Box-Behnken optimized copper oxide nanoparticles from Thymus vulgaris potentiate efficacy against multidrug-resistant bacterial pathogens and exhibit anticancer activity.

The dual crises of antimicrobial resistance and cancer demand innovative therapeutic platforms that overcome conventional treatment limitations. This study uniquely combines systematic Box-Behnken optimization of green-synthesized copper oxide nanoparticles from Thymus vulgaris with comprehensive evaluation of their synergistic antimicrobial and anticancer activities. HPLC profiling identified quercetin (55.92%), chlorogenic acid (15.33%), and gallic acid (12.28%) as principal phytochemical reducing and capping agents. Statistical optimization (R2 = 0.9886) established copper acetate concentration (F = 670.48, p < 0.0001) and incubation time (F = 124.11, p < 0.0001) as critical synthesis determinants, yielding monodisperse spherical nanoparticles (19-25 nm TEM; Z-average 119.2 nm, PDI 0.22; ζ-potential - 45.8 mV). XRD confirmed a crystalline monoclinic CuO phase, while FTIR validated phytochemical surface functionalization. TE-CuONPs exhibited concentration-dependent bactericidal activity (MIC 250-950 μg/mL; MBC/MIC ≤ 0.58) against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis as well as inhibition of biofilm formation in S. aureus and P. aeruginosa, with BIC₅₀ of 299 and 315 μg/mL, respectively. Critically, checkerboard assays revealed strong synergy with gentamicin (FICI 0.13-0.28), achieving eightfold dose reduction for both agents against S. aureus and P. aeruginosa. Time-kill kinetics demonstrated accelerated bacterial eradication, with combination therapy achieving ≥ 3-log₁₀ reduction 8-12 h faster than monotherapies, a clinically significant advantage for acute infections. Furthermore, TE-CuONPs showed moderate antiproliferative activity (IC₅₀ = 117.26 μg/mL) against MCF-7 breast cancer cells, with limited selectivity over normal fibroblasts (SI = 1.85), representing a sixfold enhancement over the crude extract. Additionally, Flow cytometric analysis revealed profound apoptotic induction, with 77.25% of cancer cells undergoing cell death (29.73% early apoptosis, 47.52% late apoptosis/necrosis). DPPH radical scavenging (IC₅₀ = 55 μg/mL) demonstrated a threefold superior antioxidant capacity versus plant extract alone. These findings advance the reproducible botanical nanoparticle synthesis and translational potential of plant-mediated nanomedicine for infectious disease management.