Green and Ecofriendly Mycosynthesis of Nanocomposite Based on Zinc and Magnesium Oxide Nanoparticles Using Endophytic Sarocladium kiliense: Characterization, Anticancer Activity, Antimicrobial and Antibiofilm Activities

ABSTRACT

Fungal species are increasingly recognized as efficient bio‐factories for the biosynthesis of nanoparticles with distinctive functionalities. In this study, zinc oxide‐magnesium oxide nanocomposite (ZnO‐MgO NCs) were biologically created employing the Sarocladium kiliense PV248633.1 fungal strain. Ultraviolet‐visible spectroscopy confirmed nanoparticle formation through characteristic absorption peaks, while Fourier‐transform infrared spectroscopy identified functional groups associated with metal‐oxygen bonds. Also, the nanoparticle size and surface characteristics Transmission electron microscopy and dynamic light scattering analyzes revealed an average particle size of 35 nm. Biological assessments demonstrate potent antitumor activity (IC₅₀ = 78.1 µg/mL) against MCF‐7 breast cancer cells, alongside minimal cytotoxicity against normal WI‐38 cells (IC₅₀ = 218.7 µg/mL), indicating selective toxicity. Antibacterial evaluations showed particularly high efficacy against Acinetobacter baumannii and Pseudomonas aeruginosa occasionally surpassing the performance of cefepime. Dose‐dependent antimicrobial activity was confirmed through well diffusion and broth microdilution techniques, with minimum inhibitory concentrations highlighting strong bactericidal potential. Biofilm inhibition assays revealed strain‐specific responses, with A. baumannii being the most susceptible. Furthermore, the synergistic combination of the biosynthesized nanocomposite with cefepime enhanced antibacterial outcomes against selected isolates, suggesting potential for addressing drug resistance. Overall, the mycogenic ZnO‐MgO NCs synthesized from S. kiliense exhibited substantial anticancer, antibacterial, and antibiofilm properties, locating this nanocomposite as a promising candidate for biomedical purposes.

Biosynthesis and multifunctional activities of ZnO–MgO nanocomposites using Sarocladium kiliense extract. The fungal extract of S. kiliense was utilized for the green synthesis of ZnO–MgO nanocomposites (NCs) using zinc nitrate and magnesium nitrate as precursors. The obtained NCs were characterized by UV–Vis, TEM, DLS, and XRD analyzes, confirming nanosized crystalline structures. The biosynthesized ZnO–MgO NCs exhibited potent antimicrobial, antibiofilm, and anticancer activities, suggesting their potential application in biomedical and pharmaceutical fields.

1. Introduction

Antimicrobial resistance represents a growing and serious public health challenge, particularly in relation to Gram‐negative bacteria. These organisms employ advanced survival strategies that undermine the efficacy of conventional antibiotics, including the production of resistance enzymes, activation of efflux systems, and the formation of protective biofilms. Biofilms, further enhance resistance and persistence, significantly complicating therapeutic efforts and limiting treatment success (ismail et al. 2025). At the same time, the global burden of cancer continues to rise, posing significant challenges to public health systems. Among all types, breast cancer remains the most diagnosed malignancy worldwide. Its increasing incidence has been attributed to a combination of hormonal factors, inherited genetic risks, lifestyle‐related behaviors, and the absence of widespread access to early diagnostic tools and effective treatment options, particularly in underserved regions (Hashem et al. 2025).

Nanotechnology has revolutionized material science by enabling the management of materials at the nanoscale, leading to the development of enhanced properties and functionalities. Nanoparticle (NP) synthesis, in particular, has emerged as a powerful approach for creating novel materials with exceptional physicochemical characteristics. Metallic and metal oxide NPs have garnered significant attention owing to their high surface‐area‐to‐volume ratio, unique surface properties, and their consequent suitability for diverse applications in medicine, agriculture, and environmental remediation (Elbas et al. 2025). Despite their potential, traditional synthesis methods such as chemical reduction and physical deposition often involve hazardous reagents, require high energy input, and generate toxic byproducts that pose risks to ecosystems and human health. Consequently, there is growing interest in sustainable or eco‐friendly approaches, including biological synthesis pathways that employ bacteria, fungi, algae, and plants (Hamed 2024). These green methods offer safer, cost‐effective, and scalable alternatives aligned with the green chemistry principles. Fungi have recently emerged as suitable and promising bio‐factories for eco‐friendly NPs synthesis. In comparison with other microbial systems, fungal strains offer several advantages, including high tolerance to metals, the ability to produce substantial biomass, simple cultivation requirements, and the release of diverse bioactive compounds that are utilized in reduction and NPs stabilization. These compounds include proteins, polysaccharides, enzymes, and phenolics that perform a crucial function in reducing metal ions and controlling NP size and morphology. Fungal‐mediated synthesis is also associated with enhanced NPs stability, uniformity, and biological activity relative to traditional fabrication techniques (Attia et al. 2023; Bano et al. 2023).

The strong antibacterial and anticancer properties of metal oxide NPs make them especially noteworthy. Because zinc oxide (ZnO) NPs can cause free radical production, bacterial cell membrane damage, and cancer cell apoptosis, resulting in well‐documented antibacterial and anticancer effects (Hashem et al. 2023). Similarly, magnesium oxide (MgO) NPs are promising candidates for biomedical applications due to their antimicrobial efficacy, biofilm inhibition, and biocompatibility. Through synergistic interactions, two metal oxides can be combined to form a BNPs system that improves their physicochemical and biological characteristics. In addition to improving stability and reducing toxicity, this integration can enhance catalytic efficiency, antimicrobial potency, and anticancer activity (Bano et al. 2024). The combined BNPs frequently perform better than individual NPs, overcoming drawbacks like low bioavailability, narrow selectivity, and structural instability to increase their potential uses in nanobiotechnology and medicine (Ramezani Farani et al. 2023; Bano et al. 2022).

