All metal salt precursors used for biosynthesis of ZnO-NPs and ZnO-Ag NPs were of analytical grade. Zinc nitrate hexahydrate (Zn(NO₃)₂.6H₂O, 98%), silver nitrate (AgNO₃, 98%) and ammonia solution (NH₄OH, 30%) were purchased from R&M Chemicals, United Kingdom. For antibacterial experiment, three Gram-positive (Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus clinical isolate and Bacillus subtilis ATCC 6633) as well as three Gram-negative strains (Escherichia coli ATCC 11775, rifampicin-resistant Escherichia coli K1 clinical isolate and Salmonella enterica ATCC 14028) were tested. Human colorectal carcinoma cell (HCT116: ATCC CCL-247), lung cancer cells (A549) and immortalized cervical cancer cells (Hela) were used for in vitro cytotoxicity assay. The cell lines were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/mL penicillin/streptomycin (Gibco).

Fresh fruit peels of P. granatum were collected from the “Green Farm” in Fars province, Neyriz, Iran. Plant extract was prepared as previously described (Sukri et al., 2019). Briefly, fresh P. granatum F. peels were dried ground into fine powder and then were subjected to aqueous extraction with distilled water at 60°C for 60 min. The crude extract was clarified by filtration followed by centrifugation at 4000 rpm for 10 min, and kept at 4°C for further experiments.

ZnO-NPs were fabricated using the same sol–gel and combustion procedure as previously described (Sukri et al., 2019). Concisely, zinc nitrate hexahydrate and the fruit peels extract were stirred at 90°C until a gel-like product was formed. After annealing process at 600°C for 60 min, ZnO-NPs, namely Z-6, were produced, in the form of a fine white colored powder. The NPs were then used for the biosynthesis of ZnO-Ag-NPs by precipitation method. For this aim, P. granatum F. peels extract and 10 mL, AgNO₃ (0.1 M) were added to 0.1 g of ZnO-NPs followed by continuously stirring in a beaker. Ammonium hydroxide (1 M) solution was added drop by drop into the beaker to reach to the desired pH level (pH 4, 7, and 9). After 60 min’ incubation at 55°C in a water bath shaker, the solutions were centrifuged at 10,000 rpm for 10 min. The precipitates were then washed three times with water and ethanol. Finally, the samples were dried at 60°C in an oven. The resulting samples are named as ZA-4, ZA-7, and ZA-9 to represent ZnO-Ag-NPs synthesized at pH 4, 7, and 9.

The crystalline phase of all samples was investigated using X-ray diffraction (X’Pert, Philips, Netherlands) using Cu Kα radiation at the angle range of 2θ (10–90°). Ultraviolet–visible (UV 1800, SHIMADZU, Japan) spectroscopy (UV-vis) was utilized in the range of 300 to 700 nm to observe the absorption peaks of each biosynthesized sample. Fourier Transform Infrared Spectroscopy analysis (FTIR Model 1650, PerkinElmer, USA) was carried out in the range of 400–4000 cm⁻¹ wavenumber to detect functional groups on the NPs. Potassium bromide (KBr) method was adopted to analyze ZnO and ZnO-Ag-NPs, while attenuated total reflection (ATR) method was used for P. granatum F. peels extract. The surface morphology and electron diffraction pattern of ZnO and ZnO-Ag-NPs were investigated using Transmission Electron Microscopy (TEM) with Energy Dispersive X-Ray Spectroscopy (EDS, JEM-2100F, Jeol Ltd., Japan).

Broth micro-dilution method was used to determine the minimum inhibitory concentration (MIC) of the NPs toward bacterial strains using the Clinical and Laboratory Standards Institute (CLSI) protocols as previously described (Ismail et al., 2019; Sukri et al., 2019). To this end, single colonies of fresh bacterial cultures on Mueller Hinton agar (MHA) plates were inoculated into sterile Mueller Hinton broth (MHB) for overnight culture (12–18 h) at 37°C prior to the experiments. Bacterial concentration was then standardized to an optical density (OD) of 600 nm (approximately 10⁸ CFU/mL) with MHB. Two-fold serial dilutions of NPs were prepared in 96-well plates to give the final test concentrations of 0, 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 μg/mL per well. Ten μl of bacterial suspension equivalent to 10⁶ CFU/mL of exponentially growing bacterial cells were added to the wells. After 18 h incubation at 37°C, OD 600_nm was determined using microplate reader (Tecan), and MIC₅₀ was then measured using an online calculator.¹ Three independent experiments were performed and the data are expressed as the mean ± standard deviation for all triplicates within an individual experiment.

