An environmentally safe approach for the facile synthesis of anti-mutagenic fluorescent quantum dots: Property investigation and the development of novel antimicrobial applications

2.1 Material preparation and reagents

The root plant material was collected at the flowering stage from the south of the Marivan area, on the plain of Rikhalan village (35.466408, 46.185039). The botanical identification was carried out by Dr. Zeinab Toluei (Department of Biotechnology, Faculty of Chemistry, University of Kashan, Kashan, I.R. Iran) using the available literature (Mozaffarian, 1996). A voucher specimen (No. M2016.12) has been recorded at the department of cell and molecular biology, university of Kashan, Iran. Silver nitrate (AgNO₃), nutrient broth medium, and nutrient agar medium were purchased from Merck. The OVCAR3 human ovarian cancer cell line was supplied by the Pasteur Institute of Iran (Tehran, Iran). The Iranian research organization for science and technology (IROST) provided standard microbial strains. The clinical strains in this study were isolated from the patient’s urinary tract infection (UTI) and provided by the microbiology laboratory at Shahid Beheshti Hospital, Kashan, Iran. Medical mask absorbable and non-absorbable suture silk were purchased in a local pharmacy. The experimental procedure was used to deionize water. The green synthesis method was used to synthesize AgQDs, where the aqueous fraction extract of the sample was employed to reduce AgNO₃ to AgQDs.

2.2 Sample preparation and extraction procedure

The plant samples were rinsed with natural water twice and dried at laboratory temperature. The dried sample was powdered and stored in a capped plastic container until extraction. The powdered sample was extracted with methanol (500 ml) using a Soxhlet extractor for 6 h. The resulting extract was filtered and evaporated (Heidolph, Rotary Evaporator, model-RE 801, Germany) at 40 °C before being dried in a vacuum oven at 35 °C. The MeOH extract (3 g dissolved in 300 ml water) was subjected to solvent–solvent partition using n-hexane (3 × 150 ml) and ethyl acetate (3 × 150 ml). After solvent–solvent distribution, the remained portion was used as a water fraction in the experiment.

2.3 Total phenol

The total phenolic content of the plant extracts was assessed using the colorimetric Folin-Ciocalteu assay (Velioglu et al., 1998). First, 0.1 ml of Folin Ciocalteu phenol reagent, 3 ml of distilled water, and 0.02 ml of extract (5 mg/ml) were combined. Three minutes later, 0.3 ml of sodium carbonate (2%) was added, and the mixture was measured at 760 nm using UV–Visible spectrophotometry. Similarly, the same procedure was repeated for all the standard gallic acid solutions (100–1000 μg), resulting in a standard curve with the following equation: Absorbance = 0.0009 × gallic acid (μg) + 0.00319

The results were reported in milligrams of gallic acid equivalent (GAE) per gram of extract.

2.4 Total flavonoid content (TFC)

Total flavonoid concentration was measured using a modified aluminum colorimetric procedure with quercetin as a standard (Sulastri et al., 2018). Ethanol was the solvent used to dilute the standard (10 mg) to 2, 4, 6, 8, and 10 μg/ml. Test tubes were filled with 1 ml of each concentration. In a subsequent stage, 1 ml of extract (1 mg/ml), 0.2 ml of potassium acetate (1 M), 3 ml of ethanol, 0.2 aluminum chloride (10%), and 5.6 ml of distilled water were added to each tube and then homogenized. All test tubes were kept at room temperature for 15 min. The absorbance was recorded at 376 nm using UV–Visible spectrophotometry. The flavonoid content of the extract was determined as milligram of quercetin equivalent (QE) per gram of extract.

2.5 Green synthesis of AgQDs

Different parameters, including pH, reaction temperature, the concentration of AgNO₃, and the concentration ratio of extract and AgNO₃ solution, were optimized to affect the synthesis rate of AgQDs. As a result, we conducted 32 trials to examine these factors (Table S01).

Effect of silver nitrate concentration: AgNO₃ solution was applied as a substrate for the reaction and silver precursor. Various concentrations of AgNO₃ (1, 2, 3, and 4 mM) were used to determine its effect on nanoparticle synthesis.

