Green synthesis nanocomposite GO/ZnFe₂O₄ with water extract of Teucrium polium and evaluation of its anti-bacterial and cytotoxicity activities

Abstract

Materials based on graphene oxide (GO) and zinc ferrite nanoparticles (ZnFe₂O₄ NPs) have attracted considerable attention due to their large surface area, structural versatility, and potential biological functionality. In this study, GO NPs, ZnFe₂O₄ NPs, and a GO/ZnFe₂O₄ nanocomposite (GO/ZnFe₂O₄ NC) were green-synthesized and systematically characterized, focusing on their structural, magnetic, optical, and biological properties. Structural and compositional features were analyzed using X-ray diffraction (XRD), Raman spectroscopy, energy-dispersive X-ray analysis (EDX), and vibrating sample magnetometry (VSM), while scanning electron microscopy (SEM) was employed to investigate the morphology of the materials. The antibacterial activity of the synthesized materials was evaluated against Escherichia coli and Staphylococcus aureus. Both ZnFe₂O₄ NPs and the GO/ZnFe₂O₄ NC exhibited notable antibacterial effects, with the nanocomposite showing superior inhibitory performance against both bacterial strains. Furthermore, the GO/ZnFe₂O₄ NC demonstrated significant cytotoxic activity against HeLa cancer cells, with an IC₅₀ value of 13.58 µg/mL, while exhibiting considerably lower toxicity toward normal fibroblast cells (IC₅₀ = 158.5 µg/mL), indicating selective anticancer potential. Gene expression analysis revealed that treatment with the GO/ZnFe₂O₄ NC significantly upregulated the pro-apoptotic genes BAX and Caspase-3, while markedly downregulating the anti-apoptotic gene Bcl-2, suggesting apoptosis as a possible mechanism underlying its cytotoxic effects.

Introduction

According to the World Health Organization (WHO), bacterial contamination has become a major threat to global public health, causing the highest mortality rates in developing countries over the past decade¹. Among available therapies, antibiotics remain the most effective option for combating bacterial infections². However, long-term misuse of antibiotics has contributed to the development of antibiotic-resistant bacteria, which threaten public health through food, water, and livestock^3,4. A global WHO report indicates that antibiotic-resistant bacteria are responsible for nearly 700,000 deaths annually, a number that could rise to 10 million per year by 2050⁵. Consequently, the global antibiotic resistance crisis demands urgent action. While the development of novel antibiotics remains a potentially effective strategy to counter multidrug-resistant (MDR) bacteria⁶, the pipeline of newly approved antibiotics is narrowing due to increasing difficulties in discovering effective antimicrobial compounds. Therefore, alternative therapeutic approaches against bacterial infections are becoming increasingly necessary. In this context, antibacterial nanomaterials have emerged as a particularly promising solution, demonstrating significant bactericidal efficacy and the potential to redefine antibiotic-free treatment paradigms^7,8.

As an oxidized form of graphene, GO exhibits a layered two-dimensional structure with outstanding physicochemical properties, including water affinity, stable dispersion in aqueous solutions, and capacity for surface modification. The presence of hydroxyl, epoxide, and carboxyl groups on GO sheets facilitates binding with biomolecules (e.g., proteins and nucleic acids), enhancing its biological compatibility and functional versatility⁹. GO has received considerable attention in drug delivery due to its extremely large surface area and high drug-loading efficiency¹⁰. The biological activities of GO and its nanocomposites have been evaluated against various bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas putida, as well as in different cell lines, including lung epithelial cells, fibroblasts, neuronal cells, and cancer cells^9,11,12.

Spinel ferrites have been widely investigated in biomedicine, optoelectronic devices, catalysis, and pharmaceutical applications due to their physical, chemical, and magnetic properties^13,14. Among these, zinc ferrite and its derivatives are commonly used in biotechnology, drug delivery, and other biomedical applications. ZnFe₂O₄ NPs have been shown to exhibit antibacterial activity against both Gram-positive and Gram-negative bacteria while remaining non-toxic to Hu02 cells¹⁵.

The subshrub T. polium is native to arid and semi-arid regions of North Africa, the Mediterranean, and Southwest Asia. Its aerial parts are traditionally used in ethnomedicine to manage gastrointestinal disorders, reduce fever, relieve constipation, and act as an antispasmodic¹⁶. Bioactive constituents in T. polium including flavonoids and terpenoids have been demonstrated to exert a wide range of pharmacological effects, such as blood-glucose lowering, anti-inflammatory, liver-protective, antifungal and antibacterial activities^17,18.

Although previous studies have reported the synthesis of GO/ZnFe₂O₄ nanocomposites using chemical reducing agents or multi-step synthesis methods, our study introduces a green, one-pot approach employing TP extract. This method enables uniform dispersion of ZnFe₂O₄ NPs on GO sheets and avoids harsh chemicals. Furthermore, we systematically evaluated the antibacterial and cytotoxic performance of the resulting nanocomposite, providing novel insights into its potential biomedical applications.

