Combating multidrug-resistant pathogens, breast cancer (MCF-7) and hepatocellular carcinoma (HepG2) using biofabricated zinc oxide nanoparticles.

The escalating global burden of multidrug-resistant (MDR) infections and cancer necessitates the development of sustainable and multifunctional therapeutic strategies. Green-synthesized zinc oxide nanoparticles (ZnONPs) have emerged as promising nanomaterials owing to their unique physicochemical properties, broad-spectrum bioactivity, and environmentally friendly production routes. In this study, ZnONPs were biosynthesized using Citrus sinensis (orange) peel waste extract as a renewable reducing and stabilizing agent. The biosynthesized nanoparticles were comprehensively characterized by UV-Vis spectroscopy, FTIR, XRD, DLS, zeta potential analysis, SEM, TEM, HR-TEM, and SAED. Their antimicrobial, Time-kill kinetics, antibiofilm, antioxidant, and anticancer activities were systematically evaluated against clinically relevant MDR pathogens and human cancer cell lines. The biosynthesized ZnONPs exhibited an absorption peak at 368 nm, a mean hydrodynamic size of 32.45 nm, low polydispersity (0.186), and a zeta potential of - 36.4 mV, confirming excellent colloidal stability and highly crystalline hexagonal wurtzite nanostructures. ZnONPs demonstrated broad-spectrum antimicrobial activity, with MIC and MBC/MFC values ranging from 120 to 512 and 240 to 1024 µg/mL, respectively. Time-kill assays revealed concentration- and time-dependent antimicrobial effects, achieving complete microbial eradication at 4MIC within 8-12 h, while C. albicans remained the least susceptible species. Moreover, ZnONPs significantly inhibited microbial biofilm formation, with inhibition rates ranging from 55.2 to 68.3% at sub-MIC concentrations. Strong antioxidant activity was observed, with DPPH and ABTS radical-scavenging IC₅₀ values of approximately 38-40 μg/mL. Importantly, ZnONPs exhibited potent, dose-dependent, and selective anticancer activity against MCF-7 and HepG2 cells, with IC₅₀ values of 58.98 and 87.93 µg/mL, respectively, while showing minimal toxicity toward normal MCF-10A cells (IC₅₀ > 250 µg/mL; selectivity index > 4.24 for MCF-7 and > 2.84 for HepG2). Confocal microscopy demonstrated preferential and concentration-dependent nanoparticle uptake by cancer cells, which closely correlated with their enhanced cytotoxicity. Mechanistic studies further confirmed apoptosis induction through activation of the intrinsic mitochondrial pathway, as evidenced by significant upregulation of the pro-apoptotic gene BAX (3.4-fold in MCF-7 and 2.9-fold in MDA-MB-231 cells) and marked downregulation of the anti-apoptotic gene BCL2 (0.4-fold and 0.3-fold, respectively; p < 0.0001). Orange peel-mediated ZnONPs exhibit potent antimicrobial, antibiofilm, antioxidant, and selective anticancer activities, underscoring their potential as sustainable multifunctional nanotherapeutics for combating antimicrobial resistance and cancer.