Optimized green zinc oxide/chitosan/amoxicillin nanocomposite against Pseudomonas aeruginosa

Abstract

Multidrug-resistant Pseudomonas aeruginosa poses a significant global health threat, highlighting the need for innovative antimicrobial strategies. This study presents the successful synthesis, optimization, and characterization of a novel zinc oxide/chitosan/amoxicillin (ZnO/CS/AMX) nanocomposite aimed at combating P. aeruginosa infections, including the ability to form biofilms. Response surface methodology (RSM) based on the Box-Behnken design (BBD) was used to optimize the production of ZnO NPs using cell-free metabolites of P. aeruginosa. The optimal parameters for biosynthesis of ZnO NPs require a mixing ratio of 1:4 v/v% between the cell-free bacterial metabolites and 30 mM Zn precursor (Zn(NO₃)₂.6H₂O) at pH 7.0 and 30 °C. The nanocomposite was synthesized by encapsulating amoxicillin (AMX) within chitosan-coated zinc oxide nanoparticles (ZnO NPs). Its properties were confirmed through various characterization techniques including UV-Vis spectroscopy (340 to 380 nm), XRD (101 plane, an average crystallite size = 59 nm), FTIR (Zn-O and proteins stretching vibrations), TEM (spherical to quasi-spherical in shape with size range = 36-98 nm), and zeta potential analyses (+ 35 ± 2.3 mV). The maximum drug loading of AMX in the ZnO/CS/AMX nanocomposite was found to be 55.7%. The antimicrobial effectiveness was thoroughly assessed against various clinical isolates of P. aeruginosa, all of which showed natural resistance to chitosan (CS) and AMX individually, as well as the reference strain ATCC 27853. Quantitative assays further confirmed the superior bactericidal potential of the nanocomposite compared to ZnO NPs and imipenem. The nanocomposite achieved exceptionally low minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) levels, as low as 10 µg/ml against resistant clinical isolates. TEM micrographs of P. aeruginosa cells treated with the nanocomposite revealed severe cellular damage. This damage included extensive separation between the cell wall and cytoplasmic membrane, complete cell lysis, and severe malformations. In addition, the nanocomposite exhibited outstanding antibiofilm activity at concentrations as low as 50 µg/ml. This activity dramatically increased in a dose-dependent manner, achieving ≤ 100% biofilm inhibition at 150 µg/ml. The cytotoxicity assessment on Vero cells demonstrated a promising safety profile, with a CC₅₀ of 292 ± 1.3 µg/ml for the nanocomposite, nearly three times higher than that of ZnO NPs (106 ± 0.9 µg/ml). This wide therapeutic window indicates that ZnO/CS/AMX can effectively combat P. aeruginosa at concentrations far below that toxic to mammalian cells. These findings demonstrate that the ZnO/CS/AMX nanocomposite acts through a synergistic, multimodal mechanism, effectively overcoming bacterial resistance and biofilm recalcitrance, while also exhibiting favorable biocompatibility. KEY POINTS: RSM-Box-Behnken optimized green biosynthesis of ZnO NPs using cell-free metabolites of P. aeruginosa Green synthesis of zinc oxide/chitosan/amoxicillin nanocomposite to combat MDR P. aeruginosa The nanocomposite markedly enhances bactericidal and antibiofilm efficacy at low doses with high mammalian cell safety and synergistic action.