Antifungal, Antibiofilm and Anticytotoxic Properties of Biogenic CuO Nanoparticles Derived From Moringa oleifera Leaves Against Vaginal Candidiasis

Mohammad Zubair,1,2 Yazeed Albalawi,3 Roba Alatawy1,2 1Department of Medical Microbiology, Faculty of Medicine, University of Tabuk, Tabuk, Kingdom of Saudi Arabia; 2Molecular Microbiology and Infectious Disease Research Unit, University of Tabuk, Tabuk, Kingdom of Saudi Arabia; 3Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tabuk, Tabuk, Kingdom of Saudi ArabiaCorrespondence: Mohammad Zubair, Email [email protected]: Moringa oleifera leaf extract can mediate green synthesis of metal oxide nanoparticles with potential antimicrobial properties. This study evaluated copper oxide nanoparticles (CuO NPs) synthesized using M. oleifera for antifungal, antibiofilm, and cytocompatibility effects in a vaginal-simulant medium.Methods: CuO NPs were synthesized using M. oleifera leaf extract and characterized by FT-IR, XRD, UV–Vis, SEM, and EDX. 10 Candida albicans isolates and ATCC 90028 were screened for biofilm production; strong biofilm producers were selected for detailed testing. Antifungal activity (viability assays, MIC50, MIC90, MFC) and dose–response relationships were determined. Effects at sub-inhibitory concentrations on EPS production, cell-surface hydrophobicity, membrane permeability, ROS generation, germ tube formation, and biofilm formation/disruption were assessed. In vitro biocompatibility was evaluated by MTT assays on L929 fibroblasts, RAW 264.7 macrophages, MCF-12F epithelial cells, PBMCs, and by hemolysis assay.Results: CuO NPs produced a concentration-dependent reduction in Candida viability with high dose–response correlation (R2 = 0.967– 0.9779). MIC50, MIC90, and MFC values ranged 44.5– 103 μg/mL (ATCC 90028: MIC50 44.5 μg/mL, MIC90 86.5 μg/mL, MFC 91 μg/mL; C. albicans 6: MIC50 51.5 μg/mL, MIC90 79.5 μg/mL, MFC 103 μg/mL). At sub-inhibitory concentrations, CuO NPs reduced EPS production and cell-surface hydrophobicity, increased membrane permeability and ROS generation, inhibited germ tube formation, and limited biofilm formation and integrity. Biocompatibility testing showed concentration-dependent cytotoxicity in fibroblasts and macrophages at higher doses, relative tolerance of MCF-12F cells, low hemolysis, and acceptable PBMC viability at lower concentrations.Conclusion: M. oleifera-mediated CuO NPs exhibit potent in vitro antifungal and antibiofilm activity against C. albicans and affect multiple virulence-related pathways, with a distinguishable therapeutic window based on cell-type–dependent cytotoxicity. These findings support further development and optimization of topical intravaginal formulations of green-synthesized CuO NPs for candidal biofilm-associated infections, with additional in vivo safety and efficacy studies needed. Keywords: Moringa oleifera, vaginal candidiasis, CuO nanoparticles, antifungal, anti-biofilm