The filamentous endophytic fungus Sarocladium kiliense (S. kiliense) is recognized for producing a wide array of secondary metabolites with notable antioxidant and antimicrobial activities. However, its application in the biosynthesis of nanomaterials, particularly NCs, remains largely unexplored despite its bioactive potential. In addition, the use of S. kiliensis unique8633.1 unique in that it secretes a variety of bioactive metabolites that function as stabilizing and reducing agents at the same time, allowing for greater biological activity, regulated particle size, and increased stability. S. kiliense is positioned as a viable bio‐factory for producing ZnO‐MgO nanostructures with selective properties by utilizing these special metabolic capabilities, which offer a unique environmentally benign pathway for nanocomposite biosynthesis (Eskander et al. 2020). Harnessing the metabolic capabilities of underutilized fungal strains offers a promising route for the green synthesis of advanced nanostructures with potential therapeutic applications. This study seeks to address this gap by employing S. kiliense PV248633.1 for the biosynthesis of ZnO‐MgO NCs and evaluating their anticancer, antimicrobial, and antibiofilm activities.

2. Material and Methods

2.1. Chemicals and Reagents

Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O) and magnesium nitrate hexahydrate (Mg(NO₃)₂·6H₂O) were obtained from Sigma‐Aldrich, Cairo, Egypt, products and utilized as a primary metal ion source. Sodium hydroxide was employed as a precipitating agent. For microbial culture, Czapek Dox broth (CDB) and Czapek Dox Agar (CDA) were procured from Himedia, Cairo, Egypt. Distilled water was used as the solvent in the procedures of most experimental procedures.

2.2. Green Synthesis of ZnO‐MgO NCs

The endophytic fungal strain S. kiliense PV248633.1, previously isolated and characterized in an earlier study (Selim et al. 2025a), was employed for the green synthesis of ZnO‐MgO NCs. Three fungal disks (each 0.8 cm in diameter), obtained after 72 h of growth, were transferred into CDB (100 ml) followed by incubation under neutral pH conditions with constant shaking at 150 rpm for 6 days at 28  ±  2°C. Afterwards, the fungal biomass was separated using Whatman filter paper No. 1, followed by thoroughly washing using distilled water for complete removal of any residual media components. Approximately 15 gm of the harvested biomass were transferred into distilled water (100 ml) and agitated for 48 h at 150 rpm to generate the fungal biomass filtrate (FBF). The obtained mixture was centrifuged for 10 min at 5000 rpm, after which the supernatant was collected and used for biocatalyst nanoparticle synthesis. For the NCs fabrication, 2.0 mM Zn(NO₃)₂·6H₂O and 3.0 mM Mg(NO₃)₂·6H₂O were added to 100 ml of the obtained FBF, followed by darkness incubation at 28 ± 2°C, 150 rpm, and pH 8 for 24 h. The formation of a white precipitate signaled the successful synthesis of ZnO‐MgO NCs, which were then collected and dried at 120°C for 24 h (Saied 2021).

2.3. ZnO‐MgO NCs Characterization

The optical properties of the biosynthesized ZnO‐MgO NCs were studied utilizing ultraviolet‐visible (UV‐vis) spectroscopy (JENWAY 6305, Staffordshire, UK) across the wavelength between 200 nm and 800 nm for detection of the characteristic peak of their surface plasmon resonance (SPR). To identify the functional groups involved in NCs formation, Fourier‐transform infrared (FTIR) spectroscopy (Cary‐660 model) was carried out with the KBr pellet technique, covering the 400–4000 cm⁻¹ spectral range. Morphological characteristics and particle dimensions were examined through transmission electron microscope imaging (TEM) (TEM, JEM‐2100 Plus, Jeol, Japan), while the obtained NCs size distribution was examined by means of the dynamic light scattering (DLS) technique (DLS, Nano ZS, Malvern, UK). Structural properties, including crystal size and degree of crystallinity, were confirmed by X‐ray diffraction (XRD) analysis (XRD‐6000, Shimadzu Scientific Instruments, Japan). Surface topology was further investigated by scanning electron microscope (SEM) imaging (SEM, ZEISS, EVO‐MA10, Germany). Additionally, the biosynthesized ZnO‐MgO BNPs' elemental composition, distribution, and purity were assessed using energy‐dispersive X‐ray (EDX) spectroscopy (EDX, Bruker, Germany).

2.4. Anticancer Activity

The cytotoxic potential of the biogenic ZnO‐MgO NCs was assessed using the MTT assay against WI‐38, a normal human lung fibroblast cell line (Van de Loosdrecht et al. 1994). Cell viability was quantified by measuring optical density (OD) at 560 nm, with background correction at 620 nm. Similarly, the antiproliferative effect of the NCs was evaluated using the same assay protocol against the MCF‐7, a breast cancer cell line. For both cell lines, the cell viability percentage and cell growth inhibition were determined using Equations 1 and 2, respectively.

$$\text{Viability}\%=\frac{\text{Treatment OD}}{\text{Control OD}}\mathrm{X}100.$$ (1)

$$\text{Inhibition}\%=100-\text{Viability}\%.$$ (2)

