Multifunctional bimetallic Ag-CuO and Ag-ZnO nanoparticles biosynthesized using Plantago ovata for biomedical...

This study investigated the green synthesis, physicochemical characteristics, and biological activities of Ag-CuO and Ag-ZnO mixed-phase nanomaterials using Plantago ovata seed extract. Monometallic AgNPs, CuONPs, and ZnONPs, the free extract, and metal-salt precursors were included as comparators. Three independently prepared extract batches showed limited batch-to-batch variability in pH, dry-solids concentration, and extraction yield, with coefficient-of-variation values below 9%. UV-vis, FTIR, XRD, HR-TEM, EDX, and elemental mapping collectively supported the coexistence of Ag and the corresponding CuO or ZnO crystalline phases. TEM-derived mean particle sizes were 72.02 ± 28.49 and 83.30 ± 32.10 nm for Ag-CuONPs and Ag-ZnONPs, respectively, while their Z-average hydrodynamic diameters were 103.8 and 124.4 nm. Their zeta potentials were -36.1 and -28.1 mV, respectively. Both mixed-phase formulations exhibited broad-spectrum antibacterial and concentration-dependent antibiofilm activities. Ag-CuONPs generally showed stronger broth-dilution activity, with minimum inhibitory concentrations of 125-500 µg mL^-1, and produced maximum biofilm inhibition of 84.91% against Staphylococcus aureus and 61.78% against Pseudomonas aeruginosa. Both formulations increased extracellular protein leakage and reduced the expression of biofilm- and virulence-associated genes, including fnbA, cna, algD, and lasB. In cancer-cell assays, Ag-CuONPs showed the greatest activity against Caco-2 cells, with an IC₅₀ of 102.89 ± 8.05 µg mL^-1, whereas Ag-ZnONPs were most active against MCF-7 cells, with an IC₅₀ of 116.98 ± 3.23 µg mL^-1. Annexin V/PI analysis confirmed substantial apoptosis in Caco-2 cells, while Ag-ZnONPs induced pronounced G₂/M-phase accumulation. Both treatments increased BAX and CASP3 expression and decreased BCL2 expression. Ag-CuONPs exhibited the strongest DPPH-scavenging activity among the nanomaterials, whereas Ag-ZnONPs showed the greatest ABTS-scavenging activity. Overall, the results demonstrate composition-dependent antibacterial, antibiofilm, radical-scavenging, and anticancer activities of the P. ovata-mediated mixed-phase nanomaterials, supporting their potential for further biomedical investigation while requiring additional mechanistic and in vivo validation.