Cytotoxic Effects of Ag-ZnO Bimetallic Nanoparticles Synthesized with Alchemilla vulgaris Extract on Colon Cancer Cell Lines HT-29 and Caco-2

Abstract

Introduction:

Nanoparticles (NPs) have attracted the attention of scientists working on nanotechnology. NPs have different therapeutic effects. The therapeutic applications of these NPs depend on the amount of metal in the target cell, particle size, and physicochemical properties. However, previous studies have shown that bimetallic and multimetallic NPs have excellent physicochemical properties, synergistic effects, and high functionality.

Materials and Methods:

In this study, Alchemilla vulgaris plant extract was used to synthesize Ag-ZnO bimetallic nanoparticles (BNPs). The Ag-ZnO BNPs produced through biosynthesis were thoroughly analyzed using UV-vis spectroscopy, STEM and EDS. [Antibacterial activity assays were conducted on both gram-negative and gram-positive bacteria, and antioxidant activity was tested using the DPPH assay. Finally, cytotoxicity on L929, HT29, and Caco = 2 cell lines was evaluated using the MTT assay].

Results:

UV-vis absorption spectrum showed maximum absorption at 437 nm and a shoulder peak at 354 nm. The particle sizes of the synthesized BNPs varied from 10 to 40 nm. Ag-ZnO BNPs did not have an antibacterial effect on gram-negative Escherichia coli bacteria. Against this, Ag-ZnO BNP had antibacterial activity against two gram-positive bacteria. Enterococcus faecalis showed an inhibition zone of 11 mm and an inhibition zone of 10 mm for Staphylococcus aureus. The antioxidant activity of synthesized Ag-ZnO BNPs was confirmed by 1-diphenyl-1-2-picrylhydrazyl. It showed a maximum antioxidant activity at 4000 µg/mL with a rate of 46.68%. The cytotoxicity of Ag-ZnO BNPs on L929, HT-29, and Caco-2 cells was evaluated using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. As a result, the Ag-ZnO BNPs showed high toxicity on HT-29 and Caco-2 cell lines. Ag-ZnO BNPs at higher concentrations killed more than 50% of cancer cells (IC₅₀: 8.43 µg/mL, IC₅₀: 6.48 µg/mL). Ag-ZnO BNPs are less cytotoxic in L929 cells at low doses (IC₅₀: 14.15 µg/mL).

Conclusion:

As a result, it has been proven that Ag-ZnO BNPs have antibacterial, antioxidant, and cytotoxic effects.

Introduction

Bimetallic nanoparticles (BNPs) are considered more intriguing than monometallic nanoparticles (MNPs) owing to their enhanced properties. BNPs are composed of two distinct metals that exhibit superior electronic, catalytic, plasmonic, optical, thermal, magnetic, and biological characteristics because of synergistic interactions between the metals.^1,2 These nanoparticles (NPs) often display improved technological relevance compared with MNPs, as they offer unique nanostructures where one metal typically forms a core while the other creates a surrounding shell.^3–6 Among bimetallic nanomaterials, silver-zinc oxide (Ag-ZnO) BNPs stand out because of their variety of applications. Ag-ZnO BNPs are particularly important due to their industrial, medical, and environmental uses.⁷ Although silver (Ag) and zinc oxide (ZnO) NPs exhibit anticancer activity on their own, the combination of these two metals in BNPs leads to synergistic enhancement of therapeutic effects. Ag is well known for its strong antimicrobial and anticancer properties, exerting cytotoxic effects by disrupting the cell membrane and damaging intracellular proteins and DNA.⁸ ZnO, on the other hand, induces cancer cell death by increasing the production of reactive oxygen species (ROS), leading to oxidative stress. When combined in Ag-ZnO bimetallic structures, ROS production is further amplified, enhancing DNA damage, mitochondrial dysfunction, and apoptosis, thereby strengthening the cytotoxic effects.^9,10 In addition, this combination retains the antimicrobial properties of Ag, reducing the risk of infection, while the biocompatibility of ZnO contributes to a synergistic effect. Consequently, Ag-ZnO BNPs offer superior therapeutic efficacy and broader biomedical applications than single-metal NPs.

