Gold Nanoparticle–Facilitated Co-Delivery of Methylene Blue and INF-55: A Dual-Mechanistic Photodynamic Strategy Against Multidrug-Resistant Bacteria

Gold nanoparticles (AuNPs) synthesized through picosecond pulsed laser ablation were systematically investigated as multifunctional enhancers in methylene blue (MB)–mediated photodynamic therapy (PDT) targeting Escherichia coli, including multidrug-resistant (MDR) strains. The AuNPs were generated at wavelengths of 532 and 1064 nm with laser repetition rates between 20 and 50 kHz, producing nanoparticles whose physicochemical characteristics depended strongly on the laser frequency and wavelength. The highest nanoparticle yield was observed at 50 kHz, accompanied by the formation of smaller particles exhibiting enhanced plasmonic resonance and stronger reactive oxygen species (ROS) generation efficiency. Optical characterization via UV–Vis and fluorescence spectroscopy confirmed nanoparticle formation and surface plasmon resonance features in agreement with transmission electron microscopy (TEM) measurements.

Photobleaching studies revealed that AuNPs substantially enhanced MB-mediated singlet oxygen generation, thereby improving the photodynamic performance. Furthermore, the addition of the efflux pump inhibitor INF-55 provided an additional antibacterial advantage, significantly increasing bacterial inactivation while maintaining a constant net ROS level. In vitro antibacterial assays demonstrated that the combined MB/AuNPs/INF-55 system achieved approximately 59% greater bacterial deactivation compared to MB/AuNPs alone, underscoring the synergistic relationship between the two components. Molecular docking simulations further revealed that INF-55 exhibited a stronger binding affinity (–9.1 kcal/mol) to the AcrB efflux pump than MB, confirming its function as a competitive inhibitor that facilitates intracellular MB retention and enhances PDT efficiency.