IntroductionNanotechnology is an emerging scientific and technological topic that is gaining attention from several sectors. Metallic nanoparticles (NPs) are often used in the biomedical sector1. The reactivity of NPs is much greater than that of bulk materials due to their extensive surface area2. Biologists are becoming increasingly interested in the utilization of NPs for various applications, including drug delivery, gene delivery, and material detection in biological samples3. Moreover, NPs also show potential in combating pathogens and cancers4. A previous investigation demonstrated the potential biological activities of biologically synthesized NPs, such as AuNPs synthesized using fresh leaves of Polygala elongata, which demonstrated potential anticancer activity against the lung cancer cell line (A549)5. Moreover, fluorinated graphene oxide nanosheets derived from Lissachatina fulica snail mucus demonstrated antibacterial efficiency against bacterial pathogens and anticancer activity against the pancreatic cancer cell line (PANC1) at low concentrations6. Another study reported that an Acorus calamus-mediated CuFe2O4/reduced graphene oxide nanocomposite showed antibacterial activity, demonstrating relative inhibition zone diameters ranging from 10 to 13 mm against the tested strains, and also low cytotoxic activity against the MCF-7 cell line with an IC50 of 360 µg/mL7. Similarly, a ZnO/g-C3N4/V2O5 heterogeneous nanocomposite revealed potential antibacterial and photocatalytic activities8. Zinc oxide nanoparticles (ZnO-NPs) offer a wide range of capabilities that make them useful in several disciplines, including chemical, physical, and biological applications9. The semiconducting and oxidizing properties of ZnO nanoparticles play an important role in their biological efficiency. The size and crystalline structure of ZnO nanoparticles determine their reactivity and chemical properties10. Furthermore, ZnO-NPs have substantial photocatalytic activity on several substances, including dyes, medicines, and toxins11. Studies have shown that ZnO-NPs are more compatible with the physiological environments of the body compared to their bulk form. This suggests that their medicinal properties may provide distinct advantages12. Green synthesis is a commonly used technique utilized by biological systems, including microorganisms, plant extracts, or their constituents, for the formation of nanoparticles13. Generally, biological-based synthesis is more advantageous than alternative chemical and physical approaches in terms of cost-effectiveness, single-step processing, and absence of chemical waste throughout the process14. Therefore, the synthesis of nanoparticles using biological means is a secure and non-toxic procedure suitable for pharmaceutical and medical applications15. The production of bio-based nanoparticles for use in the pharmaceutical, cosmetic, culinary, and environmental industries has seen significant growth in recent decades16. A previous investigation demonstrated that biogenic ZnONPs synthesized using Pleurotus sajor-caju extract demonstrated antibacterial activity against Staphylococcus aureus (6.2 ± 0.1 mm), Streptococcus mutans (5.4 ± 0.4 mm), and Bacillus subtilis (5.2 ± 0.1 mm)17. Moreover, Hypsizygus ulmarius-derived ZnO nanoparticles revealed potential antibacterial activity with relative inhibition zone diameters of 9.0 ± 0.1 mm and 14.2 ± 0.2 mm against K. pneumoniae and S. aureus, respectively, and also potent anticancer activity against MCF-7 breast cancer cells with relative LC50 values of 29.07 µg/mL18. Zinc oxide nanoparticles are very promising nanomaterials that have been shown to possess excellent physiochemical stability and unique surface attraction capabilities. As a result, they may be effectively used for many biomedical applications, including antifungal and antibacterial treatments19. Moreover, ZnO NPs have been acknowledged by the FDA-USA as the safest kind of nanoparticles20. Multiple investigations have been conducted to assess the fungicidal effects of ZnO NPs on various fungal pathogens21,22. These studies have shown that ZnO NPs exhibit little toxicity towards human cells23. The toxicity mechanisms of ZnO-NPs towards bacteria or fungi mostly rely on the size, shape, and concentration of ZnO-NPs, as well as the specific kind of medium used. In general, as the size of a particle is smaller, its surface area to volume ratio increases, resulting in stronger antimicrobial actions24. The physical interaction between ZnO-NPs and the fungal cell wall results in the disruption of the cell wall’s structure25. This interaction also triggers the excessive creation of reactive oxygen species (ROS), including hydroxyl groups, superoxide anion radicals, and hydrogen peroxide, inside the cells. Consequently, these ROS may cause the demise of the cells. The amount of ROS produced is directly correlated with the extent of the organism’s surface area that is in contact with the ZnO NPs. In addition, several studies have shown that ZnO-NPs have dose-dependent effects, wherein larger concentrations of ZnO-NPs result in increased antifungal