Inhibition of biofilm formation and virulence factors of Pseudomonas aeruginosa by ciprofloxacin-loaded ZnO@lignin@chitosan nanoparticles.

Abstract

The rapid development of antimicrobial resistance (AMR) in bacterial infections leads to increased mortality and reduced treatment effectiveness. Given the urgent need for multifunctional therapeutic agents capable of overcoming AMR, we developed a novel ZnO@lignin@chitosan nanocomposite loaded with ciprofloxacin (CIP), in which the synergistic combination of ZnO, lignin, and chitosan enhances antibacterial, antibiofilm, antivirulence, and antioxidant performance. The synthesized nanocomposite was characterized using various techniques, including Fourier transform infrared spectroscopy (FT-IR), UV-visible spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX) analysis, elemental mapping (MAP), field emission transmission electron microscopy (FE-TEM), and X-ray photoelectron spectroscopy (XPS). The ZnO@lignin@chitosan@CIP nanocomposite exhibited pH-responsive drug release, with accelerated release in acidic environments mimicking infection sites. The synergistic combination of its components enhanced multifunctional performance: lignin acted as an antioxidant, chitosan improved drug loading, biocompatibility, and inherent antibacterial activity against Gram-positive, Gram-negative, and the fungal strain Candida albicans, while ZnO nanoparticles (NPs) contributed additional antimicrobial effects. Furthermore, the nanocomposite effectively inhibited Pseudomonas aeruginosa (P. aeruginosa) biofilm formation and suppressed virulence factors, including protease, pyocyanin, and pyoverdine at sub-minimum inhibitory concentrations (sub-MICs). Moreover, gene expression analysis revealed downregulation of key quorum-sensing regulators (lasI, lasR, rhlI, and rhlR), indicating the composite's molecular antivirulence potential. These findings demonstrate that the unique synergy of ZnO, lignin, and chitosan provides multifunctional advantages, making ZnO@lignin@chitosan@CIP a promising candidate for combating drug-resistant and biofilm-associated infections.