The concentration proportion of the plant extract and the silver nitrate solution: The concentration proportion of the extract and AgNO₃ solution is mutable for the optimum production of AgQDs. In this research, the reaction was performed using varied proportions of the extract and AgNO₃ solution (1:20, 1:50, 1:100, and 1:200).

Effect of temperature: The synthesis of AgQDs was performed at 30, 40, 50, and 60 °C. The water bath is used to maintain the reaction temperature.

Effect of pH: The optimization of pH was accomplished by adding the reaction mixture to various pH values (pH: 5, 7, 9, and 11). The pH was sustained with 0.1 N NaOH and 0.1 N HCl.

The AgQDs were synthesized by the following process:

A water fraction of the extract in a ratio of 1:50 was added to the aqueous solution of AgNO₃ (3 mM/l) at pH 7 and kept in the water bath (60 °C) for 12 h. The colour of solution turned dark brown, then centrifuged three times (Eppendorf, Centrifuge, model-5810R, Germany) at 9,000 rpm for 30 min (25 °C). It was then rinsed with distilled water and ethanol before being placed in the vacuum oven for 24 h.

2.6 Characterization

The UV–Vis analysis (Shimadzu, UV Spectrophotometer, model-UV1800, Japan) was accomplished to determine the absorption maxima by scanning the sample from 360 to 700 nm. X-ray diffraction (Philips/Panalytical, XRD, model X'Pert Pro, Netherlands) with a Cu Kα-radiation (λ = 1.54 Å) in the 2θ range from 10 to 80° was used to investigate the crystal structure of AgQDs. Fourier transform infrared spectroscopy (Nicolet Magna IR-550 FTIR) of the dry powder was recorded in 4000–400 cm⁻¹ using a KBr pellet technique. Dynamic Light Scattering (DLS)-Horiba nanoparticle analyzer SZ-100 with a 532 nm laser was employed to measure particle size distribution. After sonicating the AgQDs for one hour in ethanol, the morphology and elemental fingerprinting of the resulting silver quantum dots were studied using electron microscopes (SEM: MIRA3 Tescan; EDXA: Oxford Link ISIS-300; TEM: Tecnai-12 FEI). The photoluminescence (PL) spectra were investigated by a Japan Hitachi 850 Spectrophotometer. The Nikon Eclipse Ti-s fluorescent microscope was used to record the fluorescence microscopy images, which used green and blue light as excitation sources.

2.7 Antibacterial and antifungal assays

In this study, the standard microorganisms purchased from IROST, including Salmonella paratyphi-A serotype (ATCC 5702), Shigella dysenteriae (PTCC 1188), Staphylococcus epidermidis (ATCC 12228), Klebsiella pneumonia (ATCC 10031), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 10536), Staphylococcus aureus (ATCC 29737), Proteus vulgaris (PTCC 1182) of bacteria, Candida albicans (ATCC 10231) of yeast, Aspergillus niger (ATCC 16404) and Aspergillus brasiliensis (PTCC 5011) of fungi, and 60 clinical strains were isolated from the patient's UTI. Sabouraud dextrose agar (SDA) was used to culture the fungus overnight at 30 °C, while the bacterial strains were cultured overnight at 37 °C in nutrient agar (NA). According to the Clinical and Laboratory Standards Institute-CLSI (2016), the antimicrobial activities of the AgQDs were assessed using the well diffusion method. The bacterial strains were introduced into a nutrient broth medium and incubated at 37 °C for two to four hours until they attained the turbidity of the 0.5 McFarland standard. Then 100 µl of bacterial suspension was cultured on a Muller-Hinton agar plate. Then AgQDs were mixed in deionized water (30 mg/ml), and a standard drug (ampicillin 125 μg/ml) was placed as a positive control. The inoculated plates were incubated at the appropriate temperatures for 24 h. The zone of inhibition (ZI) was evaluated using a Vernier calliper to determine the antibacterial properties of the synthesized silver quantum dots.