In the present work, TP hydroethanolic extract was used to synthesize GO/ZnFe₂O₄ NC by co-precipitation. The resulting nanocomposites were characterized and tested for cytotoxic effects on HeLa and fibroblast cell lines, as well as antibacterial activity against antibiotic-resistant S. aureus and E. coli isolated from food.

Materials and methods

Materials

In this study, all chemicals and reagents were of analytical grade and were obtained from Merck (Germany), Sigma‑Aldrich (USA), or HiMedia (India). Fibroblast and HeLa cell lines were procured from the Pasteur Institute (Tehran, Iran).

Aqueous extraction of TP and synthesis of GO/ZnFe₂O₄ NC

TP was acquired from a spices and herbal market in Kashan, Iran. The dried aerial parts of TP were ground into a fine powder using a mixer grinder. Aqueous extracts were prepared by macerating 100 g of the dried plant material in 500 mL of distilled water for 48 h. The resulting solution was filtered to obtain a uniform extract, which was used as a surfactant in subsequent nanoparticle synthesis¹⁹. The TP extract acts as both a reducing and stabilizing agent, facilitating the formation of ZnFe₂O₄ nanostructures and preventing particle agglomeration.

GO NPs were synthesized using a modified Hummers’ method²⁰. Initially, 5 g of graphite and 5 g of sodium nitrate were mixed in 240 mL of sulfuric acid and stirred magnetically at room temperature for 30 min. Subsequently, 30 g of potassium permanganate was added gradually over 30 min while carefully controlling the temperature. Next, 800 mL of distilled water and 30 mL of 30% hydrogen peroxide (H₂O₂) were introduced, and the reaction was stirred for an additional 25 min. H₂O₂ served as an oxidizing agent to promote complete formation of GO. The resulting suspension was filtered and repeatedly washed with distilled water until neutral pH was attained. The solid residue was dried in an oven at 60 °C. For GO dispersion, the dried material was sonicated in water using an ultrasonic processor (Hielscher, Germany), and undispersed particles were removed by centrifugation at 3000 rpm for 30 min, yielding a GO dispersion at 1 g/L.

ZnFe₂O₄ NPs were prepared via co‑precipitation without subsequent calcination. In this procedure, iron(III) chloride hexahydrate (FeCl₃·6 H₂O) and zinc sulfate (ZnSO₄) were each dissolved separately in 50 mL of deionized water and stirred at 50 °C for 60 min. The FeCl₃ solution was then combined with 20 mL of TP extract and stirred for 30 min prior to the addition of the ZnSO₄ solution. The precipitate formed was collected and dried at 80 °C for 2 h, yielding ZnFe₂O₄ NPs. Finally, the synthesized ZnFe₂O₄ NPs were composited with GO NPs in a 1:10 ratio to form the GO/ZnFe₂O₄ NC.

Characterization techniques

The phase and crystal structure of the prepared material were characterized via powder XRD analysis performed on a Rigaku MiniFlex600 instrument, employing monochromatic Cu Kα₁ radiation (λ = 1.5406 Å) for data collection.

The surface morphology of the synthesized photocatalysts was investigated by scanning electron microscopy (SEM) using a LEO (Carl Zeiss) GEMINI–1430–VP microscope (Oberkochen, Germany). The XRD instrument was complemented by energy‑dispersive X‑ray spectroscopy (EDX) to assess the elemental distribution in selected regions of the sample. Magnetic characterization was performed using a custom-built vibrating sample magnetometer to obtain the magnetization curves (VSM, Meghnatis Kavir Kashan Co., Kashan, Iran). Using a vibrating-sample magnetometer, the specimen was subjected to harmonic oscillations in a uniform magnetic field. The periodic movement produced a time-varying magnetic field around the sample, inducing an electromotive force in the pickup coil. The magnitude of this induced voltage was then used to deduce the magnetic moment of the sample²⁰.

For compositional analysis, Raman spectra were recorded using a Senterra Raman microscope (Bruker, Germany). The samples, dispersed in alcohol and mounted on microscope slides, were examined under the optical microscope, and Raman spectra were acquired on selected particle clusters. Excitation was provided by a 633 nm laser, with each measurement running for 30 s. The laser was focused using a 50× objective, and the power delivered to the sample was 0.21 mW (≈ 3.2% of the maximum).

Antimicrobial activity assay

In this study, two bacteria isolated from food samples, S. aureus and E. coli, were investigated for their susceptibility to various antimicrobial agents. Initially, each bacterial strain was cultured on nutrient agar (NA) for 18 h at 37 °C. Following incubation, an inoculum was prepared by diluting the bacterial cultures to a concentration of 0.5 MacFarland units, approximately 1 × 10⁸ CFU/mL, using sterile saline²¹.