2.5. Antimicrobial Activity

2.5.1. Preliminary Screening

The antimicrobial activity of the biosynthesized ZnO‐MgO NCs, expressed as inhibition zone diameter (IZD), was evaluated against Escherichia coli (E. coli) ATCC 25922, Acinetobacter baumannii (A. baumannii) ATCC 17978, Klebsiella pneumoniae (K. pneumoniae) ATCC 700603, and Pseudomonas aeruginosa (P. aeruginosa) ATCC 25668 using the well diffusion assay (WDA) as described in (Elkady et al. 2025a). Briefly, each tested bacterial strain suspension of 0.5 McFarland standard concentration equivalent to 1.5 × 108 colony‐forming units (CFU)/ml was transferred into Muller Hinton agar (MHA) surface under restricted aseptic conditions to obtain lawn growth form. A sterile cork borer was then used to make 6 wells on the MHA surface. The tested ZnO‐MgO NCs (100 μg/mL), ZnSO₄ (100 μg/mL), MgSO₄ (100 μg/mL), fungal filtrate, cefepime (50 µg/mL) representing the positive control (PC), or dimethyl sulfoxide (DMSO) solvent 0.6% (vol/vol) serving as the negative control (NC) was aseptically inoculated into each well. The plates were pre‐incubated at 4°C for 30 min to allow diffusion of the test materials into the agar, followed by incubation at 37°C for 24 h. The resulting IZDs around each well were measured, and the percentage inhibition (PI) of ZnO‐MgO NCs was calculated according to Equation 3.

$$\mathbf{Percent}\mathbf{Inhibition}\left(\mathbf{PI}\right)=\frac{\mathbf{ZnO}-\mathbf{MgO}\mathbf{BNPs}\mathbf{IZD}}{\mathbf{Cefepime}\mathbf{IZD}}\mathbf{X}\mathbf{100}\mathbf{.}$$ (3)

2.5.2. Quantitative Screening

The capability of the biosynthesized ZnO‐MgO NCs to terminate the growth of the tested Gram‐negative bacterial strains was quantitatively assessed based on the broth microdilution method in 96‐well microtiter plate. This assay determines the NCs minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and tolerance level as described by (Gatou 2024). Briefly, DMSO (0.6 V/V with sterile water) was used to dissolve the tested ZnO‐MgO NCs stock concentration, followed by its two‐fold serial dilution, using Muller Hinton broth (MHB), in each row to obtain NCs ranging from 512 to 16 µg/ml. Subsequently, the tested strain suspension (10 µL) at a concentration of 1.5 × 10⁸ CFU/mL, was inoculated into the wells of each microtiter plate row with a final well volume of 200 µL. Following aerobic incubation for 24 h at 37°C, resazurin (0.015%: 40 µL) was added to each well with subsequent further incubation at 37°C for 2 h. Afterwards, the lowermost concentration of ZnO‐MgO NCs causing no resazurin blue appearance shift towards the red color was reported as the MIC of NCs for the specific strain. Bacterial inoculum from all wells with blue color was then transferred into MHA plate and subsequently incubated at 37°C for 24 h. The minimum ZnO‐MgO NCs concentration exhibiting no visible bacterial colonies was recorded as the MBC of NCs for the tested strain. Finally, the bacterial tolerance level to the biosynthesized ZnO‐MgO NCs was determined based on Equation 4:

$$\mathbf{Tolerance}=\frac{\mathbf{MBC}}{\mathbf{MIC}}.$$ (4)

Subsequently, the findings were interpreted as ZnO‐MgO NCs bactericidal or bacteriostatic effects at tolerance levels ≤ 4 or > 4, respectively.

2.5.3. Biofilm Inhibitory Assay

The crystal violet (CV) staining method for assessing biofilm inhibition entails a multi‐step protocol. The process commences with the preparation of an overnight bacterial culture, which is normalized to a specific OD for use as a standardized inoculum. This bacterial suspension was then introduced into sterile microtiter plates. The sub‐inhibitory concentration of the biosynthesized ZnO‐MgO NCs was then added. Appropriate PC and NC wells were included for reference. The plate was subsequently subjected to static incubation at 37°C for a period of 48 h, a duration optimized for species‐specific biofilm formation. Following incubation, non‐adherent planktonic cells were meticulously removed by gentle aspiration and sequential washing of the wells with sterile phosphate‐buffered saline (PBS) followed by methanol fixation. To each well, 0.1% CV solution was then applied, followed by incubation at room temperature for 15 min, allowing CV dye selective binding to the biofilm matrix. Unbound dye was subsequently removed through 3 washes with PBS, and the plate was air‐dried. The bound CV was then solubilized with 95% ethanol, and the microplate reader was employed for their absorbance quantification at 595 nm. The measured absorbance values, which were directly proportional to the total biofilm biomass, were then utilized to determine the rate of biofilm inhibition in relation to the BNPs untreated samples (Elkady et al. 2024).

2.5.4. Biofilm Clearance Assay

The determination of pre‐formed bacterial biofilm clearance by ZnO‐MgO NCs was quantified through the multi‐stage CV staining assay. The test began by establishing robust and mature biofilms over a 48 h incubation period in sterile 96‐well microtiter plates, with the duration optimized for the specific microorganism. Following this, non‐adherent planktonic cells and spent medium were removed, and the biofilms were subjected to a gentle washing with sterile PBS. The established biofilms were then challenged with a range of concentrations of the ZnO‐MgO NCs for a defined treatment interval, typically 24 h, in parallel with untreated positive biofilm and sterility controls. After the treatment phase, the medium containing the ZnO‐MgO NCs was aspirated, and the wells were rigorously washed to eliminate residual nanomaterials and any detached cellular components. The remaining sessile biomass was then stained for 15 min with a 0.1% CV solution, which selectively binds to the negatively charged constituents of the biofilm's extracellular matrix and bacterial cells. After the unbound CV was meticulously washed away and the plates were air‐dried, 95% ethanol was used to solubilize the bound dye, and its absorbance was determined using a microplate reader at a wavelength of 595 nm. The efficacy of the nanomaterials in clearing the pre‐formed biofilm structure was ultimately quantified by calculating the percentage of biofilm clearance, which was derived from a comparison between the absorbance of NCs exposed and unexposed positive biofilm controls (Elkady et al. 2025b).