2.8 Time-killassay

S. epidermidis and C. albicans were employed for the growth kinetic test because of their inhibition activities with AgQDs. At first, 1 ml each of microbial suspensions modified to 0.5 McFarland (≈1 × 10⁸ CFU/ml) was added to Trypticase soy broth (TSB) medium, including AgQDs at the minimum bactericidal concentration (MBC) level. Also, as a control, the medium containing these microbial suspensions (without AgQDs) was prepared and incubated on a rotary shaker (130 rpm) at 37 °C. The cultures were dispersed onto tryptone soya agar (TSA) agar plates at 0, 30, 90, 210, 270, 375, 435, 565, and 1260 min. The colonies were counted after incubating the plates at 37 °C for 24 h. Time-kill assay was performed three times for each strain, and the number of colony-forming units (CFU) per milliliter was measured. The growth curves with and without AgQDs were drawn according to CFU per milliliter.

2.9 DPPH radical scavenging activity

The antioxidant potential of AgQDs was evaluated using DPPH free radical scavenging (Ebrahimabadi et al., 2010). Concentrations ranging from AgQDs (0.5, 5, 50, 100, 250, 500, 800, and 1000 μg/ml) were prepared, and butylated hydroxytoluene (BHT) was applied as well as a standard for the investigation. Then, 3 ml of methanol solution of DPPH (0.1 mMol/l) was mixed with each concentration using a vortex. The reaction mixture was incubated in the dark for 30 min (room temperature), recording the activity at 517 nm. The activity was determined and reported as a percentage of inhibition based on the following equation: Inhibition (%) = A control–A sample /A control × 100

A sample is the absorbance of the test sample (extract or AgQDs), and A control is the absorbance of the control reaction (which includes all reagents except the test compound). The half-maximal inhibitory concentration IC₅₀ value was computed.

2.10 Cytotoxicity studies

2.11 AgQDs coating on the chosen materials

This research selected eight items (absorbable and non-absorbable suture silk, medical mask fabric, yarn, filter paper, cotton, toothpick, and cotton fabric) to develop the antimicrobial activity. The selected materials were soaked in the silver solution and then subjected to a UV lamp (k = 365 nm, t = 10 min) to induce the synthesized AgQDs on their surface (Pollini et al., 2008).

2.12 Cellular uptake and bioimaging

The AgQDs solution was mixed with cell culture media and OVCAR3 cells and then incubated for 2 h to see if they could be used as fluorescent markers for cellular imaging. Before the cells were fixed on the slide for examination under a fluorescent microscope, the excess AgQDs were eliminated by washing them three times in phosphate-buffered saline (PBS).​ Afterward, live-cell imaging was performed under bright fields, green (480 nm), and blue (360 nm) excitations.

2.13 Statistical analysis

The measurements were conducted in triplicate, and the results were presented as mean values ± SD (standard deviations). Statistical software SPSS for windows (Version 21) was employed for analysis.

3.1 Bioactivity of extract:

The analytical findings for the DPPH radical-scavenging capacity, phenolic, and flavonoid content of the extract are shown in Table 1. The extract illustrated significant antioxidative activity in the DPPH test (IC₅₀ = 56.23 ± 2.85 μg/ml), which was more than the synthetic antioxidant butylated hydroxyl toluene (BHT: IC₅₀ = 18.15 ± 1.33 μg/ml). The total phenolic and flavonoid content was 97.12 ± 3.15 and 45.86 ± 1.56 mg/g, respectively. The results demonstrate that the extract has total phenolic and flavonoid content. It is demonstrated that the antioxidant activity of T. polium extract is attributed to flavonoids and phenolic compounds (Bahramikia and Yazdanparast, 2012). Several phenolic components from T. polium extract have been identified, such as isorhoifolin, cirsilineol, apigenin, cirsiliol, and poliumoside (Venditti, 2017).

3.2 Characterization of silver quantum dots

The colorless AgNO₃ solution slowly turned a brown or deep red after adding the extract. UV–Visible data showed that some designed experiments could synthesize AgQDs and give intense peaks ranging from 410 nm to 460 nm (SPR) (Kashyap et al., 2019). The absorption spectra revealed an intense peak, indicating increased AgQDs production. The intense peak at 419 nm was obtained in pH 7, 1 ml of the extract in 50 ml of AgNO₃ solution (3 mM) at 60 ^◦C. Based on the findings, this condition was identified as the most effective for synthesizing green AgQDs Therefore, we chose these conditions for further analysis (Fig S1-S4). Fig. 1 summarizes the effects of contact time on nanoparticle formation under the same conditions.