activity26. Candida spp. are the predominant fungal pathogens that opportunistically infect people. They are often acquired in hospitals and rank as the fourth most prevalent cause of nosocomial infections27. C. albicans is the primary factor responsible for almost all forms of Candidiasis28. Conventional antifungal medications work by either suppressing the development of fungal cells or causing their death. Both scenarios involve medications that impose significant selection pressure and may readily result in the development of drug resistance29. Moreover, the challenge of managing candidiasis is profoundly magnified in individuals with disabilities. This population often experiences compromised immune function, prolonged hospitalization, frequent use of broad-spectrum antibiotics, and the presence of medical devices like catheters and ventilators, all of which are significant risk factors for fungal colonization and subsequent infection. Furthermore, the high prevalence of drug-resistant Candida strains, such as fluconazole-resistant C. albicans creates a critical therapeutic impasse. Recurrent and persistent infections in this vulnerable demographic lead to increased morbidity, extended rehabilitation times, and a substantial additional burden on both patients and healthcare systems, underscoring an urgent need for novel, effective, and safer antifungal strategies30. Recently, researchers have focused on targeting fungal virulence as a potential therapeutic target31. Suppressing the conversion of C. albicans into its disease-causing state by reducing the virulence of the fungus has emerged as a successful approach for combating fungal infections32. A previous investigation demonstrated that biogenic ZnO NPs from L. sativum seeds extract exhibited strong antimicrobial activity, with higher efficacy against S. aureus (23 ± 1.25 mm inhibition at 120 mg/mL) than E. coli (16 ± 1.00 mm), while remaining hemocompatible at low doses33. Another previous investigation demonstrated that biogenic ZnONPs formulated using L. sativum seed extract exhibited broad-spectrum antibacterial activity against both Gram-positive (S. aureus: 18 ± 1.1 mm, Micrococcus luteus: 15 ± 1.2 mm) and Gram-negative pathogens (Salmonella enterica serovar Typhi: 15 ± 1.2 mm, Salmonella Setubal: 18 ± 1.4 mm, Enterobacter aerogenes: 14 ± 1 mm) at 4 mg/mL. The NPs showed comparable efficacy to the antibiotic cefexime (e.g., S. aureus: NPs 18 mm vs. cefexime 19 mm), while the crude plant extract alone displayed no significant inhibition ( 0.05). Furthermore, no significant difference was found for ZnO-NPs combined with ketoconazole, itraconazole, and amphotericin B against C. albicans strain (p > 0.05).The FICI results quantitatively confirmed the interactions that were detected by the disk diffusion assay. The most potent synergistic effects were observed for the combination of ZnONPs with nystatin against both C. albicans (FICI = 0.38) and C. tropicalis (FICI = 0.25). Additive interactions were confirmed for ZnONPs with fluconazole and terbinafine against C. albicans (FICI = 0.75 and 0.63), and with ketoconazole and fluconazole against C. tropicalis (FICI = 0.75 and 0.63). For other combinations, such as those with itraconazole and amphotericin B against both species, the indifferent FICI scores aligned with their low, non-significant IFA values. Crucially, the FICI score formally identified an antagonistic interaction for the combination of terbinafine and ZnONPs against C. tropicalis (FICI = 4.00).The antimicrobial effects of ZnO-NPs are attributed to multiple mechanisms, including the production of reactive oxygen species (ROS)78compromising the integrity of cell membranes79releasing toxic zinc ions (Zn²⁺)80, interacting with microbial DNA and proteins81and triggering oxidative stress82. Zinc oxide nanoparticles can produce ROS like hydroxyl radicals (•OH), hydrogen peroxide (H₂O₂), and superoxide anions (O₂•−) when exposed to light or specific conditions83. These ROS are extremely reactive and can cause damage to microbial cell membranes, lipids, proteins, and DNA, ultimately resulting in cell death84. Moreover, ZnO-NPs can directly interact with microbial cell membranes, leading to membrane damage. This disturbance can enhance membrane permeability, causing the leakage of cellular materials and, ultimately, cell death and lysis85. The biosynthesized ZnO-NPs can break down and release zinc ions (Zn²⁺) that bind to microbial proteins and enzymes, disrupting their normal functions and hindering cell growth. Additionally, Zn²⁺ can interfere with crucial metal ion transport systems, adding further stress to the candidal cells86. Due to their small size, ZnO-NPs can infiltrate microbial cells and interact with internal components such as DNA and proteins. This interaction may result in DNA damage, proteins losing their structure, and enzymes becoming inactive, ultimately disrupting essential cellular functions87.The notable difference in synergy between nystatin and amphotericin B (AmB), both polyene antifungals, can be attributed to their distinct physicochemical properties and mechanisms. While