2.5.5. Drug Synergy Assay

A checkerboard microdilution experiment was employed for assessment of the ZnO‐MgO NCs combined effect with cefepime against Gram‐negative bacterial strains. The assay utilized fractional inhibitory concentration (FIC) calculated on the basis of the MIC of the individual compound. To prepare the plates, Mueller Hinton broth (MHB) containing a range of fractional doses of ZnO‐MgO NCs and cefepime was prepared and inoculated with the bacterial suspension of 0.5 McFarland standard, followed by plate incubation for 24 h at 37°C. Next, the minimum combined drug concentration that prevents bacterial growth was determined. Consequently, the FIC and FIC index (FICi) were calculated from earlier literature as follows: “FICi = FICA + FICB,” in which FIC_A = compound A MIC in the combination/compound A MIC separately, and FIC_B = compound B MIC in the combination/compound B MIC separately. The tested compound interaction was identified on the basis of FICi and categorized as synergistic when FICi ≤ 0.5, additive when 1 ≥ FICi > 0.5, or antagonistic when FICi > 1 (Fadwa et al. 2021).

2.6. Statistical Analysis

Statistical analyzes were performed using GraphPad Prism software (version 8.0). One‐way analysis of variance (ANOVA) was applied to compare treatment groups, followed by Tukey's post hoc test for multiple comparisons. All experiments were conducted in triplicate, and data are expressed as the mean ± SD. A confidence interval of 95% was maintained, and differences were considered statistically significant at p < 0.05.

3. Results and Discussion

3.1. Nano Mycosynthesis

Fungal strains have gained recognition as efficient systems for synthesizing NPs with distinct properties. For instance, Hassan et al (Hassan et al. 2021). reported the biosynthesis of MgO NPs utilizing Rhizopus oryzae. According to (Jhansi et al. 2017)., employed Arachis hypogaea L. for the green synthesis of MgO NPs. The biosynthesis of ZnO NPs has also been explored with various fungal strains, including Aspergillus fumigatus JCF (Rajan et al. 2016) and Aspergillus niger, as demonstrated by Shamim et al (Shamim et al. 2019). Mohamed et al (Mohamed et al. 2019). produced hexagonal ZnO NPs using Fusarium keratoplasticum strain A1‐3, while the A. niger strain G3‐1 was employed for the generation of nanorod‐shaped ZnO NPs. In a study by Kumaravel et al (Kumaravel et al. 2021)., the entomopathogenic fungus Metarhizium anisopliae was used for the biosynthesis of ZnO–TiO₂ BNPs, which were investigated for their effectiveness against Spodoptera frugiperda. Similarly, Hashem et al (Hashem and El‐Sayyad 2024). demonstrated the biosynthesis of Ag–ZnO nanocomposites utilizing the reducing and stabilizing agent from pomegranate peel extract.

3.2. Characterization

The color change confirmed the fabrication of ZnO‐MgO NCs. A white precipitate indicated the successful synthesis of NCs. The investigation of ZnO‐MgO NCs was conducted using both physicochemical and surface characterization methods. The UV–vis spectrum of ZnO–MgO NCs exhibited a characteristic absorption peak at 300 nm (Figure 1), indicating the formation of a metal oxide heterojunction and confirming the successful mycosynthesis of the NCs. The optical properties of nanomaterials are strongly influenced by their size, shape, and distribution (Raza et al. 2016). Kamal et al (Kamal 2023a). reported the reflectance spectra of mycosynthesized ZnO NPs at room temperature that revealed an optical activity at a wavelength range of 360–380 nm. Likewise, Agaricus bisporus‐mediated biosynthesis of ZnO nanorods displayed a maximum absorption peak (λmax) at 331 nm (Rilda et al. 2023). Sharma et al (Sharma et al. 2021). observed that the optical characteristics of biosynthesized ZnO NPs showed a redshift, with an absorbance peak near 349 nm, reflecting their nanoscale dimensions. Gaber et al (Gaber et al. 2024). reported the UV‐vis spectral properties of myco‐produced ZnO‐CuO BNPs with a prominent peak at 375 nm for ZnO‐CuO BNPs, 320 nm for bio‐produced CuO NPs, and 400 nm for ZnO NPs, confirming their distinct optical behaviors. Kumar et al (Kumar et al. 2022). emphasized that UV photoluminescence studies of ZnO NPs in aqueous solutions revealed a strong absorbance peak at 270 nm, validating their formation. Hassan et al (Hassan et al. 2021). investigated MgO NPs synthesized through mycological methods and observed a SPR peak at 282 nm. Similarly, MgO NPs produced by Aspergillus terreus S1 showed an SPR peak at 280 nm (Saied 2021). Egbewole et al (Egbewole et al. 2024). examined Ag, Ni, and Ag‐Ni BNPs biosynthesized using Mallotus oppositifolius leaf extract, reporting SPR peaks at 454, 420, and 437 nm, respectively. Furthermore, UV‐vis analysis of Ag NPs revealed an absorption peak at 390 nm, while Se NPs displayed a characteristic peak at 460 nm, and Ag‐Se BNPs showed a characteristic peak at 395 nm (El‐Behery et al. 2023).

Figure 1.

Biosynthesized ZnO‐MgO NCs and S. kiliense extract UV‐vis spectra.

Analysis based on FTIR spectroscopy was conducted to identify the biosynthesized ZnO‐MgO NCs associated functional groups (Figure 2). Within the spectral range of 400 to 4000 cm⁻¹, multiple absorption peaks, corresponding to different functional groups, were recorded. The existence of phenolic or alcoholic groups was suggested based on the presence of a prominent absorption band around 3689 cm⁻¹, indicative of O─H stretching (Wang et al. 2024). A distinct peak assigned to C═C stretching vibrations was observed at 2228 cm⁻¹ (Kato et al. 2021), while signals at 2150 and 2027 cm⁻¹ were attributed to asymmetric and symmetric C─H stretching in methyl groups (Kamal 2023a). Additional bands at 1367 cm⁻¹ and 1070 cm⁻¹ were ascribed to N─O stretching specific for nitro containing compounds and C─H bending characteristic of alkanes, respectively (Nawaz et al. 2024). Importantly, absorption peaks detected between 400 and 1000 cm⁻¹ confirmed metal–oxygen bonding, confirming the successful formation of ZnO‐MgO NCs (Abdel‐Samad et al. 2024).

