Mechanistic insights and metabolic profiling of sodium hydroxide-treated green synthesized silver nanoparticles: Optimization of structural properties and assessment of antibacterial activity

<h2>Abstract</h2> This study evaluates the effects of sodium hydroxide treatment on AgNPs synthesized using <i>Fagonia indica</i> (Fa-AgNPs) and <i>Origanum vulgare</i> (Org-AgNPs). Sodium hydroxide concentrations ranging from 33 mM to 266 mM were applied post-synthesis to the nanoparticles, resulting in significant structural changes. UV–Vis spectroscopy showed a blue shift in the surface plasmon resonance (SPR) peaks, with Fa-AgNPs shifting from 436 nm to 420 nm and Org-AgNPs from 414 nm to 402 nm, indicating smaller particle sizes. Dynamic light scattering (DLS) analysis confirmed this size reduction, with the Z-average size of Fa-Na-AgNPs decreasing from 116.96 nm to 94.7 nm and that of Org-Na-AgNPs decreasing from 130.4 nm to 29.9 nm at 33 mM NaOH. Zeta potential values improved for both Fa-Na-AgNPs (−21.73 mV to −33 mV) and Org-Na-AgNPs (−30.5 mV to −40 mV), suggesting enhanced colloidal stability. Despite these structural improvements, antibacterial assays revealed reduced activity, with zones of inhibition for Fa-Na-AgNPs decreasing from 12.83 mm to 12.01 mm against <i>Escherichia coli</i> and from 11.5 mm to 8.05 mm against Methicillin-resistant <i>Staphylococcus aureus</i>. Mechanistic studies suggest that Fa-Na-AgNPs produced lesser ROS, triggered lower defense response, and inhibited efflux pump at a lower intensity than Fa-AgNPs. XPS analysis showed significant differences in the surfaces of Fa-Na-AgNPs with sodium-knocking essential compounds. This was further confirmed through metabolic profiling of Fa-AgNPs and Fa-Na-AgNPs, which showed a clear difference in phytochemicals associated with Fa-AgNPs compared to Fa-Na-AgNPs. These findings demonstrate that while NaOH treatment enhances the physical properties of AgNPs, it may compromise their antimicrobial activity, necessitating careful optimization for practical applications.