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Fig. 1

UV–vis absorption spectra of AgQDs: T. polium extract + AgNO₃ solution at different time intervals (a = 1 min, b = 30 min, c = 180 min), AgNO₃ solution (Ag⁺) (d), T. polium extract (e).

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The Infrared spectrum of the eco-friendly synthesized silver quantum dots was analyzed to identify the functional group using FTIR spectroscopy. In Fig. 2, the peak at 3439 cm⁻¹, 1384 cm⁻¹, and 824 cm⁻¹ display the formation of quantum dots. The strong stretching vibrations of the O–H functional group were identified in the IR peak spectra at 3439. The sharp peak observed at 1324 cm⁻¹ corresponds to nitro N-O bending, and the peak of 824 cm⁻¹ indicates the C–H, C–C phenyl ring substitutes (Mahadevan et al., 2017).

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Fig. 2

FT-IR spectra of biosynthesized AgQDs.

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The XRD pattern (Fig. 3) of silver quantum dots revealed the noticeable peaks at 2θ = 38.11◦, 44.09◦, 64.52◦, 77.37◦, representing the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) Bragg’s reflections of the face-centered cubic structure of silver, respectively. The average crystalline size (D) of the AgQDs was determined using the Debye-Scherrer formula, which is given as D = 0.9λ/βcosθ. Where θ is the Bragg angle (in degrees), β is the angular line width at half maximum intensity (in radians), and λ is the X-ray wavelength (in nm). For maximum intensity at 2 θ ≈ 38.11, β = 0.00824 rad, λ = 1.54˚A, and θ ≈ 19.05, Scherrer's equation was used to estimate the average size of the Ag nanoparticles, which is estimated to be around 17.90 nm (Nazari and Jookar Kashi, 2021).

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Fig. 3

X-ray diffraction patterns of AgQDs.

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The small and uniformly spherical nanoparticles are confirmed by the SEM images (Fig. 4a) of the sample obtained from the colloidal Ag solutions. The SEM images show that larger particles of AgQDs are organized due to the assemblage of nanoparticles, which solvent evaporation might induce during sample preparation. This could have played a key role in the particle size change. An SEM equipped with an EDAX detector was used to evaluate the elemental composition of the sample. The energy dispersive X-ray analysis (EDAX) in Fig. 4b showed a strong signal in 3 keV related to silver and indicated the successful formation of AgQDs. Following the literature, metallic AgQDs show strong surface plasmon resonance (SPR) at 3 keV (Ismail et al., 2018). As shown in Fig. 4(b), the appearance and percentages of C (0.77%), N (2.37%), O (7.48%), and Ag (89.39%) confirmed that the formations of metallic AgQDs are free of impurities. A transmission electron microscope (TEM) was used to investigate the size and shape of the organized nanoparticles. Fig. 4c shows that silver quantum dots exhibited considerable uniformity with a narrow distribution. The TEM image of silver quantum dots indicates spherical, with an average size of about 12 nm under their size distribution histograms (Fig. 4d). A DLS instrument was employed to analyze the size distribution of the AgQDs. The results demonstrated that the particle size for silver quantum dots synthesized using a 3 mM sample was around 4 nm. The DLS method is commonly used to determine particle size distribution in colloidal solutions. The TEM result and the size distribution of AgQDs obtained by DLS are comparable. In 2020, Hashemi et al. synthesized silver nanoparticles using the water extract of T. polium, which was collected from Kerman, Iran. Compared to the obtained AgQDs in our study, the average size of the nanoparticles was greater (range 70–100 nm) (Hashemi et al., 2020). Pugazhenthiran et al. synthesized AgQDs using aqueous extracts of sweet lime peels in another research study. They dissolved 5 ml of extract in 20 ml of AgNO₃ solution (2 mM/l) at 80 ^◦C. In contrast to our report, the synthesized nanoparticles had lower absorption at 415 nm. Moreover, in the current research, AgQDs were synthesized at 60 ^◦C in an affordable and eco-friendly neutral environment (Pugazhenthiran et al., 2021).