Figure 2.

Mycosynthesized ZnO‐MgO NCs FTIR spectrum.

The TEM image (Figure 3A) illustrates ZnO‐MgO NCs synthesized through Aspergillus‐mediated biosynthesis, displaying aggregated clusters with a crystalline structure. The dark regions in the image suggest dense, stable zones, likely resulting from the surrounding capping agents from fungal metabolites. This natural stabilization influences the nanoparticle's size and structure, as evidenced by the 200 nm scale and the size ranges between 25 and 50 nm, where the average size was 35 nm. The observed NPs characteristics underscore their potential use in diverse applications, from biocatalysis to environmental remediation, emphasizing the success of the mycosynthesis method (Kumar et al. 2021). In agreement with our findings, MgO NPs synthesized by Aspergillus terreus TFR exhibited complete transformation of the precursor into spherical shaped NPs with a polydispersity index (PDI) of 0.236 and an average diameter of 10 nm (Saied 2021). Likewise, Hassan et al. (Hassan et al. 2021). described R. oryzae‐assisted biosynthesis of uniformly distributed, crystalline, and spherical MgO NPs showing an average particle size of 20.38 ± 9.9 nm. Kamal et al (Kamal 2023b). reported the irregular shaped iron and ZnO NPs formation with a mean particle size of 16.8 nm. Moreover, Sheema et al. (2025). documented MgO‐ZnO NCs synthesized using Curcuma zedoaria oily extract had particle sizes in the range of 4.01–8.12 nm.

Figure 3.

Biogenic ZnO‐MgO NCs TEM image (A) and DLS graph (B).

The biosynthesized ZnO‐MgO NCs size and size distribution were determined, on the basis of hydrodynamic diameters, employing the DLS analysis. The particle size distribution histogram (Figure 3B) revealed an average hydrodynamic diameter of 45 nm. The size observed via DLS was larger than those measured through XRD and TEM, likely due to the presence of surface‐coating metabolites that stabilize and cap the obtained NPs (Vignesh et al. 2025). The PDI value of the ZnO‐MgO NCs was 0.248, indicating a homogeneous colloidal solution. Similarly, Kumaravel et al (Kumaravel et al. 2021). reported DLS analysis for bimetallic ZnO‐TiO₂ BNPs synthesized using Spodoptera frugiperda, with size distributions ranging from 80 to 137 nm.

The morphological analysis of ZnO‐MgO NCs biosynthesized from biomass filtrate of S. kiliense PV248633.1 was conducted using SEM imaging (Figure 4A). The obtained SEM image obviously illustrates an aggregated, layered structure with flake‐like formations. The attained image was depicted at SEM magnification of 20,000×, with a scale bar of 5 µm, indicating the nanoscale dimensions of the material. The observed morphology suggests the presence of tightly packed particles, possibly due to the interaction of stabilizing agents or capping metabolites during synthesis (Rosa et al. 2024). Malik et al (Malik et al. 2023). reported that SEM images revealed irregularly shaped clusters with coarse surfaces and slight agglomeration, predominantly exhibiting a quasi‐spherical morphology. The study also highlighted the effective capping and stabilization of the ecofriendly synthesized Ag–Fe BNPs by phytochemicals from Salvia officinalis leaves aqueous extract. The EDX was employed to support the morphological investigation of the biosynthesized BNPs and verify their elemental composition (Piergiovanni et al. 2024). This technique provided valuable information regarding the elemental purity and spatial distribution within the nanoparticles, contributing to a comprehensive understanding of their structural makeup (Patil et al. 2022).

Figure 4.

Biosynthesized ZnO‐MgO NCs SEM graph (A) and EDX analysis (B).

The integrated SEM and EDX investigation offered complementary insights into both the morphological and elemental features of the biosynthesized NCs. As illustrated in Figure 4B, the EDX spectrum confirmed the presence of Zn, Mg, O, C, and N. The detection of Zn and Mg in substantial proportions substantiates the ZnO‐MgO NCs successful fabrication. Specifically, the observed weight percentages were 25.1% for Zn, 24.6% for Mg, 44.1% for O, and 3.1% for both C and N. In terms of atomic percentages, the distribution was C (5.6%), N (4.8%), O (59.5%), Mg (21.9%), and Zn (8.3%). The high oxygen content is consistent with the expected oxide nature of the nanomaterials. Meanwhile, C and N occurrence is mostly related to organic molecules from the fungal extract, which functioned as a reducer and stabilizer during NPs biosynthesis. Such findings affirm the critical role of fungal metabolites in mediating a green, eco‐friendly synthesis process that not only facilitates NPs formation but also contributes to their capping and stability.

