Structural insights and biomedical potential of biosynthesized silver nanoparticles: antibacterial activity, anti-biofilm and cancer cell inhibition

Structural analysis

In this study, X-ray diffraction (XRD) was employed to assess the structural properties of the synthesized AgNPs. As a non-destructive technique, XRD provides critical insights into both the crystallite size and the overall nanoparticle structure. X-ray diffraction analysis was performed using a D8 Advance X-ray diffractometer, equipped with a graphite monochromator and a CuKα radiation source (λ = 1.5418 Å), operating at 45 kV and 40 mA. Crystallite size was determined using the Debye-Scherrer equation D = kλ/ βcos θ, ensuring precise evaluation of nanoparticle dimensions. In this equation: D represents the crystallite size; λ is the wavelength of the X-ray source; β is the full width at half maximum (FWHM) of the diffraction peak (in radians); k the Scherrer constant, typically ranges from 0.9 to 1; and θ is the Bragg angle (in radians) (Singh et al., 2018).

Optical properties

The optical properties of the synthesized AgNPs were assessed using UV-Vis spectroscopy (Shimadzu-1800, Kyoto, Japan). The absorption of radiation occurs in both the visible range (400–800 nm) and the ultraviolet range (200–400 nm). It is widely recognized that silver nanoparticles exhibit a yellowish-brown color. Further confirmation of this observation was obtained using a UV-Vis spectrophotometer. The UV spectra show absorption band which correspond to the surface plasmon absorption of the AgNPs. This absorption band is also a result of the oscillation of free conduction electrons within the AgNPs, triggered by the absorption of electromagnetic radiation. Furthermore, this technique was employed to determine the optical band gap energy (Eg) of the biosynthesized silver nanoparticles, providing valuable insights into their electronic structure and potential applications.

Infrared spectroscopy

Fourier Transform Infrared Spectroscopy (FTIR) is a powerful analytical technique that identifies functional groups in a sample by analyzing its interaction with infrared radiation. In the synthesis of AgNPs, FTIR offers several benefits such the identification of the molecules that contribute to the reduction and stabilization of the nanoparticles, the determination of the functional groups that facilitate the binding of biomolecules to the nanoparticles, and the assessment of the chemical stability of the nanoparticles over time or under varying environmental conditions. When analyzing FTIR spectra to identify functional groups, it is crucial to consider several key factors. Examining spectral regions can aid in identifying the functional groups within a molecule. Additionally, the intensity of the absorption peaks may differ based on the concentration of the functional group and the strength of the interactions between molecules. Moreover, the shifting of absorption peaks can occur due to solvation effects, molecular interactions, or alterations in molecular structure.

In this study, FTIR spectroscopy (Shimadzu IR, Prestige 21, Nakagyo-Ku, Japan) was performed to characterize the functional groups of synthesized AgNPs (Almayouf et al., 2024). Additionally, FTIR was used to monitor conformational changes and chemical group interactions during vancomycin loading onto the surface of AgNPs, within the wavelength range of 4,000–400 cm⁻¹ (Jadoun et al., 2021).

Particle size analysis

DLS is a commonly employed technique for analyzing the size distribution, polydispersity index (PDI), and zeta potential of nanoparticles in suspension. It assesses the Brownian motion of particles within a liquid medium and correlates it with their hydrodynamic diameter using the Stokes-Einstein equation. The PDI serves as a measure of the uniformity of particle size distribution. In this study, the DLS analysis was conducted using a Zetasizer (HT Laser, ZEN3600, Malvern Nano series) from Malvern Instruments (Worcestershire, UK), to determine the hydrodynamic sizes (Almayouf et al., 2024) and correlation coefficients of the synthesized AgNPs. The measurements were performed at 25 °C using water as the dispersion medium. The analysis was carried out under the following conditions: refractive index (RI) of 1.33, dispersion viscosity of 0.887 cP, and dielectric constant of 78.5. These parameters ensured accurate characterization of nanoparticle size distribution and stability in the aqueous medium.