Bimetallic nanostructure synthesis can be carried out using physical top-down, chemical bottom-up, and green-mediated methods.^11,12 Physical and chemical methods for NP synthesis are costly, hazardous, and time-consuming. On the contrary, there is more focus on green-mediated NP synthesis using natural resources such as plants, viruses,¹² yeasts and fungi,¹³ and algae¹⁴ known as “green synthesis.”^12–15 Plant extracts have been found to act as environmentally friendly reducing agents rather than hazardous reducing agents and are also responsible for stabilizing growing NPs instead of chemicals.¹⁶ Therefore, green synthesis of metal NPs is preferred. This involves the use of phytochemicals with antioxidant or reducing properties as potential precursors for synthesizing NPs and reducing metal ions. Biological synthesis of NPs is an eco-friendly, single-step bioreduction method that requires less energy and produces eco-friendly NPs.^17,18 A range of plant species has been utilized in the production of AgNPs, ZnONPs, and Ag-ZnO BNPs through biosynthesis.¹⁹

Alchemilla vulgaris (also known as Lady’s mantle) is a well-known example of a potential herbal therapeutic agent used to treat various diseases, including cancer. It belongs to the family Rosaceae and is characterized by a perennial herbaceous growth habit; it is widely distributed across Europe, western Asia, and North America. Among biologically active compounds, such as phenolic compounds, flavonoids, tannins, and antioxidants, A. vulgaris has demonstrated notable anticancer properties, making it valuable in cancer research and treatment. These bioactive compounds can inhibit cancer cell proliferation and induce apoptosis, thereby contributing to their therapeutic potential. Furthermore, the plant serves as an effective natural reducing agent for NP synthesis. Its antioxidant properties facilitate the reduction of metal ions and stabilization of NPs, offering an eco-friendly and non-toxic alternative to chemical synthesis methods. The inclusion of A. vulgaris in Ag-ZnO NP synthesis not only supports green synthesis by minimizing environmental impact but also enhances the biocompatibility, anticancer, antimicrobial, and antioxidant properties of the NPs, making it an ideal candidate for sustainable and therapeutic NP production.^20–22

This study aims to investigate the properties and effects of Ag-ZnO BNPs synthesized using Alchemilla vulgaris plant extract. Specifically, the objectives are to characterize the physicochemical properties of the synthesized BNPs, evaluate their antibacterial activities, assess their antioxidant properties, and examine their cytotoxic effects on HT-29 and Caco-2 colon cancer cell lines.

Materials and Methods

Preparation of A. vulgaris extract

A. vulgaris was purchased dry from a local market. A. vulgaris extract was made by boiling 2.5 g of dried herb in 200 mL of bidistilled water for 2 minutes in a microwave (900 W). A. vulgaris extract was dark yellow. After cooling, the extract was filtered using Whatman No. 1 filter paper. The extract was centrifuged at 1500 rpm for 1 minute.²¹ A. vulgaris extract was collected in a clean tube and stored at +4°C for later use.

Synthesis of BNPs from A. vulgaris extract

The synthesis of silver and zinc NPs used precursor salts of silver nitrate (AgNO₃) and zinc acetate (Zn(O₂CCH₃)·2(H₂O)₂). Anjum et al.’s (2022) method was used to synthesize Ag-ZnO BNPs.¹ The synthesis of Ag-ZnO BNPs (0.1/0.1) involved the combination of 1 mL of leaf extract with 50 mL of 0.1 M zinc acetate, followed by heating at 70°C for 5 minutes; 0.1 M silver nitrate (5 mL) was added to the Ag-ZnO BNPs, and the pH was adjusted to 12 by gradually adding 2 M NaOH. The mixture was constantly stirred at 400 rpm and incubated at 70°C for 2 hours. The progress and synthesis of all BNPs were confirmed using a UV-vis spectrophotometer. Following this, the solutions of all BNPs were centrifuged at 10,000 rpm for 15 minutes, and the supernatant was discarded and re-suspended in distilled water. Finally, the BNPs were dried overnight at 40°C, and their biological activities were characterized.

