and INF: A Dual-Action Strategy Against Multidrug-Resistant Bacteria*

Gold nanoparticles (AuNPs) fabricated by picosecond pulsed laser ablation were evaluated as modulators of methylene blue (MB)–based photodynamic therapy (PDT) against Escherichia coli. Particles generated at 532 and 1064 nm with repetition rates of 20–50 kHz exhibited frequency- and size-dependent behavior, with 50 kHz producing the greatest nanoparticle yield and smaller particles promoting enhanced reactive oxygen species (ROS) generation. Characterization by UV–Vis absorption and fluorescence spectroscopy confirmed plasmonic features consistent with TEM results. Photobleaching experiments indicated that AuNPs markedly improved MB singlet oxygen production, while addition of the efflux pump inhibitor INF-55 further increased bacterial inactivation without changing the net ROS output. Cell culture assays demonstrated that combining INF-55 with MB/AuNPs resulted in approximately 59% greater bacterial deactivation compared to MB/AuNPs alone. Molecular docking revealed that INF-55 binds more strongly to the AcrB efflux pump (−9.1 kcal/mol) than MB, confirming its function as a competitive inhibitor that facilitates intracellular MB retention. Collectively, these results present a dual-mode PDT approach in which AuNPs boost ROS generation and INF-55 enhances antimicrobial efficiency by suppressing efflux activity.