Analysis of XRD

The XRD analysis of the biosynthesized AgNPs revealed four prominent peaks, corresponding to the (111), (200), (220), and (311) crystal planes (Fig. 1). Utilizing the PANalytical X’Pert High Score Plus Software, these peaks verified the face-centered cubic (fcc) crystal structure of the AgNPs, as indicated by JCPDS file No. 03-065-2871 for Tp-AgNPs and Pg-AgNPs, and JCPDS file No. 01-087-0718 for Tm-AgNPs. The experimental diffraction angles closely aligned with the standard values, further validating the crystal structure. The stability of the AgNPs could be attributed to the presence of multiple reducing agents in the solution, which also influenced their crystalline characteristics-a phenomenon widely reported in biosynthesized NPs (Khane et al., 2022). Additionally, unidentified peaks in the diffraction pattern may indicate the presence of bioorganic compounds on the AgNPs surfaces. The size of the AgNPs plays a significant role in shaping the XRD peaks. The broader diffraction profiles, compared to bulk silver, suggested the formation of larger AgNPs. These observations highlighted the impact of biosynthesis conditions on the structural properties of AgNPs.

The interplanar d-Spacing and the lattice parameters for Tp-AgNPs, Tm-AgNPs, and Pg-AgNPs, which are tabulated in Table 1, were derived based on observed diffraction patterns. The estimated values were found to be in close agreement with the standard values of d = 2.35911 Å and a = 4.0861 Å for the crystal plane (111), which have been commonly reported in previous studies, specifically JCPDS file No. 03-065-2871 for Tp-AgNPs and Pg-AgNPs, as well as JCPDS file No. 01-087-0718 for Tm-AgNPs (with d = 2.35403 Å and a = 4.0773 Å). The size of the AgNPs’ crystallites was also calculated using Scherer’s equation D = K λ/β cosθ 18, where D represent the crystallites size (nm), K = 0.9 the Scherrer constant, λ = 0.15406 nm was the wavelength of the X-ray sources, β represents the full width at half maximum (FWHM in radians) of the main peak, and θ was the peak position in radians (Table 1).

DLS analysis

DLS is a highly versatile technique for measuring and analyzing NP size. The method is based on detecting fluctuations in light scattered by dispersed NPs, which result from their random motion, known as Brownian motion. These fluctuations are directly influenced by the hydrodynamic diameters of the NPs, providing insights into their size distribution. A critical parameter in DLS analysis is the polydispersity index (PDI), a dimensionless value that evaluates the uniformity of particle size distribution and the extent of NP aggregation. The PDI ranges from 0 to 1, with values offering important clues about the sample; a PDI ≤ 0.5 indicates a primarily monodisperse and homogeneous particle distribution while a PDI > 0.7 suggests a highly polydisperse system, characterized by a wide range of particle sizes. This distinction makes PDI a key factor in assessing the quality and stability of NPs in a given dispersion (Danaei et al., 2018). The Zeta size as a function of the intensity (%) for Tp-AgNPs, Tm-AgNPs, and Pg-AgNPs were shown in Figs. 2A, 2B and 2C, respectively. The particle size, the PDI, and the intercept value were determined for all samples (Table 2). The results indicated that both Tp-AgNPs, Tm-AgNPs exhibited monodisperse distributions and demonstrated long-term stability. In contrast, the Pg-AgNPs showed a wide range of particle sizes, suggesting that these NPs were semi-uniform in nature. This variation in size distribution highlighted the differences in the synthesis processes and the resulting stability of the NPs.

The XRD patterns indicated that the size of all three AgNPs was smaller than their hydrodynamic diameters. This discrepancy could be attributed to the fact that XRD analysis measures the crystallite size, which reflects the dimensions of the crystalline regions within the NPs. In contrast, the hydrodynamic diameter, as determined by techniques like DLS, accounted for the overall size of the particle, including both the nanoparticle core and anysurrounding solvation shell or macromolecular components. Therefore, the hydrodynamic diameter typically appeared larger than the crystallite size observed in XRD analysis (Stetefeld, McKenna & Patel, 2016).

As previously stated, DLS analysis evaluates the strength of scattered light by particles present in a liquid or suspension to establish their size distribution. The correlation coefficient, also known as the autocorrelation function, plays a crucial role in DLS analysis. It offers insights into the movement of particles over a period, enabling the estimation of their size. The behavior of the autocorrelation function reflects the variations in scattered light intensity over time. In the case of monodisperse systems, where all particles are of the same size, the autocorrelation function typically demonstrates a gradual decrease following an exponential pattern. However, for polydisperse systems with particles of varying sizes, the decay of the autocorrelation function becomes more intricate and can be represented by a combination of exponential terms. The rate of decay in the autocorrelation function is directly linked to the diffusion coefficient of the particles. Smaller particles exhibit faster diffusion, resulting in a more rapid decline in correlation. Conversely, larger particles diffuse at a slower pace, resulting in a more gradual decay of the correlation.