Characterization of BNPs

The formation of Ag-ZnO BNPs was observed by visual evaluation of the solution based on color change. After visual observation, the biosynthesis of the Ag-ZnO BNPs in the solution was monitored by measuring the UV-vis spectrophotometer (T60, PG Instruments Ltd., Japan) of the reaction mixture. UV-vis spectroscopy is commonly used to identify various types of NPs that can absorb electromagnetic radiation in the UV-vis spectral range. For instance, Ag-ZnO BNPs have a UV-vis absorbance range of ∼300–600 nm.⁴ Scanning transmission electron microscopy (STEM) was employed to examine the surface morphology, size, and shape of the Ag-ZnO BNPs. For STEM characterization, the samples were dried at room temperature, dispersed on a carbon-coated copper grid, and viewed under a 30 kV Zeiss GeminiSEM 500 electron microscope. The chemical composition was studied using X-ray energy dispersive spectrometry (EDS) technique.

Disk diffusion method

The in vitro antibacterial activity of the Ag-ZnO BNPs was assessed by the Kirby–Bauer disk diffusion method using Mueller–Hinton agar (MHA) with the measured inhibition zones in millimeters (mm). The disk diffusion method determines the capacity of antibacterial agents to spread onto agar and produce a bacterial inhibition zone.²³ Antibacterial activity was tested against Escherichia coli, Enterococcus faecalis, and S. aureus. All bacterial strains (S. aureus, E. fecalis, and E. coli) were cultured overnight at 37°C. Next, 20 µL of the bacterial cell suspension was spread onto the MHA plates. In addition, 6 mm Whatman paper sterile discs saturated with the positive control (penicillin/streptomycin), negative control (sterile water), and Ag-ZnO BNPs (200 µg/mL) were placed on each plate and incubated at 37°C for 24 hours. The antibacterial activity of Ag-ZnO BNPs was measured as clean zone inhibition (mm) using a vernier caliper.

1-Diphenyl-1-2-picrylhydrazyl scavenging assay

The antioxidant activity of the synthesized BNPs was determined by scavenging free radicals from 1-diphenyl-1-2-picrylhydrazyl (DPPH). The present test is designed to evaluate the hydrogen-donating potential of a given sample extract. This is accomplished through the examination of the decolorization process of a methanol solution comprising DPPH. DPPH, initially exhibiting a violet/purple hue in methanol, undergoes a color change to various yellow shades with the introduction of antioxidants.²⁴ To analyze antioxidant activity, 0.025 g of ZnO-Ag BNP was dissolved in 25 mL of methanol (1 mg/1 mL methanol). For positive control, 0.025 g of L-ascorbic acid was dissolved in 25 mL of methanol. The samples were prepared at various concentrations. (50–4000 μg/mL). DPPH was added to all concentrations. The reaction mixture contained 500 µL of each concentration and 1000 µL of a DPPH methanol solution (1:2). The mixture was incubated in a shaker for 1 hour. The DPPH radical scavenging activity was quantified by measuring the absorbance at 517 nm using a UV-vis spectrophotometer. The radical scavenging activity was then computed using the following formula²⁵: $% Inhibition = (Control - Sample) / Control * 100$ where %Inhibition is the percentage of the radical scavenging activity, control: is the absorbance of the control sample (DPPH solution without test sample), and sample is the absorbance of the test sample (DPPH solution with test compound).