Cell Titer-Glo 2.0 Cell Viability Assay (#G9241, Promega) was used to determine the cytotoxic effect of NPs on HCT116 colorectal, A549 lung cancer cells and Hela cells according to the manufacturer’s instruction (Yusefi et al., 2021). Briefly, 5 × 10³ cells were seeded in 96-well plate (100 μL/well) and incubated overnight at 37°C in a 5% CO₂, 95% humidified incubator. The next day, two-fold serially diluted samples at the concentrations of 0–500 μg/mL (100 μL/well) were added into the cells. After 72 h’ incubation at 37°C in a 5% CO₂ incubator, 20 μL of the MTS reagent was added into the wells and incubated for an additional 3 h at 37°C in a 5% CO₂ incubator. The optical density (OD) was then measured at 490 nm using a multimode microplate reader (Tecan). The dose–response graph was plotted by calculating the percent cell viability using an equation below:

Inhibitory concentration causing 50% growth inhibition (IC₅₀) was determined using an online calculator. Three independent experiments were performed and the data are expressed as the mean ± standard deviation for all triplicates within individual experiments.

The cytotoxicity and antibacterial data was analyzed by t-test; statistical significances were set at p < 0.05, the results were expressed as means ± standard deviation and the graphs were drawn by GraphPad Prism 9.5.0. The histogram of particle size distribution, which was sized according to the TEM images and by the ImageJ-Fiji software, was drawn by SPSS 16.0 software.

Successful synthesis of ZnO-NPs and ZnO-Ag-NPs was confirmed by XRD. As shown in Figure 2, the pristine ZnO-NPs (Z-6) revealed narrow and intense peaks, indicating high crystallinity of the nanoparticle; the diffraction peaks showed 2θ values of 32.00°, 34.66°, 36.48°, 47.77°, 56.76°, 63.06°, 66.53°, 68.13°, and 69.27° corresponding to the crystal planes of (100), (002), (101), (102), (110), (103), (200), (112), and (201), respectively. These peaks are indexed to the ZnO hexagonal wurtzite phase structure supported by JCDPS Cardno. 89-1397 data (Karaköse et al., 2017). No other peaks related to any foreign compounds were detected in the Z-6 samples.

Similar ZnO peaks were detected in the ZnO-Ag-NPs (ZA-4, ZA-7 and ZA-9), confirming the successful formation of the crystalline structures. Ag diffraction peaks were represented with 2θ values of 38.07°, 44.42°, and 64.59 corresponding to the crystal planes of (111), (200), and (220), respectively. These peaks can be indexed to face centered cubic phase metallic silver supported by JCDPS Cardno. 001-1167 (Basavalingiah et al., 2019). The absence of other diffraction peaks verifies the successful fabrication of ZnO-Ag-NPs with P. granatum F. peels extract as a mediating solvent. The diffraction peaks were found to be narrower and more intense for ZA-9 as compared to ZA-4 and ZA-7, which might refer to the increased crystallinity of ZA-9 in basic environment.

UV-vis spectroscopy was also used to confirm the biosynthesis of the NPs. As shown in Figure 3A, compared to ZnO-NPs (Z-6), the ZnO-Ag-NPs revealed a shift in the excitonic absorption peaks from 370 nm toward longer wavelength at 420–430 nm (red-shift) due to localized surface plasmon resonance (LSPR). This shift indicates successful Ag modification through substitution of Ag⁺ ions into the Zn²⁺ sites on the ZnO lattice, resulting in band-gap energy changes (Zare et al., 2019). To calculate the optical band-gap value of all ZnO-Ag-NPs samples, Tauc’s plot was plotted, where a significant decrease in band-gap value was observed in ZnO-Ag-NPs samples when compared to the pristine ZnO-NPs, as presented in Figure 3B. The band-gap value was projected to be 2.541, 2.485, and 2.515 eV for the ZA-4, ZA-7, and ZA-9, respectively. Higher band-gap value (3.283 eV) was detected for the Z-6.