According to the data presented in Figs. 3A–3C, the correlation coefficient peaks at t = 0, indicating a strong initial correlation between the scattered light intensity and its subsequent values. As time progressed, the correlation coefficient decreased, with the rate of decay influenced by the particle size. For Tp-AgNPs and Tm-AgNPs (Figs. 3A and 3B, respectively), the autocorrelation function showed a rapid decay, suggesting the presence of smaller particles with higher diffusion coefficients. In contrast, the slower decay observed in the autocorrelation function of Pg-AgNPs (Fig. 3C) pointed to larger particles with lower diffusion coefficients. These observations were consistent with the earlier results from the Zeta size measurements, which also indicated intensity variations corresponding to particle size.

Analysis of UV-vis spectra

Figure 4 illustrated the UV–vis spectra for the extracts of Tm-AgNPs, Tp-AgNPs, and Pg-AgNPs. The UV spectra showed absorption bands at 390 nm, 402 nm, and 404 nm for Tp-AgNPs, Tm-AgNPs, and Pg-AgNPs, respectiveley, which were indicative of the surface plasmon resonance of the AgNPs. These absorption bands arised from the collective oscillations of the free electrons at the surface of the NPs. The biosynthesis process was monitored visually by noting the color change, which signified the reduction of AgNO₃ and the subsequent formation of AgNPs. The results demonstrated that the synthesized samples produced AgNPs with unique physiochemical properties, as reflected in their distinct absorption spectra. Furthermore, the absorption peaks of Tp-AgNPs and Tm-AgNPs exhibited significantly higher intensities compared to Pg-AgNPs, suggesting a tendency for monodispersity. This observation was in excellent agreement with the DLS analysis, which further corroborated the uniform size distribution of these NPs. The optical band gaps (Eg) of Tp-AgNPs, Tm-AgNPs, and Pg-AgNPs were calculated using the equation Eg = 1,240/λ_max (Awad et al., 2021), yielding values of 3.18 eV, 3.08 eV, and 3.07 eV, respectively. Notably, these Eg values were within a suitable range for optical applications, suggesting that these biosynthesized AgNPs had potential for use in optoelectronic devices.

Analysis of FTIR spectra

By employing FTIR spectroscopy to analyze the AgNPs, we confirmed the presence of specific functional groups and substantiated the plant extract’s role as both a reducing and capping agent. Variations in peak intensity or shifts in wavenumber suggested the involvement of these functional groups in the binding processes.

The FTIR spectrum of Tm-AgNPs (Fig. 5) displayed absorption bands at the following wavelengths: 3,414.23, 3,238.15, 3,037.73, 2,929.02, 2,859.84, 2,374.74, 2,342.07, 2,121.96, 1,774.47, 1,740.49, 1,704.08, 1,620.12, 1,543.83, 1,460.00, 1,342.22, 1,319.10, 1,112.19, 877.38, 831.74, 624.91, 479.07, and 423.81 cm⁻¹, while the FTIR spectrum of Tm-AgNPs showed absorption bands at 3,470.14, 3,414.35, 3,238.15, 2,929.61, 2,858.92, 2,373.52, 2,103.66, 1,775.19, 1,738.81, 1,703.45, 1,619.14, 1,544.06, 1,460.34, 1,117.43, 825.17, 617.74, 483.75, and 423.80 cm⁻¹. Figure 5 also depicted the FTIR spectrum of Pg-AgNPs which revealed absorption bands at 3,803.32, 3,749.73, 3,673.94, 3,417.47, 3,238.15, 2,929.41, 2,856.13, 2,365.27, 2,339.11, 2,027.64, 1,739.10, 1,636.05, 1,620.97, 1,543.78, 1,460.42, 1,112.59, 803.75, 622.52, 487.05, and 425.16 cm⁻¹. There was a slight difference in the binding sites of the synthesized AgNPs, as indicated by their IR spectra. Shifts in absorption peaks within an IR spectrum could result from various factors, including solvation effects, which suggest that interactions between the molecule and solvent may lead to these shifts. This can occur due to polarization effects or hydrogen bonding. Additionally, molecular interactions—such as hydrogen bonds, van der Waals forces, or π − π interactions—can also contribute to shifts in absorption peaks (Almayouf et al., 2024).