For the assay, a 10% (v/v) dimethyl sulfoxide (DMSO) solution in Mueller‑Hinton Broth was prepared to serve as growth medium. In the microplate setup, each row of the 96‑well plate was organized with the last two wells designated as positive control and negative control. To each well, 5 µL of a 0.5 McFarland suspension, representing a bacterial concentration of approximately 1 × 10⁸ CFU/mL, was added, followed by 95 µL of MHB. Subsequently, 100 µL of serial 2-fold dilutions of GO NP, ZnFe₂O₄ NP, and GO/ZnFe₂O₄ NC were transferred into the respective wells. The 96-well plates were incubated in a shaking incubator at 37 °C for 20 h to facilitate bacterial growth. After the incubation period, each well was examined for turbidity, indicating bacterial growth. The degree of turbidity across the wells provided insights into the antimicrobial effectiveness of the nanoparticles tested, aiding in the assessment of their potential for use in therapeutic applications against bacterial pathogens.

The Minimum Inhibitory Concentration (MIC) was determined by identifying the lowest concentration of GO NP, ZnFe₂O₄ NP and GO/ZnFe₂O₄ NC that resulted in no turbidity in the wells, indicating effective bacterial growth inhibition. Furthermore, the Minimum Bactericidal Concentration (MBC) was defined as the lowest concentration at which no visible growth was observed on Mueller Hinton Agar (MHA) after subculturing from the wells that exhibited no turbidity. This determination of both MIC and MBC provides important insights into the antibacterial efficacy of the nanoparticles²².

The disc diffusion assay was performed as described in a previous study²³. Briefly, bacterial cultures (approximately 1 × 10⁸ CFU/mL) were spread onto MHA plates. 6 mm diameter of sterile Whatman No. 1 paper discs were impregnated with different concentrations of GO NPs, ZnFe₂O₄ NPs, and GO/ZnFe₂O₄ NC, each diluted in 10% (w/v) DMSO, and placed on the inoculated plates. The plates were incubated for 24 h at 37 °C. The inhibition zone (IZ) widths were measured in millimeters using a ruler. DMSO 10% served as the negative control, while antibiotics such as Amoxicillin/clavulanic acid (25/10 µg), Penicillin (6 µg), Erythromycin (30 µg), Fosfomycin (10 µg), Colistin (50 µg), Cefotaxime (30 µg), Ampicillin (10 µg), Ticarcillin (75 µg), Vancomycin (30 µg), and Methicillin (5 µg) were used as positive controls.

Cytotoxic activity

The MTT assay was performed on HeLa and fibroblast cells to evaluate the cytotoxicity of GO NPs, ZnFe₂O₄ NPs, and ZnFe₂O₄/GO NC²⁴. The growth medium consisted of basal medium enhanced with 10% heat‑inactivated FBS, 1% penicillin–streptomycin (10000 U penicillin and 10 mg streptomycin per 0.9% NaCl) and 2 mM L‑glutamine. Cell cultures were maintained at 37 °C under 5% CO₂ with adequate humidity. Cells were passaged in 75 cm² flasks every three days with fresh medium. For cytotoxicity assays, different concentrations of nanoparticles (0–1000 µg/mL) were tested. Cells were plated at 10,000 cells/well in 96‑well microplates and grown until ~ 90–95% confluence prior to exposure. Each well received 100 µL of medium containing the NPs at specified concentrations, followed by a 24-hour incubation. Subsequently, 20 µL of MTT solution (5 mg/mL) was added, and plates were incubated for an additional 4 h. Following incubation, the medium was aspirated and 200 µL DMSO was added to each well in order to solubilize the formazan precipitate. The optical density was then recorded at 570 nm using a microplate reader (Stat Fax 3200, USA). The percentage of viable cells was subsequently determined using the standard formula:

The selectivity index (SI) was calculated as the ratio of IC₅₀ in normal fibroblast cells to IC₅₀ in Hela cancer cells:

Expression of apoptosis-regulatory proteins

Total RNA was isolated from HeLa cells untreated or treated for 24 h with GO NPs, ZnFe₂O₄ NPs, or the GO/ZnFe₂O₄ NC using the RNeasy Mini Kit (Qiagen). RNA yield and purity were assessed with a Nanodrop 8000 spectrophotometer (Thermo Scientific). First‑strand cDNA synthesis was carried out employing MLV reverse transcriptase (GE Healthcare). Quantitative PCR targeting apoptotic markers (BAX, Cas‑3) and the anti‑apoptotic gene Bcl‑2 was conducted on a Roche LightCycler 480 system (96‑well format) using SYBR Green I dye (Roche Diagnostics). Each 20 µL reaction contained 75 ng cDNA and 5 µM of each primer. The thermocycler protocol consisted of an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 20 s²⁵. Expression levels were normalized to GAPDH as the internal control.

Data analysis

Each experimental condition was performed in triplicate (n = 3), and data are presented as mean ± standard deviation (SD). Statistical analysis was conducted using one-way ANOVA, and differences were considered statistically significant at p < 0.05.