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Fig. 4

SEM (a), EDX spectrum (b), TEM (c) images, and the size distribution histogram (d) of AgQDs.

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3.3 PL

Photoluminescence has been observed in metal nanoparticles such as Au and Ag (Khalil et al., 2012; Vinay et al., 2019). Fig. 5a displays the room temperature PL emission spectrum of AgQDs in the wide range of 320–450 nm. The excitation peaks were 210 to 310 nm (ten by ten). The strong band emission from 340 to 430 nm was repeatably recorded. This range belongs to the violet emission region. The emission band was reported at 487 nm upon excitation at 432 nm for synthesized AgQDs using Tamarind fruit extract (Jayaprakash et al., 2017). Fig. 5 shows the fluorescence microscopic images of OVCAR3 cells treated with AgQDs for 4 h under a bright field (Fig. 5. b1) and different excitation wavelengths of 480 nm (Fig. 5. b2) and 360 nm (Fig. 5. b3). Microscopic images of AgQDs-labeled OVCAR3 cells as a fluorescent probe demonstrated prominent green and blue emissions. The penetration of the AgQDs into the cells with their fluorescent properties in the cellular environment was demonstrated by green and blue-colored luminescence in the nuclei. In contrast, the luminescence in the cytoplasm was extremely feeble. The AgQDs obtained solution was strongly fluorescent under UV-A (365 nm), whereas the AgNO₃ solution and empty vial as control had no fluorescent light (Fig. 5c).

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Fig. 5

(a) PL emission spectra of the AgQDs obtained by excitation wavelengths from 210 to 310 nm. (b) b1 (A bright-field microphotograph of the OVCAR3 cells), b2 (A fluorescence microphotograph of OVCAR3 cells labeled with the AgQDs (λex: 480 nm)), b3 A fluorescence microphotograph of OVCAR3 cells labeled with the AgQDs (λex: 360 nm)). (c) Samples under UV-A (365 nm) light: c1 (AgNO₃ solution), c2 (AgQDs solution), c3 (empty vial).

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4.1 Antimicrobial activity

The minimum inhibitory concentration (MIC) values were measured for samples and antibiotics with notable inhibiting effects on the growth of bacteria. The biosynthesized AgQDs were prepared with dilutions ranging from 31.25 to 500 μg/ml. The MIC results of the samples and antibiotics for standard and clinical strains are shown in tables 2 and 3, respectively. These results showed that S. epidermidis and C.albicans were more sensitive than gram-negative bacteria. K. pneumonia, S. dysenteriae, and S. paratyphi-Aserotype have shown more resistance to AgQDs. The antimicrobial activity of AgQDs against E. coli was reported by Ibrahim. Their results displayed that the zone of inhibition was 17 mm, which is better than the synthesized AgQDs in our investigation (Ibrahim, 2015).

The results showed that clinical strains were more resistant than standard strains. Further, AgQDs indicated high antimicrobial activity compared with AgNO₃. The inhibition zone diameter of AgQDs and AgNO₃ was (10–35 mm) and (8–14 mm), respectively. AgQDs have a higher surface-to-volume ratio than silver nitrate. At the nanoscale, the optical, electrical, and catalytic properties change. Due to their unique properties, these materials manufacture products for targeted drug delivery, diagnosis, imaging, and antimicrobial agents. Therefore, the characteristics of silver nanoparticles have made them suitable for use in medical and health products and the treatment or prevention of infections (Bruna et al., 2021). Moreover, AgQDs have deeper contact with microorganisms because of their vast surface area, and AgQDs are a source of Ag +. For these reasons, AgQDs might cause high antimicrobial activity.