MTT assay

The effect of BNPs on the susceptibility of normal fibroblast cell line L929, human colorectal adenocarcinoma cell line HT29, and colon cancer cell line Caco-2 was evaluated using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Cells were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum, L-glutamine, and sodium pyruvate, 100 IU/mL penicillin, and 100 g/mL streptomycin. They were then maintained at 37°C in a humid environment with 5% CO₂ concentration in a cell culture flask. After 24, 48, and 72 hours of culturing cancer cells in microplates, Ag-ZnO BNPs at concentrations of 0, 0.1, 0.5, 1, 5, 10, 20, and 50 μg/mL were added to each well. After 4 hours of incubation, 10 μL MTT dye at a concentration of 5 mg/mL was added to each well. Next, 100 μL of DMSO (Dimethylsulfoxide) was added to each well and stirred for 30 minutes at 37°C in the dark. The optical absorbance of the samples was measured using a Multiskan GO spectrophotometer (Thermo Fisher Scitific, Waltham, Massachusetts, USA), and the findings were recorded at a wavelength of 570 nm. The percentage of live cells treated relative to untreated was used in the following calculation to calculate the results²⁶: $Cell viability (%) = Abs o f Sample / Abs o f Control \times 100$

The IC₅₀ value (half maximal inhibitory concentration) was calculated using GraphPad Prism 9 software. The data were fitted to a four-parameter sigmoidal dose–response curve, where % cell viability was calculated for each drug concentration. The IC₅₀ value was derived from this fitted curve.

Statistical analysis

The statistical analysis of data was expressed as the mean ± SEM. The Student’s t test was used for statistical comparison, and differences were considered significant at p ≤ 0.05. All experiments were performed at least three times. GraphPad Prism 9 program was used to calculate IC₅₀ and SD (Standard deviation).

Results and Discussion

Synthesis and characterization of Ag-ZnO BNPs

Main Ag-ZnO BNPs were synthesized by A. vulgaris aqueous extract (Fig. 1). Formation of Ag-ZnO BNP’s first observation can be easily monitored by the color change of the solution which showed absorption in the visible range of electromagnetic radiation. The reaction mixture for ZnO NPs showed a light brown color, while the AgNP reaction mixture showed a greyish-black color.^27,28 In this study of BNP synthesis, the color of the reaction mixture was black (Fig. 2A). As mentioned in the methodology section, a standard sample of Ag-ZnO BNPs was characterized. Ag-ZnO BNP formation was confirmed using a UV-vis spectrophotometer. The absorbance of the biosynthesized Ag-ZnO BNPs was measured in the wavelength range of 300–600 nm. The UV-vis absorption spectrum showed a maximum absorption at 437 nm and a shoulder peak at 354 nm (Fig. 2B). These peaks might be the result of electrons moving from the lower valence band to the higher conduction band, which induces absorption. Furthermore, the existence of particles in the nanoscale realm is what causes these abrupt peaks. In addition, it might be a sign of a restricted particle size distribution.²⁹ The synthesis of Ag-ZnO BNPs was confirmed by the absorption spectra at 354 nm, which corresponds to ZnO NPs, and 437 nm, which corresponds to AgNPs.

FIG. 1.

Green synthesis of Ag-ZnO BNPs. Ag-ZnO BNPs, silver-zinc oxide bimetallic nanoparticles.

FIG. 2.

Color change of Ag-ZnO BNPs (A); UV-vis spectrum of Ag-ZnO BNPs (B).

STEM was used to determine the morphological properties of the NPs. The biosynthesized Ag-ZnO BNPs were mostly spherical; however, some irregularly shaped particles were also identified. A few agglomerations (Fig. 3A) were observed, revealing that the average individual size of the NPs was ∼10 nm. The sizes of the individual NPs ranged from 10 to 40 nm (Fig. 3). Figures 3B and 3C present the image of Ag-ZnO BNPs, showing that the individual grain size of Ag-ZnO BNPs is ∼10 nm. The degree of purity of the Ag-ZnO BNPs was confirmed by EDS. The EDS profile of the BNPs showed weak signals for silver atoms and strong signals for zinc atoms. The EDS spectra of the catalysts are shown in Figure 4. Well-defined peaks for Ag, Zn, and O elements are shown for Ag-ZnO. The main components were zinc (67.9%), oxygen (18.7%), and silver (13.4%). The highest peaks for zinc ions were observed between 0.5 and 1 keV using an EDS spectrum. Strong peaks were observed at 3 keV, which are characteristic of AgNPs.