Results

Characterization

Figure 1 shows the XRD pattern of the synthesized GO/ZnFe₂O₄ NC obtained using Cu Kα radiation (λ = 1.5406 Å) over a 2θ range of 10°–80°. The diffraction peaks at 2θ ≈ 18.2°, 30.1°, 35.4°, 36.9°, 43.1°, 53.5°, 56.9°, 62.6°, and 74.0° correspond to the (111), (220), (311), (222), (400), (422), (511), (440), and (533) planes of the cubic spinel ZnFe₂O₄ phase (JCPDS no. 01-077-0011). The absence of additional peaks confirms the formation of a single-phase ZnFe₂O₄ structure with high crystallinity. XRD results, together with SEM and Raman analyses, suggest the successful incorporation of GO within the composite matrix, while slight peak broadening indicates nanoscale crystallite size. Cu does not constitute any component of the synthesized nanocomposite, and its mention refers solely to the Cu Kα radiation employed during XRD analysis.

Fig. 1.

XRD pattern of GO NP (A), ZnFe₂O₄ NP (B), and GO/ZnFe₂O₄s NC (C). Different intensity scales are used for clarity.

SEM analysis revealed quasi-spherical nanoparticles with an average diameter of 10–20 nm, exhibiting a relatively uniform size distribution and slight agglomeration due to interparticle interactions. Additionally, thin sheet-like 2D structures with wrinkled and folded surfaces were observed, confirming the coexistence of particulate and layered morphologies within the synthesized nanomaterials (Fig. 2).

Fig. 2.

SEM of GO NP (A), ZnFe₂O₄ NP (B), and GO/ZnFe₂O₄s NC (C).

EDX analysis confirmed the presence of Fe and Zn as the major elements, along with a minor oxygen signal, indicating the possible formation of oxide phases such as ZnO or Fe₂O₃. The corresponding peaks at approximately 0.5–0.6 keV (O Kα), 0.7 keV (Fe Lα), 6.4 keV (Fe Kα), 7.0 keV (Fe Kβ), 1.0 keV (Zn Lα), 8.6 keV (Zn Kα), and 9.6 keV (Zn Kβ) confirm the coexistence of Fe and Zn species (Fig. 3). The strong Fe peaks and moderate Zn peaks suggest a Zn-Fe oxide composition with high elemental purity.

Fig. 3.

EDX of GO/ZnFe₂O₄ NC. EDX spectrum recorded in the range of 0.05–10 keV.

Raman analysis (400–3400 cm⁻¹) of the GO/ZnFe₂O₄ NC revealed two prominent peaks at approximately 1350 cm⁻¹ (D band) and 1580 cm⁻¹ (G band), corresponding to disordered and graphitic sp² carbon structures, respectively. The moderate intensity ratio indicates partial structural disorder and defect-rich carbon domains. The absence or weak appearance of the 2D band around 2700 cm⁻¹ suggests limited graphitic stacking and a predominantly amorphous carbon structure (Fig. 4). ZnFe₂O₄ NP exhibits weak Raman signals compared to GO NP due to its spinel structure and lower Raman scattering efficiency, which is consistent with previous reports. A slight increase in baseline intensity at higher wavenumbers may be attributed to minor fluorescence from surface-bound organic species.

Fig. 4.

Raman of GO NP (A), ZnFe₂O₄ NP (B), and GO/ZnFe₂O₄s NC (C).

Magnetic analysis at room temperature revealed a narrow S-shaped hysteresis loop within the applied field range of − 15,000 to + 15,000 Oe, exhibiting a saturation magnetization (Mₛ) of about 15 emu/g with negligible coercivity (H_c) and remanence (M_r) (Fig. 5). These characteristics confirm the soft magnetic or superparamagnetic nature of the synthesized material, indicating reversible magnetization with minimal hysteresis loss.

Fig. 5.

VSM of GO/ZnFe₂O₄s NC.

Antibacterial properties

In this study, the antibacterial activities of GO NP, ZnFe₂O₄ NP and a GO/ZnFe₂O₄ NC (each at 30 mg/mL) were evaluated by the disk-diffusion method against E. coli and S. aureus. The data showed that GO NP produced modest IZs (8 ± 1 mm for E. coli; 10 ± 1 mm for S. aureus), while ZnFe₂O₄ NP exhibited similar activity against E. coli (9 ± 0.5 mm) but substantially more effectiveness against S. aureus (25 ± 2 mm). The GO/ZnFe₂O₄ NC outperformed the individual components, with IZs of 10 ± 1 mm and 14 ± 2 mm for E. coli and S. aureus, respectively, suggesting a synergistic effect (Table 1). Compared with conventional antibiotics tested, the nanoparticle systems show promise but require further optimization to match clinical standards.

Table 1.

Inhibition zone (mm) of GO NP, ZnFe₂O₄ NP, GO/ZnFe₂O₄ NC, and reference antibiotics against E. coli, and S.aureus isolated from food samples.