Mahadevan et al. synthesized silver nanoparticles from Atalantia monophylla (L) Correa leaf extract, which illustrated that the nanoparticles had antimicrobial activity against S. aureus (37 mm), B. cereus (36 mm), and C. albicans (34 mm) (Mahadevan et al., 2017). Furthermore, in research by Bapat et al., the antimicrobial activity of green silver nanoparticles was evaluated using the disc diffusion method. Based on the findings, the nanoparticles displayed feeble antibacterial activity against the chosen bacterial strains (Bapat et al., 2022). In another research, the antibacterial activity of biosynthesized AgNPs displayed an inhibition zone of 14.93 ± 0.57 mm against S. aureus (Das et al., 2019). Moreover, green synthesized AgNPs using Cicer arietinum showed moderate antibacterial activity against pathogenic Gram-negative (E. coli (14 ± 0.5 mm), S. aureus (13 ± 1 mm)) and Gram-positive (P. aeruginosa (13 ± 0.5 mm), B. cereus (10 ± 0.5 mm)) (Mouriya et al., 2023). Kordzangeneh and Jookar Kashi evaluated the antibacterial effect of Ag/AgCl composite against bacterial strain. Their results showed that the composite had antibacterial and antibiofilm activities (Kordzangeneh and Jookar Kashi, 2022). The results of other study revealed that P. aeruginosa (14.18 ± 0.02 mm) was the most sensitive to AgNPs, rather than S. aureus (12.18 ± 0.02 mm) and E. coli (12.16 ± 0.03 mm) (Singh et al., 2023). The results showed that silver nanoparticles synthesized using different fruits have antimicrobial effects on S.aureus, B.cereus, E.coli, and C. krusei (Wasilewska et al., 2023). Rakhshan et al. reported that green synthesized nanoparticles have antimicrobial activity against B.subtilis (Rakhshan et al., 2022). Antimicrobial effects are probably due to the attachment of AgQDs to bacterial cell walls, membranes, and proteins and the change in DNA conformations, leading to the death of the bacterial cell. Moreover, we proposed that AgQDs produce free radicals, which are also considered an important factor in the mechanism by which the cell dies. Scheme 1. demonstrates our suggested mechanisms for the antibacterial activity of AgQDs.

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Scheme 1

The proposed mechanisms for the antibacterial activity of AgQDs.

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The colony counter assay has been used to estimate the density of microorganisms in liquid culture by counting individual colonies on an agar plate. In our study, a colony counting assay was performed to determine the number of viable bacteria cells in a plate after being treated with the AgQDs. The minimum bactericidal concentration (MBC) of nanoparticulate for S. epidermidis and C. albicans was the same (62.5 μg/ml). Using time–kill assays, populations of bacteria organisms tested in the presence of 62.5 μg/ml AgQDs were increased from 0 to 400 min (Fig. 6). In the presence of AgQDs (62.5 μg/ml), S. epidermidis populations were decreased to zero in 565 min. Populations of S. epidermidis and C. albicans were reduced to % 27.27 and % 21.9, respectively, from 375 to 435 min.

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Fig. 6

Growth curves of S. epidermidis and C. albicans in the absence and presence of AgQDs at concentration of MBC (62.5 μg/ml).

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4.2 Brine shrimp

The number of brine shrimp larvae that survived after 24 h was counted, and the LC₅₀ value was calculated. The brine shrimp lethality assay (LC₅₀) for AgQDs and the positive control (vincristine sulfate) was 6.21 μg/ml and 0.751 μg/ml, respectively. The green synthesis AgQDs of T. Polium L. showed considerable brine shrimp larvicidal activity. This research is the first report on brine shrimp lethality activity of fluorescent silver quantum dots using T. Polium L. Our results illustrated that the brine shrimp cytotoxicity assay could be an easy bioassay to screen nanoparticles. Using the brine shrimp assay to indicate cytotoxicity and antitumor activity is conceivable. The positive correlation of the brine shrimp cytotoxicity experiment suggests that the nanoparticles could be a source of cytotoxicity, which may support their usage as an anticancer agent or carrier. As a result, the cytotoxic effect of nanoparticles can be related to their size and shape. However, AgQDs can cure a wide range of infections and prevent cancer. In research by Kummara et al., the anticancer activity of green and chemically synthesized AgQDs was evaluated using the brine shrimp method. They demonstrated that silver nanoparticles had high cytotoxicity and anticancer activity (Kummara et al., 2016). Furthermore, in research by Alam, cytotoxicity assessment of green synthesis silver nanoparticles using strawberry fruit pomace extract showed high cytotoxic activity with an LC₅₀ value of 200 μg/ml (Alam, 2022). In another research, Nazari et al. employed Marinospirillum alkaliphilum to synthesize AgNPs and examined its anticancer properties using the brine shrimp assay. According to their studies, the nanoparticles were highly toxic (LC₅₀ = 1 μg/ml) (Nazari and Jookar Kashi, 2021). We believe that our results are accurate.