FTIR spectroscopy data analysis, as shown in Figure 4, determined functional groups in ZnO-NPs and all ZnO-Ag-NPs. Characteristic peaks of hydroxyl groups, due to atmospheric moisture absorption, were detected between 3332 and 3385 cm⁻¹. This stretching mode of O-H groups can be due to the formation of inter- and intra-molecular hydrogen bonds in the sample (Abou Oualid et al., 2017). Broad band ranging from 401 cm⁻¹ to 530 cm⁻¹, highlighted the red dotted box, can be attributed to the stretching vibration of ZnO (Rohith et al., 2018). Compared to the strong absorption Zn-O peak in the ZnO-NP (Figure 4B), the intensity of Zn-O peak was slightly reduced in the ZnO-Ag-NP, due to the successful binding of Ag on the surface of the nanoparticle (Basavalingiah et al., 2019).

Characteristic peaks in the range of 900 to 1500 cm⁻¹ (yellow box) are most probably referring to the presence of oxygen stretching and bending. It might also display the peaks of remaining compounds from the peel extract. All FTIR peaks and the functional group assigned to them are listed in Table 1. Bands found between 1343 and 1392 cm⁻¹ corresponds to C=O bending, and peaks at 1529 and 1552 cm⁻¹ represent O-H bending vibrations (Satdeve et al., 2019). Sharp peaks between 1529 and 1552 cm⁻¹ might also belong to the stretching peak of C=O group present in the polyphenol content of the plant extract, for example gallic acid (Pandiyan et al., 2019). Weak band of C-N group were also detected in the range of 1080 to 1360 cm⁻¹ (Das et al., 2016). The Ag⁺ corresponding peak was also detected around 1029 cm⁻¹ in ZnO-Ag-NPs (Pandiyan et al., 2019).

Morphology and particle distribution in the nanoparticles were assessed by HR-TEM. TEM images (Figures 5A–C) showed the presence of irregular agglomerated particles in the ZnO-Ag-NPs. Ag-NPs were detected as the darker spherical particles with the mean diameter of 15.08, 16.06, and 14.00 nm for ZA-4, ZA-7, and ZA-9, respectively, as shown in the particle distribution histograms. Smaller particle size of the Ag-NPs in the samples prepared at pH 9 (Figure 5C) might be due to the effect of higher concentration of ammonia in the reaction. Several publications have reported the size-controlling role of ammonia in high pH level, as it can help fasten the reaction rate of silver nucleation as well as stabilizing the Ag-NPs (Suwatthanarak et al., 2016; Pimpan and Ritthichai, 2018; Marciniak et al., 2020).

Further characterization of the ZnO-Ag-NPs (ZA-9) using selected area electron diffraction (SAED), as shown in Figure 6A, revealed clear rings indexed to both hexagonal wurtzite phase structure of ZnO (yellow rings) and face centered cubic phase of Ag (orange rings). From the magnified HR-TEM image (Figure 6B), an interplanar lattice spacing value of 0.247 nm, corresponding to the (1 0 1) plane of hexagonal ZnO, was measured. It indicates that one of the growth planes of the ZnO-NPs is along the (1 0 1) plane (Sharma et al., 2018; Marciniak et al., 2020). A d-spacing value of 0.23 nm, corresponding to the hkl lattice plane of (1 1 1) of cubic Ag phase (Le et al., 2019), was also determined in the magnified image. These results are consistent and in complete agreement with the XRD results discussed in Figure 2.

Energy Dispersive X-Ray Spectroscopy (EDS) was also carried out to determine the elemental composition in the ZA-9, where the presence of zinc (Zn), oxygen (O) as well as silver (Ag) elements was detected in the sample (Figure 7). The elemental mapping images revealed uniformly distribution of Ag over the surface of the ZnO-NPs. Quantifiable EDS weight ratios of 63, 36.8, and 0.2% were measured for Zn, O and Ag, respectively, in the sample.

ZnO-NPs has been shown to significantly inhibit bacteria growth (Raghupathi et al., 2011). In this study, we investigate if plant-mediated synthesized ZnO and ZnO-Ag-NPs could inhibit bacterial growth. Our data revealed a broad range of antibacterial activities of ZnO-Ag-NPs against both gram-positive and gram-negative bacteria, as shown in Figures 8A–F. The growth of both groups of bacteria was found to be affected by the NPs in a concentration-correlated manner. Significantly less concentration of the ZA-4, ZA-7, and ZA-9 nanoparticles was needed to reach more than 50% inhibition of bacterial growth when compared to ZnO-NP Z-6.