Antimicrobial agent	E. coli	S.aureus
GO NP (30 mg/mL)	8 ± 1aA	10 ± 1 aA
ZnFe2O4 NP (30 mg/mL)	9 ± 0.5 aA	25 ± 2 bB
GO/ZnFe2O4s NC (30 mg/mL)	10 ± 1 aA	14 ± 2 bC
Amoxicillin-clavulanic acid (20 µg/10 µg)	14 ± 1 aB	24 ± 2 bB
Fosfomycin (200 µg)	32 ± 2 aC	29 ± 2 aD
Ticarcillin (75 µg)	22 ± 1 aD	25 ± 1bB

Different lower-case letters in each row and upper-case letters in each column to indicate significant difference (P < 0.05). Data is reported as mean (n = 3) ± standard deviation.

The MIC and MBC assays against E. coli and S. aureus showed that the GO/ZnFe₂O₄ NC exhibited the strongest antibacterial effect among the tested nanomaterials. For E. coli, the composite achieved an MIC of 3.75 mg/mL and an MBC of 7.5 mg/mL (compared with GO NP at 7.5/15 mg/mL and ZnFe₂O₄ NP at 7.5/15 mg/mL). For S. aureus, the composite reached an MIC of 0.9 mg/mL and an MBC of 1.8 mg/mL, outperforming both GO NP (MIC/MBC: 3.75/7.5 mg/mL) and ZnFe₂O₄ NP (3.75/7.5 mg/mL). These results indicate that the GO/ZnFe₂O₄ NC composite offers enhanced bactericidal potency, particularly against the gram‑positive S. aureus.

Cytotoxicity

In the MTT assay, the GO/ZnFe₂O₄ NC exhibited substantially higher cytotoxicity toward fibroblast cells compared with its individual components. Specifically, the IC₅₀ values were approximately 2896 µg/mL for GO NP alone, ~ 377.8 µg/mL for ZnFe₂O₄ NP, and ~ 158.5 µg/mL for the GO/ZnFe₂O₄ NC. These results indicate dose-dependent cytotoxicity of the composite toward fibroblast cells, suggesting that the material affects normal cells at higher concentrations while highlighting the need for careful evaluation of its safety profile (Fig. 6).

Fig. 6.

Cytotoxicity effects of GO NP, ZnFe₂O₄ NP, and GO/ZnFe₂O₄ NC on fibroblast cells.

The cytotoxicity of GO NP, ZnFe₂O₄ NP, and the GO/ZnFe₂O₄ NC against HeLa cells was evaluated using MTT assays, and the corresponding IC₅₀ values were determined (Fig. 7). GO NP alone exhibited relatively low cytotoxicity with an IC₅₀ of 1020 µg/mL, while ZnFe₂O₄ NP showed moderate toxicity with an IC₅₀ of 21.98 µg/mL. Notably, the GO/ZnFe₂O₄ NC demonstrated significantly enhanced cytotoxicity, with an IC₅₀ of 13.58 µg/mL, indicating a synergistic effect between the components. These results suggest that the composite markedly increases cellular susceptibility compared to individual materials, highlighting its potential as a bioactive agent.

Fig. 7.

Cytotoxicity effects of GO NP, ZnFe₂O₄ NP, and GO/ZnFe₂O₄ NC on HeLa cells.

The calculated SI values were 2.84 for GO NP, 17.18 for ZnFe₂O₄ NP, and 11.67 for the GO/ZnFe₂O₄ NC (Table 2). These results indicate that all materials exhibited some degree of selectivity toward cancer cells over normal fibroblasts. Notably, the ZnFe₂O₄ NP and GO/ZnFe₂O₄ NC demonstrated high SI values, reflecting pronounced preferential cytotoxicity against HeLa cells, whereas GO NP showed relatively low selectivity. This analysis underscores the potential of the GO/ZnFe₂O₄ NC as a bioactive material with enhanced cytotoxicity toward cancer cells while maintaining lower toxicity toward normal cells.

Table 2.

IC₅₀ values of GO NP, ZnFe₂O₄ NP, and GO/ZnFe₂O₄ NC in fibroblast and HeLa cells, and corresponding selectivity indices (SI).

Sample	IC₅₀ (Fibroblast) µg/mL	IC₅₀ (HeLa) µg/mL	Selectivity Index (SI)
GO NP	2896	1020	2.84
ZnFe₂O₄ NP	377.8	21.98	17.18
GO/ZnFe₂O₄ NC	158.5	13.58	11.67

Expression of BAX, Cas-3, and Bcl-2 Genes

To evaluate the impact of treatments on apoptosis regulation, the mRNA expression levels of BAX, Cas-3, and Bcl-2 were analyzed in four experimental groups: Untreated, GO NP, ZnFe₂O₄ NP, and GO/ZnFe₂O₄ NC.

BAX: Treatment with ZnFe₂O₄ NP induced a significant upregulation of BAX expression compared to untreated cells (P = 0.01). GO NP alone did not elicit a notable change in BAX levels. Notably, the GO/ZnFe₂O₄ NC resulted in a marked increase in BAX expression (P < 0.0001 vs. untreated), which was significantly higher than the effects observed with both ZnFe₂O₄ NP (P = 0.003) and GO NP alone (P = 0.0005) (Fig. 8A).