The bands detected in the IR spectra at approximately 3,238, 3,400, 2,929, 2,860, 1,620, 1,460, and 1,112 cm⁻¹ suggested the presence of various functional groups. The peaks at around 3,238 and 3,414 cm⁻¹ were likely associated with the stretching vibrations of the hydroxyl group (-O–H), a hallmark of alcohols. The bands at 2,929 and 2,860 cm⁻¹ might correspond to the stretching of the N–H bond typical of amines, as well as indicating the -CH stretching associated with alkyl groups. Furthermore, the band near 1,620 cm⁻¹ could represent the C=O bond found in carboxylic acids or esters, while the peak at 1,460 cm⁻¹ was likely indicative of the amide I bond (N–C=O) present in proteins, esters, or ethers. Lastly, the absorption band at 1,112 cm⁻¹ might highlight the C–N bond found in aliphatic amines or in alcohols and phenols. Additionally, the emergence of new peaks at around 877, 830, and 803 cm⁻¹ suggested the potential attachment of CH groups to AgNPs. These interpretations aligned with findings from previous research (Manikandan et al., 2017).

Antibacterial effect

Agar well diffusion assay.

The inhibition zone diameters demonstrated varying antibacterial effects of the different AgNPs (Tp-AgNPs, Tm-AgNPs, and Pg-AgNPs) against both Gram-positive and Gram-negative bacteria (Table 3). These effects were compared to chloramphenicol, which produced larger inhibition zones ranging from 30 to 35 mm across the bacterial strains. For the Gram-positive strain S. epidermidis, Tp-AgNPs and Tm-AgNPs demonstrated significant antibacterial activity, producing inhibition zones of 20 mm and 19.5 mm, respectively, though these were smaller than those observed for chloramphenicol (Fig. 6). In contrast, Pg-AgNPs exhibited no visible antibacterial effect when evaluated using the agar well diffusion method (Table 3). S. aureus exhibited moderate susceptibility, with inhibition zones of 11.5 mm and 12.5 mm for Tp-AgNPs and Tm-AgNPs, respectively, while Pg-AgNPs showed no effect. Among Gram-negative strains, E. coli and S. marcescens displayed mild inhibition zones of 14 mm and 10 mm with Tp-AgNPs and Tm-AgNPs, respectively, and a 10 mm inhibition zone for Pg-AgNPs. For P. aeruginosa, Tp-AgNPs produced a notable inhibition zone of 19 mm, which was significantly reduced for Tm-AgNPs (10 mm) and Pg-AgNPs (15 mm) (Fig. 6). In contrast, Tm-AgNPs showed only a 10 mm zone, and Pg-AgNPs exhibited no inhibition (Table 3).

Table 3:

Inhibition zone diameters, minimum inhibitory concentration MIC and minimum bactericidal concentration MCB values of AgNPs (Tp-AgNPs, Tm-AgNPs, and Pg-AgNPs) against Gram-positive and Gram-negative bacteria.

Strains	Gram	Tp-AgNPs	Tm-AgNPs	Pg-AgNPs	Chloramphenicol
Inhibition zone diameter (mm)
Staphylococcus epidermidis	+	20 ± 0	19.5 ± 0.7	0	30 ± 0
Staphylococcus aureus	+	11.5 ± 0.7	12.5 ± 0.7	0	32 ± 0
Escherichia coli	–	14 ± 0	10 ± 0	10	35 ± 0
Pseudomonas aeruginosa	–	19 ± 0	10 ± 0	15	30 ± 0
Serratia marcescens	–	14.5 ± 0.7	10 ± 0	0	35 ± 0
MIC (mg/mL)
Strains	Gram	Tp-AgNPs	Tm-AgNPs	Pg-AgNPs
Staphylococcus epidermidis	+	0.15	5	5
Staphylococcus aureus	+	0.15	5	5
Escherichia coli	–	0.31	5	1.25
Pseudomonas aeruginosa	–	0.039	1.25	2.5
Serratia marcescens	–	0.31	5	0
MBC (mg/mL)
Staphylococcus epidermidis	+	0.31	5	5
Staphylococcus aureus	+	2.5	5	5
Escherichia coli	–	0.62	5	1.25
Pseudomonas aeruginosa	–	0.62	1.25	2.5
Serratia marcescens	–	0.62	5	5