FIG. 3.

STEM micrographs of biosynthesized Ag-ZnO BNPs assisted by aqueous extract of Alchemilla vulgaris. STEM, scanning transmission electron microscopy.

FIG. 4.

EDS spectra of Ag-ZnO BNPS. EDS, energy dispersive spectrometry.

Antibacterial activity of synthesized Ag-ZnO BNPs

The efficacy of BNPs, measured in terms of zones of inhibition (mm), was tested against three different bacteria (S. aureus, E. faecalis, and E. coli) using the disk diffusion method. The antibacterial effect of biosynthesized Ag-ZnO BNPs (200 µg/mL) was also compared with both negative and positive controls. The results showed that the negative control did not exhibit any antibacterial activity. Furthermore, E. coli demonstrated resistance to the Ag-ZnO BNPs, indicating that Ag-ZnO BNPs did not have an antibacterial effect on the gram-negative E. coli. In contrast, the Ag-ZnO BNPs displayed antibacterial activity against the two gram-positive bacteria (Fig. 5). E. faecalis showed an inhibition zone of 11 mm, while S. aureus had an inhibition zone of 10 mm (Table 1).

FIG. 5.

Inhibition zone of Ag-ZnO BNPs against Enterococcus faecalis (A), Staphylococcus aureus (B), and Escherichia coli (C).

Table 1.

Zone of Inhibition of Biosynthesized Ag-ZnO BNPs from Alchemilla vulgaris Selected Human Pathogens

Diameter of zone of inhibition (mm)
	Enterococcus faecalis	Staphylococcus aureus	Escherichia coli
Ag-ZnO BNP (200 µg/mL)	11	10	0
Sterile water	0	0	0
Penicillin/streptomycin	26	24	0

Ag-ZnO BNP, silver-zinc oxide bimetallic nanoparticle.

In a study by Chan et al., evaluating ZnO NPs, copper oxide (CuO) NPs, and ZnO-CuO nanocomposites for antibacterial activity, it was reported that ZnO NPs exhibited significant antibacterial potential (S. aureus = Bacillus subtilis [15.63 μg·mL⁻¹] > E. coli [62.50 μg·mL⁻¹] > Klebsiella pneumoniae [125.00 μg·mL⁻¹]).³⁰ Similarly, in a study by Selvanathan et al., ZnO NPs showed antibacterial activity against two gram-positive bacteria (B. subtilis and S. aureus) and two gram-negative bacteria (K. pneumoniae and Pseudomonas aeruginosa), with inhibition zones ranging between 8 and 15 mm.³¹ Anjum et al. investigated the antibacterial potential of green-synthesized BNPs against various bacterial species using the well diffusion method. They reported that Ag-ZnO BNPs inhibited the growth of B. subtilis, P. aeruginosa, and Pseudomonas fluorescens, forming inhibition zones of 12, 12.5, and 9.5 mm, respectively.¹

The well-known inhibitory properties of Ag and ZnO NPs have been utilized in several therapeutic applications, particularly in the inhibition of gram-positive and gram-negative bacterial strains.³² Previous studies have shown that nano-sized particles induce cytotoxicity and promote cell death, including apoptosis and necrosis, by generating ROS or increasing intracellular oxidative stress. ROS are a critical trigger for DNA alterations and the breakdown of cell membranes.³³ BNPs provide antibacterial activity by disrupting bacterial membranes, inducing oxidative stress, and causing damage to bacterial DNA and proteins.¹ It has also been suggested that zinc kills bacteria by activating electrons, while silver acts as an antibacterial agent, and that the ions of both metals have the potential to function as antibacterial agents. Consequently, BNPs demonstrate enhanced activity even when synthesized using different methods.^32–35