4.3 Ames

Cancer has risen as a global health concern that involves over 100 different kinds of abnormal cell division with the property of metastasis (Barabadi et al., 2020; Vahidi et al., 2020). Nanomaterials could be used to develop next-generation anticancer agents. Hence, newly produced nanoparticles should be thoroughly investigated. The Ames test is one of the methods used to determine if nanoparticles have anticancer effects. Therefore, bacterial mutagenicity and antimutagenicity of AgQDs were assessed. The experiment was organized only in the absence of S9 since AgQDs were metal, and it was improbable that S9 would metabolize them. Strain TA98 and strain TA100 detected mutagens that caused various frameshift and base-pair substitutions. The AgQDs were nontoxic for S. typhimurium strains at the performed concentration. The mutagenicity and antimutagenic effects of the AgQDs are summarized in Table 4. A substance with an induction factor of two or greater is considered a mutant. The number of returned colonies depends on the concentration and has a dose-dependent effect. The sample is not mutagenic if the induction factor is less than 1.5, and between 1.5 and 2 represents poor mutagenicity (Dashtizadeh et al., 2021). In addition to this, the anti-mutagenicity effect of the sample with three levels of weak, medium, and strong was assumed to be below 20%, between 40% and 25%, and more than 40%, respectively (Espanha et al., 2014). It was discovered that strain TA98 produces the best response from the tested nanoparticles. For the highest AgQDs concentration (400 μg), the largest number of reverted colonies was discovered, which displayed a maximum induction factor (IFmax) of 1.18 and 1.13 for strains TA98 and TA100, respectively. Although, none of the tested nanoparticle concentrations led to any mutation induction. Moreover, statistical data evaluation showed they were significantly higher than the control induction. The results of colony counting in the Ames test using silver quantum dots showed a significant anti-mutagenic effect on colony growth. In addition, prevention percent was used to determine the prevented reverted mutations of AgQDs in the anti-mutagenicity test. The AgQDs (400 μg) represented the highest prevention percentage (89.47%). The findings prove that the AgQDs have anti-mutagenicity activity, confirming the brine shrimp test. Nikouharf Fakher et al. determined the mutagenic effects of synthesized Ag/AgCl nanoparticles on strains TA100 and TA98. They have demonstrated that the nanoparticles did not show a mutagenic effect, and their percent inhibition of mutagenicity was more than 97% (Fakher and Kashi, 2021). In another study, Khan et al. evaluated the mutagenic properties of synthesized AgNPs using an aqueous extract of Pinus wallichiana stem against strains TA98 and TA100. They concluded that the nanoparticles were nontoxic and nonmutagenic (Khan et al., 2020).

4.4 MTT

AgQDs as therapeutic agents is limited, although it has shown antimicrobial activities and biological applications (Mohanty et al., 2012). Our research determined the sensitivity of OVCAR3 cells to AgQDs through an MTT assay. As displayed in Fig. 7, AgQDs considerably decreased the proliferation of OVCAR3 cells in a time- and dose-dependent manner. The IC₅₀ of AgQDs was 5.9, 2.3, and 0.8 µg/ml in OVCAR3 cells after 24, 48, and 72 h, respectively. It was proven that AgQDs concentrations ranging from 0 to 0.25 μg had no prominent cytotoxic effect on the OVCAR3 cells. The AgQDs indicated no considerable cytotoxic effects at 0.5 μg/ml concentration after 24 h. On the other hand, approximately 23% of the cells were viability dosed at 8 μg/ml after 72 h. Also, trypan blue viability assay results were consistent with the MTT assay. The cell viability was equal after 48 and 72 h at 16 μg/ml. The biosynthesized silver nanoparticles by (Hamelian et al., 2018) showed that the viability of Hela cells was even up to 200 μg/ml after 48 h. Previous research on green synthesized AgNPs from Piper longum extract demonstrated that viability in vitro cytotoxic properties against Hep-2 cells was observed at 49% in 31.25 μg/ml concentration (Justin Packia Jacob et al., 2012). Furthermore, increased cytotoxicity of AgNPs on the MCF-7 cell line was displayed with increasing concentration (Selvi et al., 2016). In another study, silver nanoparticles indicated a selective cytotoxic effect against cancer cells (IC₅₀ = 82.016 μg/ml) and a non-toxic effect in normal fibroblast cells (Mouriya et al., 2023). Moreover, the study of the cytotoxicity of AgNPs against MCF 7, Hela, and OP9 cell lines demonstrated synergistic effects on cytotoxicity (Ghosal et al., 2020).

