Silver nitrate was purchased from Rankem brand, India. The Hi-cDNA synthesis kit (MBT076-10R) was purchased from HiMedia, the master mix for PCR from Thermofisher Scientific and the specific primers for the gene ZHE1 & ZHE2, beta-actin (Sigma Aldrich) from BioSource & Surgical. The A549 cell line and L132 cell line were purchased from NCCS, Pune. Dulbecco’s Modified Eagle Medium (DMEM), antibiotic solution, diethylpyrocarbonate (DEPC), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was from HiMedia; bovine serum (FBS) was purchased from Gibco; dimethyl sulphoxide, acridine orange (AO), ethidium bromide (EtBr), phosphate-buffered saline (PBS) pellets were purchased from Rankem brand, India. Green tea was purchased from Korakundah Estate.

AgNPs were synthesized by the chemical reduction method described earlier [11] using trisodium citrate as a reducing agent. In 100 ml of distilled water, 25 ml of aqueous 5mM of Silver nitrate solution was added and boiled to heat. 5 ml of trisodium citrate solution (1 %) was quickly added to the boiling solution. Heating was continued until the colour changed to light yellow, followed by stirring. The working concentration of AgNPs was 1 mM, diluted for further studies using water (for zebrafish embryo study) and complete DMEM (for cell culture studies).

An amount of 2 g of green tea was soaked overnight in 100 ml of distilled water and in another beaker, 10 mM of silver nitrate solution was prepared. 0.5 ml of 10 mM of silver nitrate solution was taken and 0.4 ml of tea extract was added. Further, 9 ml of distilled water was added and kept at room temperature until the colour changed to dark brown, indicating the formation of silver nanoparticles. The working concentration of GT/AgNPs was 0.5 mM, diluted for further studies using water (for zebrafish embryo study) and complete DMEM (for cell culture studies).

The as-synthesized AgNPs and GT/AgNPs were characterized using different photophysical tools as described earlier [12]. The nanoparticle samples were diluted in distilled water and proceeded to measure the absorption spectrum using a Shimadzu UV-1800 absorption spectrophotometer. Further, to measure the particle size of the nanoparticle and surface charge distribution, a Malvern nano ZS90 zeta sizer instrument was used. The scattered intensity data was processed in the instrumental software, and the hydrodynamic diameter (nm) and surface charge were obtained. Bruker alpha FT-IR spectrometer was used in the transmittance mode to record the FTIR spectra, which infers the chemical bonds and their stretching, bending vibrations, etc., present in the compound. The samples were liquid, so the attenuated reflectance (ATR) mode was used to record the transmittance percentage with respect to the wavenumber in cm^-1.

Since the study investigates the low-concentration effect of silver nanoparticles, it is important to know whether, at this concentration, the synthesized AgNPs could exert any antimicrobial activity or not [13]. The overnight grown culture of the laboratory E. coli K12 strain MG1655 was sub-cultured in 2 ml of freshly prepared LB broth taken in four different test tubes labelled as T1, C1, T2, C2 (T1-AgNPs, C1-reducing agent trisodium citrate, T2-GT/ NPs and C2-reducing agent green tea) and allowed to grow aerobically for about 90 min at 37 °C. When the culture reached the mid-log phase, the NPs (0.2 nM) and reducing agents (equal volume to the NPs solution) were added to the respective tubes and incubated again. Around 0.1 ml of the culture was withdrawn at regular intervals (0, 3 and 6 h), serially diluted up to 10^-6 in saline. From each dilution, 0.01 ml was taken and sequentially spotted on LB agar plates. The plates were incubated at 37 °C for about 24-36 h, the colonies were counted and the cfu/ml was calculated. The antibacterial activity was assessed based on the colony-forming unit.

Two types of cells, A549 (lung cancer cells) and normal cells (L132) were used to study the toxicity of the synthesized AgNPs and GT/AgNPs at low concentrations. To determine the cell viability, an MTT assay was done according to Metkar et al. 2022 [14]; in brief, in a 24-well plate, 1.15×10⁴ of A549 and 1.4×10⁴ L132 cells, respectively, were grown in DMEM with 10 % FBS and antibiotics (1%) supplementation. The plate was incubated for 24 h at 37 °C in a humidified atmosphere with 5 % carbon dioxide. Then, both A549 and L132 cells were treated with AgNPs and GT/AgNPs at the doses of 0.05, 0.1, 0.2, 0.3 and 0.5 nM, respectively. After 24 h of treatment, 25 μl of MTT (10 mg/ml) was added to each well in dark condition and kept for 4 h incubation at 37 °C in a CO₂ incubator for the development of formazan crystals. Further, 1 ml of DMSO was added to each well and mixed properly to solubilize the formazan crystals formed and then the optical density (O.D.) was noted at 570 nm. The cell viability (%) was then calculated using Equation (1):

The experiment was repeated thrice.