In alignment to the findings presented in Figure 8, calculated MIC₅₀ (Table 2) revealed much lower antibacterial efficacy of ZnO-NPs (Z-6) against the bacterial strains when compared to the ZnO-Ag-NPs. The data also showed higher resistance of the tested Gram-positive bacteria toward the pristine ZnO-NPs (Z-6), most probably due to its thicker cell wall, making it harder to permeate (Mthana et al., 2022). Interestingly, the ZnO-Ag-NPs (ZA-4, ZA-7, and ZA-9) showed better antibacterial activities against the Gram-positive strains. This might be due to strong attraction of positively charged Zn²⁺ and Ag⁺ ions dissociated from ZnO-Ag-NPs toward the negatively charged surface of Gram positive bacteria, leading to damage of the cell membrane. ZA-7 displayed the best growth inhibition activity against S. aureus, while ZA-4 could remarkably inhibit the growth of MRSA and B. subtilis. ZA-7 also exhibited the best antibacterial activity against the Gram-negative bacteria, especially S. enterica. Higher inhibition activities of ZnO-Ag-NPs could also be contributed by the surface coating by P. granatum F. peels extract as they are proven to have antimicrobial properties as well (Singh et al., 2018).

The exact mechanism of killing bacteria by ZnO-NPs and ZnO-Ag-NPs is still not fully understood. Generally, it seems that these NPs directly interact with the bacterial cell wall or membrane by releasing metal ions that disrupt the cell permeability, causing damage to the first line of defense (Gupta et al., 2018). Upon entry into the cells, the NPs and free metal ions will affect the bacteria’s biochemical processes by causing DNA damage and protein denaturation (Khan et al., 2018). This will finally trigger cell death as the bacteria fails to normally replicate. Figure 9 illustrates the possible antibacterial mechanism once in contact with NPs.

The possible explanation for the higher antibacterial activity of ZnO-Ag-NPs as compared to ZnO-NP might be due to structural changes of ZnO-NP after being coupled with Ag; introduction of Ag atoms onto the surface of ZnO-NPs lead to the formation of lower electron state in the band gap which can trap photogenerated charge carriers (Sukri et al., 2021), leading in an increase in electron–hole pairs which could react with oxygen and water to produce reactive oxygen species (ROS), as a critical element in triggering cell death.

To examine the cytotoxicity effect of the nanoparticles in vitro, colorectal (HCT116) lung (A549) and cervical (Hela) cancer cells were treated with different concentrations of the nanoparticles, and anti-proliferative effect was investigated by MTT assay. The results, summarized in Figure 10, showed that the nanoparticles could dose-dependently inhibit the proliferation of the cells. We found that the ZnO-Ag-NPs (ZA-4, ZA-7, and ZA-9) had significantly better anti-proliferative effect on cancer cells when compared to the ZnO-NP (Z-6), indicating the enhancement of cytotoxic activities of ZnO-NPs after conjugation with Ag. In contrast to the pristine ZnO-NPs, the ZnO-Ag-NPs could completely inhibit cell growth at the low concentration of 31.25 μg/mL. Based on the calculated IC₅₀ values, presented in Table 3, ZnO-Ag-NPs exhibited notably higher cytotoxic activities against the cancer cells 72 h after the treatment when compared to ZnO-NP. The best anti-proliferative effect was observed in the ZA-9-treated cells with the lowest IC₅₀ values of 15.12–16.11 μg/mL toward the cancer cells. This may be related to the smaller size of ZA-9 as compared to ZA-4 and ZA-7, leading to increased surface chemistry of the NPs with cancer cells (Mthana et al., 2022).

Similar to antibacterial mechanism, Zn²⁺ ions and free radicals released from Ag have been reported to induce ROS production, triggering cells death via ROS-mediated apoptotic process. An interesting study from Sachdev et al. (2015) investigated the cellular uptake of carbon dots-silver@zinc oxide nanocomposite (CD Ag@ZnO NC) inside cancer cells, revealing cytoplasm and nuclear localization of the NCs in a dose-dependent manner. In lower concentration of 20 μg/mL, the NC was found mostly in the cytoplasm whereas nuclear localization was also observed in higher concentrations of 50 and 70 μg/mL.