DOI: 10.7717/peerj.19608/table-3

These findings indicated that Tp-AgNPs were generally more effective across various bacterial strains, although their antibacterial activity still fell short of the broad-spectrum inhibition exhibited by chloramphenicol. Gram-negative bacteria were more sensitive to AgNPs than Gram-positive bacteria, likely due to their thinner cell membranes, which allow easier nanoparticle diffusion. In contrast, Gram-positive bacteria possess a thicker peptidoglycan layer that impedes nanoparticle entry. The enhanced permeability in Gram-negative bacteria enables AgNPs to reach the cytoplasm, where they interact with sulfur-containing enzymes and proteins, disrupt cellular functions, and interfere with DNA replication (Durán et al., 2016). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for Tp-AgNPs, Tm-AgNPs, and Pg-AgNPs against various bacterial strains revealed distinct antibacterial efficacies (Table 3). Tp-AgNPs exhibited the highest potency, particularly against P. aeruginosa (MIC: 0.039 mg/mL, MBC: 0.62 mg/mL) and S. epidermidis (MIC: 0.15 mg/mL, MBC: 0.31 mg/mL), indicating strong bactericidal activity. For S. aureus, Tp-AgNPs showed low MIC values (0.15 mg/mL) but required higher MBC (2.5 mg/mL) to achieve bactericidal effects. Among Gram-negative bacteria, E. coli demonstrated moderate sensitivity to Tp-AgNPs (MIC: 0.31 mg/mL, MBC: 0.62 mg/mL), while S. marcescens required slightly higher concentrations (MIC: 0.31 mg/mL, MBC: 0.62 mg/mL). Notably, Tm-AgNPs and Pg-AgNPs exhibited reduced efficacy across most strains, requiring higher concentrations to achieve similar inhibitory and bactericidal effects. These results underscored the variability in antibacterial activity depending on the type of nanoparticle and bacterial strain, with Tp-AgNPs emerging as the most effective overall.

AgNPs exert their antibacterial effects through multiple proposed mechanisms. One mechanism involves the attachment of AgNPs to the bacterial cell membrane, leading to structural and functional disruption. This interaction is followed by the invagination of the cell and the release of silver ions, which possess potent antibacterial activity, particularly effective against Gram-negative bacteria due to their thinner cell walls (Singhal et al., 2021). Another pathway suggests that AgNPs anchor to the bacterial cell wall, penetrate the cell, and disrupt internal processes, ultimately leading to cell death. Furthermore, AgNPs can generate free radicals, which exacerbate their bactericidal effects by damaging cellular components such as proteins, lipids, and DNA, and inducing oxidative stress. These combined actions underline the multifaceted antimicrobial potential of AgNPs, making them effective against a broad spectrum of bacterial pathogens (Singhal et al., 2021).

Several studies highlight the synthesis of AgNPs using P. granatum peel extract, demonstrating significant antimicrobial efficacy against both Gram-positive and Gram-negative bacteria. AgNPs derived from the aqueous peel extract exhibited notable inhibition zone diameters, with concentrations of 100 µg/mL effectively inhibiting the growth of E. coli and P. aeruginosa. Remarkably, even at lower concentrations of 25 and 50 µg/mL, the AgNPs inhibited Klebsiella pneumoniae, producing inhibition zones of nine mm and 14 mm, respectively. Similarly, against S. aureus, inhibition zones of six mm and 14 mm were observed at these concentrations, underlining the potent antimicrobial activity of these AgNPs (Devanesan et al., 2018). Moreover, AgNPs synthesized using P. granatum extract demonstrated higher susceptibility in Gram-negative bacteria compared to Gram-positive ones, with MIC values ranging from 16 to 512 µg/mL (Aygün et al., 2022). In a comparative study, the antibacterial activity of silver nanoparticles synthesized with T. polium extract and a 96% ethanol extract was tested against E. coli K-12 and S. aureus. The results revealed that the plant extract significantly enhanced the antibacterial efficacy of the silver nanoparticles, with a 4.2-fold increase in activity against Escherichia coli and a 6.6-fold increase against S. aureus (Kazaryan, Rshtuni & Hovhannisyan, 2021). These findings emphasized the enhanced antimicrobial potential of plant extract-mediated AgNPs and their synergistic effects when combined with bioactive plant compounds.