Fig. 8.

Effects of GO NP, ZnFe₂O₄ NP, and GO/ZnFe₂O₄ NC on the expression of BAX, Cas-3, and Bcl-2 Genes.

Cas-3: Exposure to ZnFe₂O₄ NP significantly elevated Cas-3 expression relative to untreated cells (P = 0.02), while GO NP treatment alone had no significant effect. The GO/ZnFe₂O₄ NC elicited the highest induction of Cas-3 (P < 0.0001 vs. untreated), with expression levels significantly surpassing those observed with ZnFe₂O₄ NP (P = 0.02) and GO NP alone (P = 0.004) (Fig. 8B).

Bcl-2: Treatment with GO NP significantly reduced Bcl-2 expression compared to untreated controls (P = 0.04). ZnFe₂O₄ NP alone did not produce a significant change relative to GO NP. The GO/ZnFe₂O₄ NC strongly suppressed Bcl-2 expression (P < 0.0001 vs. untreated), with levels significantly lower than those observed with both ZnFe₂O₄ NP (P = 0.01) and GO NP alone (P = 0.03) (Fig. 8C).

These results indicate that the GO/ZnFe₂O₄ NC exerts a pronounced pro-apoptotic effect by upregulating pro-apoptotic genes (BAX and Cas-3) while downregulating the anti-apoptotic gene Bcl-2, suggesting a synergistic role in apoptosis induction.

Discussion

The successful fabrication of the GO/ZnFe₂O₄ NC is well supported by the structural, morphological, compositional, and magnetic characterization data. In the XRD analysis, the appearance of characteristic peaks assigned to the spinel ZnFe₂O₄ phase confirms the formation of the target crystalline architecture. Simultaneously, the diffraction peak observed at a lower 2θ angle in the precursor GO material was either strongly attenuated or absent in the composite, indicating effective exfoliation of the GO sheets and subsequent integration into the ZnFe₂O₄ matrix. This observation is consistent with previous studies; for instance, Tong et al. (2022) reported that the GO peak becomes nearly undetectable after hybrid formation, which was attributed to structural rearrangement and intimate integration between GO and ferrite phases²⁶.

From a morphological perspective, SEM images reveal a uniform distribution of ZnFe₂O₄ NP across the surfaces of GO sheets. Such homogeneous decoration can partially suppress severe nanoparticle agglomeration and help preserve a higher effective surface area, which is advantageous for functional applications. In previous studies reduced GO/ZnFe₂O₄ composites showed nanoparticles anchored on rGO sheets and exhibited superior catalytic activity compared with free ZnFe₂O₄²⁷.

It should be noted that the crystallite size calculated from XRD reflects the size of coherently diffracting crystalline domains, whereas the particle size observed in SEM corresponds to larger structural entities that may consist of multiple crystallites. The larger particle dimensions observed in SEM images can therefore be attributed to nanoparticle aggregation as well as the anchoring of ZnFe₂O₄ nanoparticles onto GO sheets. Such agglomeration effects are common in ferrite-based nanocomposites and do not necessarily contradict the nanoscale crystallite size obtained from XRD analysis. Although SEM images qualitatively confirm the successful formation and distribution of nanoparticles, a detailed particle size distribution analysis based on statistical measurements (n ≥ 100) was not performed in this study and is considered a limitation of the current work^28,29.

Elemental composition data from EDX further support the successful synthesis: Zn and Fe characteristic peaks appear at the expected atomic ratios for the spinel phase, and the detected oxygen peak confirms the oxide nature of the material. These findings corroborate structural data from XRD and Raman spectroscopy, providing a comprehensive verification of composite formation. This multi-technique validation mirrors the approach adopted by Alsulami et al. (2022) in engineering GO/ZnFe₂O₄/polyaniline composites for supercapacitor application³⁰.

Raman spectral analysis offers insight into the carbon-based component’s structural integrity and defect density. The G band (~ 1580 cm⁻¹) reflects the in‑plane vibrations of sp² carbon atoms, while the D band (~ 1350 cm⁻¹) indicates defects, edges, or structural disorder. Accordingly, our Raman data indicate that GO in the composite is no longer pristine, but structurally modified in a way that supports the intended functional architecture. From the magnetic measurements, the M–H loop of the composite shows an S‑shaped curve with low coercivity (H_C) and near‐zero remanence (M_r), and a saturation magnetization (M_s) in the order of ~ 15 emu/g, indicating superparamagnetic behaviour at room temperature. Superparamagnetism is especially desirable in certain applications (e.g., biomedical or magnetic separation) because it enables rapid magnetization under an external field and virtually zero residual magnetization when the field is removed, thus avoiding unwanted aggregation. Similar behavioural trends have been documented for ZnFe₂O₄/graphene systems designed for electromagnetic shielding and catalysis³¹.