Antioxidant activity of synthesized Ag-ZnO BNPs

Antioxidants are natural or synthetic substances that can prevent or delay damage to cells caused by oxidants. Oxidative stress and other health problems are typically caused by free radicals. One of the most frequently used methods for determining antioxidant capacity is the DPPH assay.^36,37 In addition, since oxidative compounds are continuously produced in cellular metabolism, it is important to counteract these oxidants to maintain the balance of intra- and extracellular homeostasis. In this study, the antioxidant capacities of the synthesized BNPs were evaluated.³⁸ Their capacity to scavenge free radicals was assessed using the free radical compound DPPH. The scavenging ability was measured at various Ag-ZnO BNP concentrations (50, 100, 250, 500, 1000, 2000, 3000, and 4000 µg/mL) by characteristic absorbance at 517 nm. As the concentration increased, antioxidant activity also increased. The maximum antioxidant activity was observed at 4000 µg/mL, with a rate of 46.68%. Ag-ZnO BNPs showed moderate antioxidant activity compared to the positive control, L-ascorbic acid (Fig. 6).

FIG. 6.

DPPH radical scavenging activity of green synthesized Ag-ZnO BNPs. DPPH, 1-diphenyl-1-2-picrylhydrazyl.

Anticancer activities of synthesized Ag-ZnO BNPs

The cytotoxic effects of Ag-ZnO BNPs on L929, HT-29, and Caco-2 cell lines were analyzed using MTT assay. The anticancer activity of BNPs was determined by measuring the percentage of cell viability in HT-29 and Caco-2 cells. The results clearly demonstrated the anticancer potential of green synthesized Ag-ZnO BNPs compared with untreated cells, which were 100% viable. The cells were treated with 0–50 μg/mL Ag-ZnO BNPs for 24, 48, and 72 hours. The viability of the HT-29 and Caco-2 cell lines decreased in a dose-dependent manner with increasing concentrations of Ag-ZnO BNPs. After 72 hours, Ag-ZnO BNPs exhibited significantly higher cytotoxicity (Fig. 7). Table 2 provides data on the cytotoxicity of BNPs against cancer and normal cells (in vitro). The table indicates that the cytotoxicity of the synthesized NPs is time- and/or dose-dependent. The IC₅₀ values were 14.15 µg/mL for the L929 cell line, 8.43 µg/mL for HT-29, and 6.48 µg/mL for Caco-2. The antiproliferative activity index of Ag-ZnO BNPs was 73.2% for L929 cells, 78.99% for HT-29 cells, and 98.22% for Caco-2 cells. These results indicate that Ag-ZnO BNPs exhibit stronger anticancer effects, especially on Caco-2 colon cancer cells. According to the findings, Ag-ZnO BNPs significantly impacted the HT-29 and Caco-2 cell lines by causing cell damage, consistent with the literature.^39,40

FIG. 7.

MTT assay graph showing the percentage of viable cells using different concentrations of Ag-ZnO BNPs (A) 24 hours, (B) 48 hours, and (C) 72 hours.

Table 2.

Maximum Cell Death and IC₅₀ Values of Ag-ZnO BNPs against L929 (Normal Fibroblast Cell Line), HT29 (Human Colorectal Adenocarcinoma Cell Line), and Caco-2 (Colon Cancer Cell Line)

	L929		HT-29		Caco-2
	MCD (%)	IC₅₀ (μg/mL)	MCD (%)	IC₅₀ (μg/mL)	MCD (%)	IC₅₀ (μg/mL)
	MCD (%)	IC₅₀ (μg/mL)	MCD (%)	IC₅₀ (μg/mL)	MCD (%)	IC₅₀ (μg/mL)
24 hours	35.75	ND	31.75	ND	36	ND
48 hours	46.04	ND	44.43	ND	65	ND
72 hours	73.2	14,15	78.99	8.43	98.22	6.48

ND, not determined.