The diffraction peaks in the biosynthesized ZnO‐MgO NCs characteristic XRD pattern (Figure 5) confirmed the crystalline nature of both the ZnO and MgO components. For ZnO, characteristic peaks were observed at 2θ values of 34.4°, 36.2°, 47.5°, 56.6°, 62.8°, and 72.6°, corresponding to the (002), (101), (102), (110), (103), and (201) planes, respectively, matching JCPDS card No. 36‐1451 (Gaber et al. 2024). For MgO, prominent peaks at 37.0°, 43.0°, 62.4°, and 78.5° indicated the (111), (200), (220), and (222) planes, consistent with JCPDS No. 89‐7746 (Saied 2021). These results clearly demonstrated the coexistence of both ZnO and MgO phases within the nanocomposite structure. Also, the calculated average crystallite size of the biosynthesized ZnO‐MgO BNPs was about 34 nm as determined based on the Scherrer equation, d = K/cos. Furthermore, the ZnO NPs exhibited a hexagonal crystalline structure, with diffraction peaks corresponding to the (100), (002), (101), (102), (110), (103), (200), (112), (201), and (202) planes, as reported by Kamal et al (Kamal 2023a). Similarly, Sharma et al (Sharma et al. 2021). identified a hexagonal phase of ZnO with reflections at (100), (002), (101), (102), (110), (200), (112), (201), (004), and (202) planes. The crystallinity of mycosynthesized MgO NPs was emphasized by Hassan et al (Hassan et al. 2021); their NPs exhibited specific diffraction planes at (111), (200), (220), (311), and (222). Furthermore, Amin et al. (2024). demonstrated MgO NPs biosynthesized employing Penicillium crustosum EP‐1 cell‐free filtrate, with specific peaks representing 2θ values of 78.62°, 74.92°, 62.36°, 42.68°, and 37.08° that correspond to the crystallographic planes (222), (311), (220), (200), and (111).

Figure 5.

Mycosynthesized ZnO‐MgO NCs XRD spectrum.

3.3. Cytotoxicity and Anticancer Activity

The findings of the biosynthesized ZnO‐MgO NCs cytotoxic effect on the Wi‐38 normal cell line (Figure 6A) illustrated its non‐toxic effect; the BNPs exhibited an IC50 of 218.7 µg/mL. This confirmed the obtained ZnO‐MgO NCs biosafety (IC₅₀ ≥ 90 μg/mL) (Ioset et al. 2009). At lesser dosages, including 31.25 µg/mL or 62.5 µg/mL, WI‐38 cell viability is around 90%–100%. Cell viability decreases significantly with higher concentrations of ZnO‐MgO NCs (250 µg/mL and above). At 500 µg/mL, cell viability drops to 20%–30%, whereas at 1000 µg/mL, it approaches zero. was assessed.

Figure 6.

Biogenic ZnO‐MgO NCs inhibitory effect against normal WI‐38 (A) and cancerous MCF‐7 (B) cell lines cells. Values are expressed as mean ± SD (n = 3). Bars with different letters are significantly different (p < 0.05). Letters a, b, c,…. Mean significance power.

Also, evaluation of ZnO‐MgO NCs anticancer activity illustrated its promising inhibitory effect towards the cancerous MCF‐7 cell line with IC50 78.1 µg/mL (Figure 6B). Furthermore, ZnO‐MgO NCs at 31.25, 62.5, 125, and 250 µg/mL showed antitumor activity of 13.98%, 41.47%, 74.58%, and 94.35%, respectively. In a previous study, Pluchea indica extract was used for the preparation of MgO‐ZnO nanocomposite with outstanding antitumor effect against MCF‐7 and HepG2 at safe dosages with IC50 of 31.25 µg/mL and 73.61 µg/mL, respectively (Selim et al. 2025b).

Moreover, ZnO‐MgO nanocomposite was prepared using the Turbinaria conoides seaweed aqueous extracts, where it exhibited anticancer activity toward the U87 cell line with IC50 12.73 µg/mL (Anbazhagi et al. 2025). Additionally, Khan et al (Rashid Khan et al. 2024). reported MgO NPs with an antitumor effect towards the MDA‐MB‐231 breast cancer cell line. The synthesized ZnO‐MgO NCs exhibited anticancer activity through proposed mechanisms, including the production of several reactive oxygen species (ROS) (Mousa et al. 2023; Gassim and Makkawi 2023). When these NPs interact with cancer cells via ROS production with subsequent DNA and intracellular protein destruction, oxidative stress, and cell death (Mousa et al. 2023). Furthermore, ZnO NPs can selectively target cancerous cells while causing minimal damage to healthy cells (El‐Shorbagy 2019). Additionally, ZnO‐MgO NCs can induce cancer cell apoptosis and modulate their key signaling pathways (Behzadi et al. 2019). Studies suggest that these NPs can disrupt mitochondrial function and enhance the efficacy of conventional chemotherapeutic drugs (Behzadi et al. 2019; Rasmussen et al. 2010) The release of zinc ions also contributes to cancer inhibition (Gassim and Makkawi 2023). Also, MgO NPs have demonstrated selective cytotoxicity against tumor cells by inducing ROS‐mediated apoptosis (Behzadi et al. 2019).

3.4. Antimicrobial Activities of ZnO‐MgO NCs

3.4.1. Initial Activity

Variable and strong antibacterial activities of the ZnO‐MgO NCs were revealed by the WDA (Figure 7) against the tested Gram‐negative bacteria (Table 1 and Figure 8). Interestingly, the ZnO‐MgO NCs were more effective than cefepime against A. baumannii (21.66 mm vs 17.34 mm, respectively) and P. aeruginosa (25.33 mm vs 24.67 mm, respectively), indicating greater efficacy against these isolates. Conversely, cefepime exhibited superior activity against E. coli (23.66 mm vs 19.34 mm, respectively) and K. pneumoniae (12 mm vs 14 mm, respectively), indicating strain‐dependent differences in susceptibility. However, statistical analysis of the results demonstrates an insignificant difference (p = 0.853) between the ZnO‐MgO NCs and cefepime. These findings were consistent with earlier studies that explored the antibacterial potential of different nanomaterials. For instance, a study by (Fodil et al. 2024) demonstrated that smaller NPs generally exhibit an enhanced bacterial inhibitory effect caused by the increased surface area and consequent reactivity. Similarly, (Raghupathi et al. 2011) found that smaller‐sized ZnO NPs were more active against E. coli and S. aureus bacterial strains. Our current results further support the notion that NPs composition and size principally determine antibacterial efficacy (Gupta and Sharma 2022). The observed strain‐dependent differences in susceptibility highlight the importance of tailoring NPs formulations to target specific bacterial pathogens (Slavin et al. 2017).

