Mechanistic Insights into the Antibacterial Action of Metallic Nanoparticles Functionalized with Ofloxacin Against Corynebacterium diphtheriae

Abstract

The emergence of multidrug-resistant Corynebacterium diphtheriae necessitates innovative antibiotic delivery systems. We present a comparative mechanistic study of silver (Ag), zinc oxide (ZnO), and copper oxide (CuO) nanoparticles surface-functionalized with Ofloxacin (OFX). Physicochemical analyses (DLS, zeta potential, TEM, FTIR) confirmed uniform OFX loading (Ag–OFX 83±2%, ZnO–OFX 78±3%, CuO–OFX 67±2%) and stable colloids (55–82 nm, –30 to –20 mV). Antibacterial assays (MIC, MBC, ZOI; CLSI) and time-kill kinetics demonstrated superior bactericidal potency of Ag–OFX (MIC 0.8±0.1 µg/mL; 4-log CFU reduction at 6 h) versus ZnO–OFX (MIC 2.2±0.2 µg/mL) and CuO–OFX (MIC 3.5±0.4 µg/mL) (ANOVA, p<0.01). Mechanistic studies revealed four synergistic pathways: enhanced uptake (confocal and flow cytometry), reactive oxygen species (ROS) generation (DCFH-DA assay), membrane poration (propidium iodide uptake; SEM), and DNA gyrase inhibition by metal ions. Statistical analysis (ANOVA with Tukey’s test) validated significance (p<0.05). Ag–OFX hybrids exhibited the highest ROS (4.2-fold increase) and membrane disruption (60% PI-positive cells). Colloidal stability tests over 30 days (4 °C) showed negligible changes (size Δ<10%). These insights inform the rational design of next-generation nanotherapeutics targeting diphtheria.