Yin et al. evaluated the cytotoxic effects of biosynthesized bimetallic Ag@ZnO nanocomposites on HeLa cancer cells and reported an IC₅₀ value of 5 µg/mL. In their study, no significant cytotoxicity was observed on the H8 normal cell line, which is in line with our findings.⁴¹ Narayanan et al. reported that biosynthesized ZnO and Ag/ZnO nanocomposites exhibited time- and dose-dependent cytotoxic effects in HeLa cancer cells and also had high antibacterial and photocatalytic activities. The highest cytotoxicity for ZnO was 42.26 ± 2.23% in HeLa cells, while Ag/ZnO showed 50.5 ± 0.15% at a concentration of 100 μg/mL after 48 hours of exposure. Compared with Ag/ZnO, ZnO was found to be less cytotoxic to HeLa cancer cells.⁴² Monem et al. focused on the synthesis and characterization of AgNPs, Ag@ZnO, and Ag@SiO2 NPs. In their study, the cytotoxic effect of Ag@ZnO on MCF-7 breast cancer cells was examined, and the IC₅₀ value was reported as 430 µg/mL. The study also highlighted that Ag@ZnO exhibits strong antibacterial effects, which increase especially in the presence of light. However, the IC₅₀ values reported were much higher compared with our results.⁴³ Balamurugan et al. reported an IC₅₀ value of 49.1 ± 2.33 µg/mL in HT-29 colon cancer cells for AgNPs synthesized using Elaeocarpus serratus fruit extract.⁴⁴ In another study conducted with AgNPs synthesized using Zingiber officinale and Curcuma longa rhizomes, an anticancer effect on HT-29 cells was observed with an IC₅₀ value of 150.8 µg/mL.⁴⁵ Kang et al. reported IC₅₀ values for ZnO NPs of different sizes synthesized to evaluate size-dependent cytotoxicity in cancer cells, with values of 15.55 ± 1.19, 22.84 ± 1.36, and 18.57 ± 1.27 μg/mL, respectively.⁴⁶ When comparing the IC₅₀ values from these studies with the current results, Ag-ZnO BNPs demonstrate greater efficacy at lower concentrations in colon cancer cells than either AgNPs or ZnO NPs alone.

Apoptosis can be identified by observing structural changes in cells through light microscopy (Fig. 8). Biosynthesized Ag-ZnO BNPs, which can alter the morphology of cancer cells, serve as an early indicator of apoptosis. Cells exposed to Ag-ZnO BNPs for 72 hours displayed typical apoptotic features such as condensed nuclei, membrane blebbing, and apoptotic fragments. These findings are consistent with previous studies.⁴⁷

FIG. 8.

Effect of Ag-ZnO BNPs on L929, HT-29, and Caco-2 cell morphology.

Conclusions

In this study, Ag-ZnO BNPs were successfully synthesized via a green synthesis method, resulting in NPs with sizes between 10 and 40 nm. These NPs demonstrated enhanced antimicrobial activity, particularly against gram-positive bacteria. In addition, they exhibited moderate antioxidant properties and significant cytotoxic effects on HT-29 and Caco-2 cancer cell lines, suggesting promising potential for anticancer applications. Compared with monometallic counterparts, Ag-ZnO BNPs showed superior performance in cytotoxicity. These findings highlight the potential of Ag-ZnO BNPs not only as effective anticancer agents but also as viable alternatives to conventional antibiotics. However, further studies are required to fully explore their mechanisms of action and broader applicability in medical and pharmaceutical fields.

Footnotes

Data Availability Statement

The data that support the findings are included in the article.

Authors’ Contributions

C.A.A.: Conceptualization, formal analysis, investigation, methodology, supervision, and writing—review and editing. B.R.S.: Formal analysis, investigation, methodology, resources, software, visualization, and writing—original draft. S.P.: Formal analysis, investigation, methodology, and writing—review and editing. F.K.: Formal analysis, methodology, software, and visualization.

Author Disclosure Statement

The authors declare no conflict of interest.

Funding Information

No funds, grants, or other support was received.

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