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Fig. 7

Cytotoxic of AgQDs synthesized against OVCAR3 cell line after 24, 48, and 72 h.

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4.5 The effect of AgQDs on normal cells

The results of the inhibitory effect of the AgQDs on normal human fibroblast cells showed that the AgQDs had an insignificant effect on normal human fibroblast cells at concentrations near IC₅₀ of AgQDs on cancer cells after 24, 48, and 72 h.

The AgQDs killed normal cells in 16%, 20%, and 24% after 24, 48, and 72 h, respectively. These findings confirmed that these doses of AgQDs have no considerable side effects (Fig. 8).

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Fig. 8

Cytotoxic of AgQDs synthesized against normal human fibroblast cells after 24, 48, and 72 h.

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4.6 DPPH

In this method, the IC₅₀ of AgQDs was 4.8 μg/ml proving that AgQDs have higher antioxidant activity than BHT (IC₅₀ = 18.15 μg/ml). Two oxidation states (i.e., Ag⁺ and Ag²⁺) can exist for silver, so silver-based nanoparticles may be able to remove free radicals by donating or accepting electrons based on the reaction environments. The experiment results found that the antioxidant activity of AgQDs is relevant to their concentrations. The findings also revealed the substantial increase in antioxidant activity of green synthesized AgQDs compared to T. polium extract. According to the results, we proposed that the AgQDs are responsible for most of the antioxidant activity. Kanipandian et al. synthesized silver nanoparticles using Cleistanthus collinus extract. They found that the DPPH radical scavenging capacity was enhanced with plant extract, similar to our research (Kanipandian et al., 2014). Yousaf et al. studied the synthesis of silver nanoparticles using methanol, ethanol, and aqueous extracts of A. millefolium. They found that silver nanoparticles synthesized from methanol extract showed significant antioxidant properties (IC₅₀ = 7.03 ± 0.31 μg/ml) compared to the ascorbic acid standard (IC₅₀ = 4.29 ± 1.74 μg/ml) (Yousaf et al., 2020). Researchers have shown that the strong scavenging ability of silver nanoparticles might be dependent on donating electrons or hydrogen ions to neutralize the unstable DPPH free radicals in the reaction environment (Bardania et al., 2020).

4.7 Coating material by AgQDs

Antimicrobial activity (agar diffusion tests) was carried out on silver-treated and untreated (as control) toothpicks, cotton, medical mask fabric, cotton fabric, non-absorbable suture silk, absorbable suture silk, yarn, and filter paper against C.albicans and S.epidermidis strains. Fig. 9 shows the results of the agar diffusion test. Hence, a well-marked inhibition zone was revealed around the silver-treated items, pointing out the significant efficacy of the materials against gram-positive (S.epidermidis) bacteria and yeast (C.albicans). However, untreated samples showed no antibacterial efficacy. The results displayed that the antibacterial silver-coated sutures developed in this investigation could be an affordable alternative to general sutures. Moreover, other silver-treated materials, such as spinning and cosmetics, can be suggested for industrial applications. Simone et al. reported that silver-treated suture silk could represent clinical advantages in preventing surgical infections (De Simone et al., 2014). In another research, Sharma et al. produced silver nano-coated fabric and evaluated antimicrobial activity against E. coli and S. aureus. They discovered that fabric treated with nanoparticles effectively against S.aureus (Sharma et al., 2018).

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Fig. 9

Antimicrobial test for untreated and silver treated on a: toothpick b: cotton c: medical mask fabric d: cotton fabric e: non-absorbable suture silk f: absorbable suture silk g: yarn h: filter paper against C. albicans and S. epidermidis.

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