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

Abstract

Abstract

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.

Keywords

Box-Behnken optimization / Copper oxide nanoparticles / Antimicrobial synergy / Antibiotic resistance / Green nanotechnology

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