Since the results of the MTT assay showed that the GT/AgNPs killed specifically the cancer cells, not the normal cells, the live dead assay using EtBr and AO was executed only for the cancer cells, according to Liu et al. [15], with slight modification. The live cells will be green in colour, whereas the dead cells will take red colour under a fluorescent microscope. The exponentially growing A549 cells were grown on a coverslip and were treated with AgNP and GT/AgNP at a dose of 0.2 nM (the dose was decided based on MTT assay results). After treatment, plates containing the coverslips were incubated for 24 h. For live dead assay, 1.2 mM of AO and 1.9 mM of EtBr were prepared. The working solution (dye mixture) was prepared by dissolving 5 μl of AO and 2 μl of EtBr in 2 ml of PBS in the dark. The coverslips containing the treated and untreated cells were incubated with the dye solution at 37 °C in the dark for 3 min and then visualized under a fluorescent microscope (Olympus, BX 51). The images were captured at different locations on the slide and the number of live and dead cells were plotted for both nanoparticle treatments. The experiment was done in triplicates and repeated two times.

The SEM analysis was done according to Girigoswami et al. [16]. The A549 cells were grown in glass coverslips placed inside petri plates for 24 h at 37 °C in a CO₂ incubator. The cells are treated with 0.2 nM concentration of AgNPs and GT/AgNPs. 24 h after treatment, the cells were washed with PBS (pH 7.4) and then fixed with acetomethanol (acetone: methanol= 1:1) at 4 °C for 4 h. The fixing solution was thoroughly evanesced. Using SCD005Pt-coater (Bal- Tec AGI, Liechtenstein), samples were coated with a thin film of platinum. With the S-4800 scanning electron microscope (Hitachi High Technologies Co., Japan), electron micrographs were obtained with an acceleration voltage between 1 and 5 kV. To avoid the sample burning, higher acceleration voltages were not used [17].

The wild-type female and male zebrafish (Danio rerio) were cultured in separate rectangular glass tanks and the adult fishes were maintained at 25 ± 2 °C with a water pH 7.0. Air pumps were attached inside the fish tank for proper air circulation for the fish. Live blood worms and standard flakes were given to the fish twice a day. For spawning, the male and female fish of 1:2 ratio were kept in the breeding tank with 12 h light-dark condition and the next day, the embryos were collected and washed in water three to four times. Animal protocols were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) 2021, Department of Animal Husbandry and Dairying, Government of India. The Institute Animal Ethics Committee (IAEC) of Chettinad Academy of Research and Education approved the Danio rerio embryos (letter No. IAEC1/Proposal:81/A.Lr:61/Dt. 11.05.2022).

Cumulative hatchability was evaluated following our previous report [17]; in brief, to assess the toxicity, in a 6 well plate, zebrafish embryos were treated with different concentrations of AgNPs and GT/AgNPs (0.05, 0.1, 0.2, 0.3 and 0.5 nM). Each well contained 30 embryos and 3 ml of E3 medium. In a separate well of another 24-well plate, the control embryo was maintained under the same conditions containing only E3 medium. The embryos were observed under a compound microscope at different hours of post-fertilization (hpf) (10, 28, 53, 60, 77, 100, 124 and 148 hpf) and the images were captured accordingly. The cumulative hatchability was calculated with the formula for the control groups (Eq. (2)) and the different nanoparticle-treated groups:

where, N_h is the number of eggs hatched and N₀ is the total number of eggs taken for treatment with nanoparticles [17]. A cumulative hatchability graph was plotted, taking the time along the abscissa and the percentage of embryos hatched along the ordinate for different concentrations. The experiment was repeated three times.

The embryos were collected in a similar way as described above for extracting the mRNA, which was converted to cDNA and taken as a template for gene analysis, according to Ghosh and Girigoswami [17] and Peterson and Freeman [18,19], with a few modifications. Briefly, at 32 hpf, 40 embryos were exposed to 0.1 nM concentration of AgNPs and GT/AgNPs, respectively, and after 24 h, the embryos were taken for RNA isolation. Control embryos were maintained only in the E3 medium without any treatment.

After treatment, the RNA was isolated according to Ghosh and Girigoswami [17] and Peterson and Freeman [18,19], with some modifications. All the glassware and plasticware were soaked in DEPC water overnight, dried and autoclaved before the initiation of RNA isolation. Briefly, from each group, the embryos were taken in separate vial and homogenized on ice after adding 100 μl of Trizol reagent. Further, 0.9 ml of Trizol was added to the tubes individually and kept for 5 min at room temperature. Chloroform (0.2 ml) was added to these tubes and mixed vigorously several times, followed by 3 min incubation at room temperature. The mixtures were centrifuged at 12000 rpm at 4 °C for 15 min. The aqueous phase in the top layer was transferred into a fresh tube without disturbing the middle layer. To precipitate the RNA, 0.5 ml of isopropanol was poured into each tube, incubated for 10 min at room temperature and centrifuged at 12000 rpm for 5 min at 4 °C. Aspiration of the supernatant was done carefully without agitating the pellet. Washing of the pellet was done with 75 % ethanol using centrifugation at 12000 rpm at 4 °C for 5 min. Ethanol was removed after centrifugation, air-dried for 10 min and dissolved in RNase-free water. The RNA concentration was calculated by measuring the O.D. at 260 nm, and the RNA purity was calculated by the ratio of O.D.s at 260 nm and 280 nm. If the ratio was above 1.8, it was processed for cDNA synthesis.