Figure 7.

Representative WDA demonstrating ZnO‐MgO NCs antimicrobial activity against P. aeruginosa ATCC 25668.

Table 1.

Biosynthesized ZnO‐MgO NCs activity by WDA.

Test Isolates	Agar well diffusion assay
	IZD* (mm)
	ZnO‐MgO NCs (50 µg/mL)	Cefepime (50 µg/mL)	PI*
E. coli ATCC 25922	19.33 ± 0.58c	23.67 ± 0.6b	81.7
A. baumannii ATCC 17978	21.67 ± 0.58b	17.33 ± 0.6c	125
K. pneumonia ATCC 700603	14.00 ± 1.0 d	12.00 ± 1.0 d	116.7
P. aeruginosa ATCC 25668	25.33 ± 1.15a	24.67 ± 1.2a	102.7

Note: Values are expressed as mean ± SD (n = 3). Values with different letters (a–d) are significantly different from each other (p < 0.05).

^* PI: percent inhibition and IZD: inhibition zone diameter.

Figure 8.

Biogenic ZnO‐MgO NCs and cefepime comparative antimicrobial activity against some Gram‐negative bacteria as revealed by WDA.

3.4.2. Inhibitory Concentrations Revised Totally Changed

The broth microdilution assay illustrated that the MIC and MBC values differed substantially among the tested isolates (Table 2 and Figure 9). Specifically, E. coli ATCC 25922 and P. aeruginosa ATCC 25668 exhibited MIC values of 64 µg/mL but higher MBCs of 512 µg/mL. These results indicated bacteriostatic effects, suggesting that bacterial growth is suppressed but not eradicated at lower concentrations. In contrast, A. baumannii has shown an equal MIC and MBC of 128 µg/mL, with a MIC index of 1, which is consistent with a bactericidal mechanism, and highlights a full bacterial killing at inhibitory concentrations. K. pneumoniae displayed intermediate behavior with MIC of 256 µg/mL and MBC of 512 µg/mL and bactericidal effects. These findings underscore the clinical relevance of ZnO‐MgO NCs as potential antimicrobial agents, particularly against some Gram‐negative isolates, with variations in susceptibility emphasizing the need for pathogen‐specific assessment. Previous study by (Al‐Khaial et al. 2024) showed that ZnO NPs can damage bacterial cells through interactions with surface proteins, penetration of the cell, and the generation of ROS. Another study by (Carofiglio et al. 2020) indicated that ZnO‐MgO NCs have potential as antibacterial agents against Gram‐negative pathogens, and their effectiveness warrants further investigation and development.

Table 2.

Inhibitory concentrations of ZnO‐MgO NCs.

Isolates* Character	ZnO‐MgO NCs
	µg/mL		MIC index	Antibiosis effect
	MIC	MBC
E. coli ATCC 25922	64	512	8	Bacteriostatic
A. baumannii ATCC 17978	128	128	1	Bactericidal
K. pneumonia ATCC 700603	256	512	2
P. aeruginosa ATCC 25668	64	512	8	Bacteriostatic

^* MIC; minimum inhibitory concentration and MBC; minimum bactericidal concentration.

Figure 9.

Heatmap describes the MIC (A) and MBC (B) of ZnO‐MgO NCs against E. coli ATCC 25922 (1), A. baumannii ATCC 17978 (2), K. pneumonia ATCC 700603 (3), and P. aeruginosa ATCC 25668 (4).

3.4.3. Biofilm Inhibitory Effect

The antibiofilm activity of the ZnO‐MgO NCs demonstrated concentration‐dependent inhibitory effects on Gram‐negative pathogens with profound strain‐specific differences. At ½ MIC, biofilm inhibition by the BNPs was 81.31%, 82.44%, 83.41%, and 91.37% for E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa, respectively. The ⅛ MIC sub‐inhibitory concentration of ZnO‐MgO NCs also maintained significant activity against P. aeruginosa towards biofilm disruption (82.90%), followed by K. pneumoniae (76.92%), A. baumannii (66.31%), and E. coli (54.31%), as shown in Figure 10. These results were consistent with previous studies on the antibacterial and antibiofilm properties of metal oxide NPs. For instance, (Udayagiri et al. 2024) reported that bio‐ZnO NPs displayed enhanced antibacterial activity against S. aureus and E. coli, indicating that ZnO NPs are more effective against Gram‐positive bacteria due to structural variations in their cell walls. Similarly, (Ahmed et al. 2017) found that synthesized ZnO NPs significantly inhibited biofilm formation, with 85% and 97% inhibition against S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 biofilms, respectively. The concentration‐dependent biofilm inhibition observed in the current study is consistent with these findings, highlighting the potential of ZnO‐MgO NCs as effective agents for disrupting biofilms and combating antimicrobial‐resistant infections. Furthermore, the higher biofilm inhibition at low doses aligns with recent studies on metal oxide NPs, where ZnO and MgO hybrids suppressed quorum sensing and extracellular polymeric substance (EPS) production in P. aeruginosa (Sivaraj et al. 2022). These findings revealed that ZnO‐MgO NCs can effectively disrupt biofilm matrices, and among these P. aeruginosa is most severely affected, perhaps due to its composition of EPS, which may be more accessible to NCs penetration (Shahed 2023).

Figure 10.

Biofilm inhibition percentages of ZnO‐MgO NCs at ½ MIC, ¼ MIC and _1/8 MIC values against E. coli ATCC 25922, A. baumannii ATCC 17978, K. pneumonia ATCC 700603, and P. aeruginosa ATCC 25668.