both target ergosterol, nystatin molecules are smaller and form smaller, more transient pores in the fungal membrane compared to the larger, more stable channels formed by AmB aggregates88. We hypothesized that the smaller nystatin-induced pores are sufficient to cause membrane leakage but are also more permissive for the subsequent influx of ZnO-NPs or Zn²⁺ ions, leading to amplified intracellular oxidative damage.The profound synergy observed between ZnO-NPs and nystatin, particularly against C. tropicalis (IFA: 0.99), is of significant clinical relevance. By combining ZnO-NPs with a conventional antifungal, the effective dose of the drug required to achieve fungicidal activity can be substantially lowered. This approach has a high potential to reduce the drug concentration below its toxic threshold, thereby mitigating dose-limiting side effects like the nephrotoxicity associated with high doses of polyenes89. This is a crucial strategy for managing chronic or recurrent infections, especially in debilitated patients.This synergy is likely multimodal: nystatin binds to ergosterol, creating pores in the fungal membrane90which potentially facilitates the increased uptake of ZnO-NPs (Fig. 13). Subsequently, the internalized NPs can induce massive ROS generation91 and release Zn²⁺ ions92leading to catastrophic oxidative damage and disruption of metabolic enzymes93. The weaker synergy with amphotericin B, another polyene, might be attributed to differences in their binding affinity to ergosterol or their aggregation state in solution. The synergy with azoles like fluconazole may stem from the combined stress of ergosterol biosynthesis inhibition (azole) and direct membrane/oxidative damage (ZnO-NPs)94.Fig. 12Antifungal activity of ZnO NPs against C. albicans and C. tropicalis. Zones of inhibition for various NP concentrations (0.125-1.000 mg/disk), with terbinafine (30 µg/disk) as positive control and methanol solvent as negative control disks.Full size imageTable 3 Antifungal activity of the biosynthesized znonps. Against C. albicans and C. tropicalis strains.Full size tableTable 4 Synergistic antifungal activity of ZnO-NPs with conventional antifungals against C. albicans and C. tropicalis. Data represent mean Inhibition zone diameters (mm ± SE, *n* = 3). Superscript letters (ᵃ, ᵇ) indicate statistical significance within columns (Tukey’s test, *p* < 0.05). IFA = Increase in fold area; ns = not significant (p ≥ 0.05). FICI (Fractional inhibitory concentration Index) interpretation: ≤0.5 synergistic, > 0.5–1.0 additive, > 1.0–4.0 indifferent, > 4.0 antagonistic.Full size tableFig. 13Proposed mechanism of synergy between ZnO NPs and nystatin: (1) membrane pore formation by nystatin, (2) enhanced uptake of ZnO NPs/Zn²⁺, (3) ROS generation, and (4) intracellular damage.Full size imageAntioxidant activityThe antioxidant activity of the biogenic ZnO NPs synthesized using L. sativum seeds extract was assessed by DPPH scavenging assay. The DPPH inhibition percentages were found to be 28.14 ± 0.87, 36.48 ± 1.14, 48.32 ± 1.35, 61.87 ± 1.56, 78.95 ± 1.64 and 86.78 ± 1.83 for the different ZnONPs concentrations of 50, 100, 200, 400, 800 and 1600 µg/mL, respectively (Fig. 14). Accordingly, the antioxidant activity of ZnO-NPs was concentration dependent. DPPH is a well-known and stable synthetic solid radical commonly used to assess the antioxidant potential of various compounds. The spectrophotometer was used to quantify the reduction of DPPH by receiving hydrogen or electrons from ZnO nanoparticles, which caused the colour to change from purple to yellow. Linear regression analysis revealed that the IC50 of ZnO NPs was 335.48 µg/mL whereas the IC50 of ascorbic acid was 131.03 µg/mL. Collectively, the biosynthesized ZnO-NPs have potential for creating potent antioxidants that could be utilized in treating numerous diseases linked to oxidative stress.Fig. 14Concentration-dependent DPPH radical scavenging activity of biosynthesized ZnO NPs. The free radical scavenging capacity of ZnO NPs (50–1600 µg/mL) was assessed and compared to ascorbic acid as a standard antioxidant control. Data are presented as mean percentage inhibition ± standard error (SE) of three replicates.Full size imageCytotoxic assay of ZnONPsThe antiproliferative activity of the biosynthesized ZnONPs was evaluated using MTT assay against HUH7 and WI38 cell lines. The cytotoxic effect of ZnONPs was found to be concentration dependent against HUH7 cell line, demonstrating relative cell viability percentages of 78.58, 64.85, 58.73 and 37.85% at the concentrations of 25, 50, 100 and 200 µg/ml, respectively. On the other hand, the bioinspired ZnONPs revealed a lower cytotoxic effect against WI38 normal cell line demonstrating relative cell viability percentages of 62.96 and 45.92% at ZnONPs concentrations of 100 and 200 µg/ml, respectively. As shown in Fig. 15, the cytotoxic effect of ZnONPs was concentration-dependent in both cell lines. Post-hoc analysis confirmed that ZnONPs exhibited significantly greater cytotoxicity in HUH-7 cancer cells compared to WI-38 normal cells at concentrations of 100 µg/ml (p