Taken together, our findings indicate that the proposed synthesis route yields a well-structured nanocomposite with the intended architecture and promising functional attributes. While the applied characterization techniques confirm the successful formation and uniform distribution of ZnFe₂O₄ NP on GO sheets, more advanced analyses, such as high-resolution TEM or XPS, would be required to comprehensively elucidate the interfacial interactions between the two phases.

Our results demonstrated that the GO/ZnFe₂O₄ NC exhibits enhanced antibacterial performance compared to the individual components. The antibacterial action of GO has been extensively documented. One comprehensive review notes that GO can exert antibacterial effects through sharp‑edge membrane stress causing disruption of bacterial cell walls, generation of reactive oxygen species (ROS), and entrapment or wrapping of cells^32,33. For example, studies have shown GO nanowalls against S. aureus and E. coli with evidence of membrane penetration and leakage of intracellular contents³⁴. In the present work, our findings indicate that GO NP alone exhibited limited antibacterial activity at the concentrations tested.

Regarding ZnFe₂O₄, fewer studies have focused on its standalone antibacterial efficacy. Morais et al. (2021) synthesized ZnFe₂O₄ NP and demonstrated moderate antibacterial activity (for S. aureus and E. coli) at relatively high concentrations (MIC > 1 mg/mL) and inhibition percentages of ~ 40‑70% after 24‑72 h³⁵.

More recently, studies of magnetically‑enhanced ZnFe₂O₄ nanocomposites show promise for antibacterial applications³⁶. In our experiment, ZnFe₂O₄ NP produced a measurable antibacterial effect by itself, supporting the notion that ferrite‑based metal‑oxide nanomaterials can disrupt bacterial viability possibly through electrostatic interactions between the negatively charged bacterial membrane (rich in functional groups such as carboxyl, hydroxyl, phosphate) and the positively charged metal ions (e.g., Zn²⁺, Fe³⁺) that may adhere, disturb respiratory enzymes, generate ROS, and cause cell death.

Our GO/ZnFe₂O₄ NC exhibited the strongest antibacterial performance, which may be attributed to synergistic effects: (i) GO providing a high‑surface‑area support and potential membrane‑disruption mechanism, (ii) ZnFe₂O₄ contributing ionic / ROS‑generating antibacterial action, and (iii) improved dispersion and contact of nanoparticles when anchored on GO sheets, thereby enhancing bacterial exposure and efficiency. A previous study showed that NiFe₂O₄/CeO₂/GO enhanced photocatalytic and antibacterial performance compared to the individual components³⁷. Our findings are consistent with previous literature.

This study is limited to in vitro testing, and additional investigations are needed to assess the potential clinical applicability of the nanoparticles relative to conventional antibiotics.

The results of the MTT assay clearly indicate that the GO/ZnFe₂O₄ NC exhibited substantially greater cytotoxicity than its individual components in both normal fibroblast and HeLa cell lines. In fibroblasts the IC₅₀ decreased from ~ 2896 µg/mL (GO NP alone) and ~ 377.8 µg/mL (ZnFe₂O₄ NP) to ~ 158.5 µg/mL for the composite; similarly, in HeLa cells GO NP alone had an IC₅₀ of ~ 1020 µg/mL, ZnFe₂O₄ NP ~ 21.98 µg/mL, while the composite reached ~ 13.58 µg/mL. These pronounced reductions in IC₅₀ suggest a synergistic interaction between GO NP and ZnFe₂O₄ NP, likely resulting from enhanced cellular uptake or intracellular disruptive mechanisms when combined.

Comparable findings have been reported in the literature. For instance, the GO–ZnFe₂O₄/DOX NC developed by Ali et al. (2022) demonstrated higher cytotoxicity in HeLa cells, along with increased ROS and apoptosis compared with GO or ZnFe₂O₄ alone³⁸.

In another study, ZnFe₂O₄ NP exhibited dose-dependent cytotoxicity against MCF-7 breast cancer cells and were found to be more toxic at concentrations exceeding ~ 125 µg/mL³⁹. Together, these independent reports strengthen the interpretation that the integration of ZnFe₂O₄ with a GO scaffold amplifies bio-reactivity beyond that achievable by the metal ferrite alone.

The SI provides a measure of preferential cytotoxicity toward cancer cells relative to normal cells, with higher values indicating greater selectivity. In this study, the GO/ZnFe₂O₄ NC exhibited an SI of 11.67, reflecting strong selective toxicity toward HeLa cells while maintaining lower cytotoxicity against fibroblasts. Similarly, ZnFe₂O₄ NP alone displayed a high SI (17.18), whereas GO NP showed modest selectivity (SI = 2.84). These findings suggest that the combination of GO with ZnFe₂O₄ enhances cancer cell-targeted cytotoxicity, which may result from synergistic interactions between the components. SI values greater than 2 are generally considered indicative of selective toxicity toward cancer cells, as reported for curcumin–zinc oxide nanoparticles exhibiting SI above this threshold in comparison with normal Vero cells⁴⁰. Similarly, iron oxide–graphene NC have demonstrated high SI (~ 10.8) against HepG2 liver cancer cells relative to healthy fibroblasts, indicating pronounced selectivity provided by the nanocomposite design⁴¹. These findings are consistent with our results.

