Mycogenic synthesis of copper oxide nanoparticles using response surface methodology and assessment of their...

Endophytic fungi represent a strategic biological template for the synthesis of metallic nanomaterials with enhanced stability and biocompatibility. This study investigates the bio-assisted synthesis of copper oxide nanoparticles (B-CuO NPs) using an aqueous extract of Alternaria alternata, an endophyte isolated from the medicinal plant Phyllanthus emblica. The synthesis was achieved through a bio-inspired chemical reduction approach using sodium borohydride, where fungal secondary metabolites, profiled by Gas chromatography-mass spectrometry, acted as critical stabilizing and capping agents. For technical precision and reproducibility, the synthesis parameters were refined using Response Surface Methodology coupled with Box-Behnken design. Comprehensive physicochemical characterization confirmed the formation of stable, crystalline, phase-pure, nanosized particles with a polydispersity index of 0.242, indicative of uniform size and colloidal stability. Biological efficacy was evaluated through diverse antioxidant assays, antibacterial analysis against V. parahaemolyticus and E. coli, and safety by hemolysis testing. Results demonstrated strong radical scavenging potential, particularly in the hydroxyl assay (77.94%), and recorded inhibition zones of 24-29 mm supported by time-dependent growth kinetics. Mechanistic investigations via 2',7'-dichlorodihydrofluorescein diacetate staining and 5,5'-dithiobis-(2-nitrobenzoic acid) assays confirmed that B-CuO NPs key contributing factors associated with oxidative stress-mediated antibacterial pathways through a significant surge in intracellular reactive oxygen species (up to 81.53%) and the subsequent depletion of reduced glutathione levels (up to 51.13%). Hemocompatibility assessment demonstrated below 9% hemolysis at 600 µg/mL, confirming safety for biomedical and intravenous applications. These findings establish Alternaria alternata-mediated B-CuO NPs as a promising, nanomaterial platform with dual antibacterial and antioxidant potential for future biomedical applications.