3.5. Biofilm Clearance Activity

The biofilm clearance activity of the biosynthesized ZnO‐MgO NCs displayed remarkable strain‐dependent variations. A. baumannii was most susceptible (79.74%–80.19% clearance, SD ± 0.23), followed by K. pneumoniae (50.85%–51.02%, SD ± 0.08), while E. coli (20.92%–21.13%, SD ± 0.10) and P. aeruginosa (26.98%–27.18%, SD ± 0.10) exhibited comparatively lower clearance rates as illustrated in Figure 11. These findings were consistent with previous research indicating that A. baumannii biofilms are particularly vulnerable to certain antimicrobial agents. For instance, studies have shown that the presence of specific outer membrane proteins in A. baumannii can influence its biofilm formation and susceptibility to antimicrobial peptides (Udayagiri et al. 2024). Additionally, the relatively lower clearance rates observed for E. coli and P. aeruginosa align with the understanding that these strains often exhibit robust biofilm structures that can impede the efficacy of antimicrobial agents (Grygiel et al. 2024). The observed variations in biofilm clearance rates highlighted the importance of strain‐specific considerations in the development and application of antimicrobial strategies. This study used CV as a convenient, high‐throughput method to quantify total biofilm biomass; however, this assay could not distinguish live from dead cells or specifically assess the EPS matrix. Consequently, the results only demonstrated reductions in overall biomass rather than confirmed killing of viable biofilm cells. Consistent with other work, the absence of additional methods such as XTT metabolic assays, CFU counting, and confocal microscopy is acknowledged as a limitation, and incorporating these techniques in future studies is recommended to better evaluate biofilm viability and structure.

Figure 11.

Biofilm clearance activity of ZnO‐MgO NCs at MIC value against E. coli ATCC 25922, A. baumannii ATCC 17978, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 25668.

3.6. Synergistic Interaction

The checkerboard assay (Figure 12) results as presented in Table 3 illustrated that the combination of ZnO‐MgO NCs and cefepime exhibited varying interactions across the different Gram‐negative bacterial strains. A synergistic effect was observed against P. aeruginosa ATCC 25668, as evidenced by a FICi = 0.375. An additive interaction was noted with K. pneumoniae ATCC 700603, which yielded an FICi of 0.75. For both E. coli ATCC 25922 and A. baumannii ATCC 17978, the combination demonstrated indifference, with FICi values of 3 and 1.5, respectively. These findings indicated that the effectiveness of the ZnO‐MgO NCs and cefepime combination was dependent on the specific Gram‐negative bacterial species being targeted. This observation is consistent with recent studies that have explored the potential of metal‐based NPs as adjuvants to enhance the efficacy of antimicrobial agents against resistant bacteria (Barber et al. 2021) and (Yang et al. 2022). The findings of this research highlight the effectiveness of ZnO‐MgO BNPs combined with cefepime as a new mechanism to combat bacterial resistance, and notably against drug‐resistant organisms. Similarly, (Williams et al. 2024) also reported that ZnO NPs greatly improved the effect of β‐lactam antibiotics against resistant Gram‐negative bacteria, which aligns with our findings. These results collectively underscore the relevance of continued investigation into NPs‐antimicrobial conjugates as a prospective approach to mitigate the global antimicrobial resistance crisis.

Figure 12.

Check board assay of ZnO‐MgO NCs and cefepime against some Gram‐negative bacteria.

Table 3.

Checkboard assay of ZnO‐MgO NCs with cefepime.

Isolates	FIC (BNPs)	FIC (Cefepime)	FIC index	Combination effect
E. coli ATCC 25922	2	1	3	Indifference
A. baumannii ATCC 17978	0.5	1	1.5	Indifference
K. pneumonia ATCC 700603	0.5	0.25	0.75	Additive
P. aeruginosa ATCC 25668	0.125	0.25	0.375	Synergism

Abbreviation: FIC, fractional inhibitory concentration.

4. Conclusion

This study demonstrates the environmentally friendly synthesis of ZnO‐MgO NCs using S. kiliense PV248633.1 fungal strains, showcasing their potential as effective antimicrobial agents. Characterization techniques confirmed the successful biosynthesis and stability of the NCs, which exhibited significant anticancer activity against MCF‐7 cells while remaining non‐toxic to normal WI‐38 cells. The findings showed significant inhibition zones, especially against A. baumannii and P. aeruginosa, more potent than cefepime. Dose‐dependent antibacterial activity was confirmed with low MIC50 values. Biofilm inhibition and removal were concentration‐ and strain‐dependent, with A. baumannii being the most sensitive. The synergistic effect of ZnO‐MgO NCs with cefepime had additive activity against K. pneumoniae and P. aeruginosa. Thus, these findings suggest that ZnO‐MgO NCs could serve as a promising alternative in the fight against antimicrobial‐resistant infections, warranting further exploration in clinical applications. Although these findings demonstrate this fungal system's potential as a bio‐factory for functional nanomaterials, the current investigation is restricted to in vitro tests. However, future research should concentrate on clarifying the biological effects that have been seen, evaluating the biodistribution and toxicity of NPs in animal models, and confirming their effectiveness in clinically relevant infection and tumor situations.

Author Contributions

Samy Selim: conceptualization, methodology. Fathy M. Elkady: methodology, writing – original draft, writing – review and editing. Ebrahim Saied: conceptualization, methodology, writing – original draft, writing – review and editing. Amr H. Hashem: conceptualization, methodology, writing – original draft, writing – review and editing. Amer M. Abdelaziz: conceptualization, methodology, writing – original draft, writing – review and editing. Mohammed S. Abdulrahman: methodology, writing – original draft. Faisal Alsenani: methodology. Omar Awad Alsaidan: methodology. Sami I. Alzarea: methodology. Yousef Alhaj Hamoud: writing – review and editing. Hiba Shaghaleh: writing – review and editing. Mohammed Aufy: writing – review and editing.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data sets used and/or analyzed during the current study available from the corresponding author on reasonable request.