The present study demonstrates that treatment with the GO/ZnFe₂O₄ NC markedly affects the expression of apoptosis-regulating genes in comparison to the individual components. Specifically, the composite induced a strong up-regulation of the pro-apoptotic gene BAX (P < 0.0001 vs. untreated, and significantly higher than ZnFe₂O₄ NP alone, P = 0.003; GO NP alone, P = 0.0005). Similarly, the effector caspase gene Caspase‑3 (Cas-3) was significantly elevated by the composite treatment (P < 0.0001 vs. untreated; vs. ZnFe₂O₄ NP, P = 0.02; vs. GO NP, P = 0.004). In contrast, the anti-apoptotic gene Bcl‑2 was strongly down-regulated by the composite (P < 0.0001 vs. untreated; significantly lower than ZnFe₂O₄ NP, P = 0.01; and GO NP alone, P = 0.03). These findings suggest the GO/ZnFe₂O₄ NC promotes a shift toward apoptosis by simultaneously increasing pro-apoptotic signalling and decreasing anti-apoptotic defence.

Similarly, NiZn ferrite NP were reported to upregulate Bax and Caspase-3/9 expression while downregulating Bcl-2 in breast cancer cells, along with inducing cytochrome-c release from mitochondria. Moreover, exposure of MCF-7 cells to ZnFe₂O₄ NP for 24 h resulted in a significant increase in lipid peroxidation and a marked decrease in glutathione levels⁴².

Mechanistically, this pattern is consistent with activation of the intrinsic (mitochondrial) apoptotic pathway, in which BAX insertion into the mitochondrial outer membrane triggers permeabilization, cytochrome c release and caspase cascade activation, while Bcl-2 serves as a major gatekeeper against that process. For instance, studies have shown that an increased BAX/Bcl-2 ratio correlates with mitochondrial outer membrane permeabilization and apoptosis onset⁴³. Thus, the observed gene-expression changes with the composite suggest a synergistic pro-apoptotic effect that may be greater than that of the individual GO NP or ZnFe₂O₄ NP treatments.

Therefore, future studies should focus on optimizing the concentration range and surface modification of GO/ZnFe₂O₄ NC to achieve selective cytotoxicity toward malignant cells while maintaining biocompatibility.

Conclusion

The present study demonstrates the successful green synthesis of GO/ZnFe₂O₄ NC, confirmed by structural, morphological, compositional, and magnetic analyses. The uniform distribution of ZnFe₂O₄ nanoparticles on GO sheets preserves high surface area and promotes superparamagnetic behavior, supporting the potential of the composite for diverse functional applications. Functionally, the GO/ZnFe₂O₄ NC exhibited superior antibacterial activity compared to its individual components, likely due to synergistic effects such as membrane disruption by GO NP and ROS/ionic-mediated bacterial inhibition by ZnFe₂O₄ NP; however, these mechanisms were not directly validated in this study. Cytotoxicity assays revealed that the composite selectively induces apoptosis in HeLa cells while maintaining comparatively lower cytotoxicity toward fibroblasts, as reflected by a high selectivity index (SI = 11.67). Mechanistically, this pro-apoptotic effect is supported by significant upregulation of BAX and Caspase‑3, and strong downregulation of Bcl‑2, indicating activation of the intrinsic mitochondrial apoptotic pathway and a synergistic action of the composite components. These results highlight the ability of the GO/ZnFe₂O₄ NC to amplify both antibacterial and anticancer bioactivity beyond the individual components. Future work should focus on further mechanistic studies, in vivo validation, and optimization of dosage and surface modifications to maximize selective cytotoxicity while preserving biocompatibility.

Abbreviations

GO

Graphene oxide

ZnFe₂O₄

Zinc ferrite

XRD

X-ray diffraction

EDX

Energy-dispersive X-ray

VSM

Vibrating sample magnetometer

SEM

Scanning electron microscopy

WHO

World Health Organization

MDR

Multidrug-resistant

TP

Teucrium polium

NPs

Nanoparticles

NA

Nutrient agar

MHB

Mueller Hinton Broth

MIC

Minimum Inhibitory Concentration

MBC

Minimum Bactericidal Concentration

MHA

Mueller Hinton Agar

IZ

Inhibition Zone

ROS

Reactive oxygen species

SI

Selectivity index

Author contributions

The initial idea for the experiment was conceived by HM and RSC. MK, IS, MN, MM and RSC participated in designing the study and performed the statistical analysis. Overall supervision of the project, manuscript submission, and correspondence were handled by HM and RSC. The tasks of conducting the experiments and drafting the manuscript were the responsibility of MK, IS and MN.

Funding

The authors received no specific funding for this work.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study received ethical approval from the Research Ethics Committee of Kashan University of Medical Sciences under the code IR.KAUMS.MEDNT.REC.1403.193.