Anti-biofilm activity.

A biofilm is a structured community of bacterial cells that adhere to a surface and are embedded within a self-produced matrix of EPS. This matrix consists of exopolysaccharides, particulates, and microbial lysates. Biofilm formation provides bacteria with enhanced resistance to environmental stresses, such as fluctuations in nutrients and oxygen levels, pH changes, and antibiotic treatments (Shahwany, Tawfeeq & Hamed, 2016). This protective mechanism significantly contributes to bacterial survival in extreme environments, both within and outside the host, and is a major factor in the persistence and chronic nature of certain infections (Singhal et al., 2021).

In this study, P. aeruginosa and S. aureus strains were exposed to biosynthesized AgNPs, and their ability to form biofilms was evaluated. The results, illustrated in Fig. 7, demonstrate the anti-biofilm activity of three types of nanoparticles: Pg-AgNPs, Tp-AgNPs, and Tm-AgNPs. The effects were assessed separately for S. aureus (Fig. 7A) and P. aeruginosa (Fig. 7B), highlighting their potential in disrupting biofilm formation and reducing bacterial resilience in challenging environments. Compared to chloramphenicol, which exhibited a 71 ± 2.5% biofilm inhibition at 2.5 mg/mL, Tp-AgNPs demonstrated higher inhibition against S. aureus, achieving over 92 ± 0.54% inhibition at concentrations as low as 1.25 mg/mL and maintaining significant effects even at reduced doses (73.64 ± 6.09% at 0.156 mg/mL) (Fig. 7A). Tm-AgNPs showed moderate inhibitory effects, peaking at 81.56 ± 2.83% at five mg/mL, with minimal activity observed at lower concentrations. In contrast, Pg-AgNPs displayed the least inhibitory activity against S. aureus, achieving notable inhibition only at five mg/mL (71.06 ± 0.023%) (Fig. 7A). For P. aeruginosa, Tp-AgNPs again exhibited the highest biofilm inhibition, reaching over 93 ± 3.86% at 0.156 mg/mL and nearly complete inhibition (99.38 ± 1.97%) at five mg/mL, surpassing chloramphenicol (82.2 ± 6.6%) (Fig. 7B). Tm-AgNPs displayed moderate inhibition, peaking at 95.2 ± 7.51% at five mg/mL but with lower efficacy at minimal concentrations. Pg-AgNPs were the least effective, showing substantial inhibition only at higher concentrations, with a peak of 82.4 ± 4.36% at five mg/mL (Fig. 7B). Overall, Tp-AgNPs demonstrated the most potent anti-biofilm activity against both bacterial strains, followed by Tm-AgNPs, while Pg-AgNPs were the least effective, especially at lower doses.

Limited studies have explored the anti-biofilm potential of AgNPs compared to their antimicrobial effects. Singhal et al. (2021) reported that AgNPs biosynthesized using P. granatum leaf extract exhibited greater inhibition of biofilm formation in P. aeruginosa compared to S. aureus at higher concentrations (100 µg/mL) (Singhal et al., 2021). Bacterial biofilm formation is initiated by the production and secretion of exopolysaccharides, which are crucial for biofilm development. Therefore, inhibiting exopolysaccharides synthesis serves as an effective strategy to restrict biofilm formation, forming the basis for evaluating the anti-biofilm activity of AgNPs (Mohanta et al., 2020).

Anti-tumoral effect

Breast carcinoma remains a major health concern with rising incidence rates, and current treatments are limited to conventional radiotherapy and chemotherapy. These therapies often lack selectivity, affecting both cancerous and healthy cells, which underscores the need for innovative treatment options (Jang et al., 2016). NPs, with their unique properties, offer a novel approach to tumor detection, prevention, and treatment by targeting cancer cells through blood vessels, penetrating tumor regions, and reaching the target cells (Devanesan et al., 2018; Jannathul Firdhouse & Lalitha, 2015).

