Exploring the antivirulence mechanisms of ZnO-PEG-quercetin nanoparticles: Biofilm disruption, attenuation of virulent factors, and cell invasion suppression against pathogenic Pseudomonas aeruginosa.

The dense biofilm architecture and efflux pump activity play critical roles in Pseudomonas aeruginosa infections by hindering the accumulation and long-term efficacy of antibacterial agents within bacterial cells. The development of engineered nanoparticles capable of penetrating the polysaccharide matrix of biofilms represents a promising strategy for addressing bacterial infections. This is the first report on the synthesis of quercetin-functionalized PEGylated ZnO nanoparticles (ZnO-PEG-QUE NPs) and the evaluation of their anti-biofilm activity against pathogenic strains of P. aeruginosa. The synthesized NPs exhibited spherical shapes with an average size of 59.52 nm. ZnO-PEG-QUE NPs demonstrated biofilm inhibitory levels between 49 % and 67 %, and significantly reduced the production of total exopolysaccharides, alginate, and pellicle by 64.61 %-71.69 %, 30.47 %-45.36 %, and 24.22 %-85.97 %, respectively. ZnO-PEG-QUE NPs not only inhibited early-stage biofilm formation but also disrupted mature biofilms, indicating a dual mode of action against both biofilm development and persistence. Based on our findings, ZnO-PEG-QUE NPs effectively eradicated mature biofilms by 67.2 %-72 % and significantly reduced the metabolic activity and viable cells of preformed biofilms to 34.12 %-55.57 % and 6.25-8.15 log CFU, respectively. Electron and fluorescence microscopy analyses also confirmed the antibiofilm potential of ZnO-PEG-QUE NPs. Furthermore, bacterial adhesion and invasion to HDF cells were significantly diminished in the NP-treated groups. The attenuation of efflux pump activity in the NP-treated strains was confirmed using the EtBr-agar cartwheel assay. Taken together, these findings highlight the therapeutic potential of ZnO-PEG-QUE NPs as a novel and effective strategy to combat biofilm-associated infections, warranting further investigation in preclinical models.