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Zinc oxide nanoparticles (ZnO-NPs) have gained attention for their anticancer and antimicrobial activity. Our study highlights a novel anti-virulence strategy against multidrug-resistant pathogens by showing that ZnO-NPs suppress bacterial virulence and quorum-sensing genes ( rmpA , fnbA , cna , and LuxS ) at sub-MIC levels. In this study, we synthesized ZnO-NPs using the chemical co-precipitation process, we confirmed their characteristics with the techniques TEM, XRD, UV-Vis spectroscopy, and measuring their zeta potential. ZnO-NPs are almost spherical, 30 nanometers in size, with a notable UV absorption at 375 nm and a zeta potential of -9.25 mV. ZnO-NPs showed impressive inhibition zones, especially against E. coli , with a zone size of 30.33 mm. The MIC of ZnO-NPs varied, with Staphylococcus aureus needing the highest concentration at 500 µg/mL, while E. coli and Pseudomonas aeruginosa needed 62.5 and 125 µg/mL, respectively. We also looked at how these particles affect cancer cells and found they reduced their growth in a dose-dependent way, with IC50 values of around 79 and 151 µg/mL for MCF-7 and HepG2 cells. Interestingly, when we examined the bacteria at the genetic level, we saw that ZnO-NPs at 62.5 µg/mL resulted in down-expression of key virulence genes like rmpA , fnbA , and cna to about 60% of normal levels, and the quorum-sensing gene luxS to 80%. This suggests that even at lower doses, the particles can weaken bacterial ability to cause disease without being fully bactericidal. Overall, our results emphasize how ZnO-NPs can be both antibacterial and anticancer agents, especially by targeting gene expression to boost their effectiveness.