The current data demonstrated the dose-dependent cytotoxic effects of Tp-AgNPs, Pg-AgNPs, and Tm-AgNPs on MCF-7 breast cancer cell lines (Fig. 8). At the lowest tested concentration of 62.5 µg/mL, cell viability remained relatively high for Pg-AgNPs and Tm-AgNPs, with values of 81.94 ± 4.34% and 70.25 ± 5.34%, respectively. In contrast, Tp-AgNPs exhibited significantly higher cytotoxicity, reducing cell viability to 52.96 ± 4.05%. As the concentration increased, all three NPs showed a dose-dependent reduction in cell viability. At 250 µg/mL, Tp-AgNPs maintained the most potent cytotoxicity, reducing viability to 30.34 ± 1.73%, while Pg-AgNPs and Tm-AgNPs displayed slightly higher viabilities of 67.03 ± 2.2% and 65.50 ± 4.33%, respectively. At the highest concentration of 1,000 µg/mL, all NPs demonstrated substantial cytotoxic effects, with Tm-AgNPs achieving the lowest cell viability at 23.79 ± 4.84%. Pg-AgNPs and Tp-AgNPs showed comparable effects at this concentration, with viabilities of 24.82 ± 0.58% and 30.50 ± 0.45%, respectively.

These results indicated that while all three AgNPs exhibit cytotoxic effects against MCF-7 cells, Tp-AgNPs are more effective at lower concentrations, with an IC₅₀ value of 45.7 ± 2.93 µg/mL. Conversely, Tm-AgNPs demonstrated enhanced cytotoxicity at higher concentrations. The observed dose-dependent cytotoxicity underscored the potential of these AgNPs as anticancer agents and highlighted the need for further investigation into their mechanisms of action. These findings reinforce the promising role of AgNPs as dose-responsive therapeutic agents against MCF-7 cell lines.

According to previous studies, the anti-tumor effects of silver nanoparticles are linked to several mechanisms, including damage to nucleic acids and organelles, induction of oxidative stress, initiation of apoptosis, and disruption of mitochondrial function (Ong et al., 2013; Amini et al., 2021). AgNPs can enter cancer cells through endocytosis and accumulate in the perinuclear area and endo-lysosomal compartments. They can also reach the mitochondria, where they interfere with the respiratory chain, leading to the production of ROS. These ROS can damage proteins, lipids, and nucleic acids, as well as compromise cell membranes, ultimately activating apoptotic pathways and resulting in cell death (Yeşilot & Aydın Acar, 2019). Current findings align with several studies investigating AgNPs synthesized using similar plant extracts. For instance, AgNPs derived from the aqueous leaf extract of P. granatum effectively inhibited HeLa cell proliferation, with an IC50 value of 100 µg/mL (Sarkar & Kotteeswaran, 2018). Similarly, P. granatum peel extract AgNPs demonstrated significant cytotoxicity against the RKO colon cancer cell line, reducing cell viability to 61% at concentrations above 12.5 µg/mL (Devanesan et al., 2018). Furthermore, AgNPs synthesized using T. polium leaf extract exhibited potent anticancer effects against the MNK45 human gastric cancer cell line, achieving an IC₅₀ value of 68.2 µg/mL (Hashemi, Tasharrofi & Saber, 2020). Amini et al. (2021), 30 also reported that T. polium-based AgNPs significantly reduced NALM-6 leukemia cell viability at concentrations ≥ 50 µg/mL by inducing apoptosis, while sparing normal peripheral blood mononuclear cells.

Moreover, a notable property of NPs is that their biological impact varies considerably based on their size, which, in turn, plays a critical role in determining their efficiency of cellular uptake, from the intracellular membrane to the nuclear membrane (Kim et al., 2012). It has been documented that the cytotoxic effects of various metallic nanoparticles, such as gold and silver NPs, on human cancer cell lines are size-dependent (Kim et al., 2012; Yuan et al., 2010). Therefore, the cytotoxic effects observed for the three AgNPs against MCF-7 cells could be attributed to their relatively larger size in comparison to that of the cells. Larger NPs have a higher bioactive compound-to-NP ratio, which facilitates the reorganization of these compounds on their surface to optimize their loading into cells (Jiang et al., 2008).

All together, these studies corroborate the cytotoxic potential of Ag-NPs derived from plant extracts on cancer cell line, highlighting their selective action and promising role as anticancer agents.