After RNA isolation, cDNA was prepared using a Hi-cDNA synthesizing kit (MBT076-10R) supplied by HiMedia. The following reagents were mixed as follows: in a RNase free PCR tube - 1 μl of Oligo (dT), 1 μl of random hexamer, 2 μl of (Random Hexamer: Oligo (dT) mix), 0.28 μg, 1.1 μg and 0.17 μg concentration of control, AgNP and GT/AgNP RNA sample was added respectively and made up to 10 μl with PCR water (molecular biology grade PCR water). The tubes were incubated for 5 min at 65 °C and immediately cooled in ice. To the above tube, the following reactants were added: RT buffer (M-MuLV) of 4 μl, 2 μl of 10X solution for M-MuLV, 1 μl of M-MuLV reverse transcriptase (RNase H), 0.5 μl of ribonuclease inhibitor, 2 μl of 10 mM of dNTP mix and finally made up to 20 μl with PCR water. Then, the tubes were mixed nicely and kept at 42 °C for 60 min, followed by 70 °C for 5 min (1 cycle). The tube was kept at 4 °C. The RT product was stored at -20 °C.

The cDNA was successfully synthesized and was utilized to study the expression of ZHE1 and ZHE2 by PCR. The housekeeping gene, β actin, was taken as control. The sequences of primer used for amplification are as follows: β actin F: 5’-CGAGCTGTCTTCCCATCCA 3’; β actin R: 5’-TCACCAACGTAGCTGTCTTTCTG 3’.; ZHE1 F: 5’-CTGAACTTCTCTACACACTGAGG-3’; ZHE1 R 5’-CCTTATCACC ATCACCTCACTTC-3’; ZHE2 F: 5’-CTCCACACACTGAGACTAAATGG-3’; ZHE2 R: 5’-GGAAATAAGAGCACGTACTGTGG-3’. The reagents for PCR were added and mixed for each 0.5 ml tube as follows: 12.5 μl of 2X Master mix, which contains 0.05 U / μl of Taq DNA polymerase, 4 mM MgCl₂, reaction buffer, and 0.4 mM of each dNTP. Then 0.5 μl of forward primer and reverse primer of a specific gene, 2.0 μl of template and 9.5 μl of molecular grade water were added. All the genes had common PCR condition: the initial denaturation was at 95 °C for 5 min, and then 35 cycles were run with each cycle containing the three steps: 30 s denaturation at 95 °C, 30 s annealing at 54 °C, and 45 s extension at 72 °C. After the 35 cycles, the final extension at 72 °C for 5 min was kept. The RT product was kept at 4 °C for 5 min and then stored at -20 °C.

Agarose gel (1 %) was prepared with tris acetate EDTA buffer, to which 4 μl of ethidium bromide was added and dissolved correctly. The gel boat was sealed and the gel was poured and allowed to solidify. 10 μl of each PCR sample was mixed with 5 μl of sample loading dye. The samples were loaded in their respective wells, and the gel was run at 100 V and visualized under a UV-transilluminator. The images were captured using Genei capture imaging software in a Gel-Doc (GeNei Imaging System). The intensity of the bands was calculated using Image J software.

Silver nanoparticles synthesized via the green route were observed by a colour change after adding green tea extract (Figure S1). The characterizations of the AgNPs and GT/AgNPs were done using different photophysical tools for measuring their absorption spectrum, hydrodynamic diameter, surface charge and functional groups. Figure S2 shows the absorption spectrum of AgNPs and GT/AgNPs. The λ_max was found to be 429 nm for AgNP and 434 nm for GT/AgNP, which corroborates with the characteristic absorption peak of silver nanoparticles. AgNP’s and GT/AgNPs’ hydrodynamic diameters and zeta potentials are given in Figure S3. The hydrodynamic diameter obtained for AgNP was 65.39 nm and for GT/AgNP it was 106.14 nm. AgNP showed a surface charge of -25.1 mV and GT/AgNP was about -16.9 mV. The characteristic FTIR peaks obtained were noted. The visible peaks are present at 3312 cm^-1 for O-H stretching, 1634 cm^-1 for N-H bending, 664 cm^-1 and 530 cm^-1 for C-Br stretching for AgNP. For GT/AgNP, visible peaks obtained at 3319 cm^-1, 1634 cm^-1, and 538 cm^-1 represent that O-H stretch, N-H bend, and C-Br stretch were present, respectively [20]. The FTIR spectra for AgNP and GT/AgNP are given in Figure S4.

Figure S5 shows the bacterial growth in AgNPs (0.2 nM) and GT/AgNPs (0.2 nM), as well as in the plates treated with reducing agents (trisodium citrate and green tea extract). There was no inhibition of E. coli growth after treatment with such a low dose of silver nanoparticles.

The main focus of our study was to observe the effect of silver nanoparticles at very low doses. Cell viability of A549 cells and L132 cells after treatment with AgNP and GT/AgNP was assessed using an MTT assay. It was evident from the result that 0.2 nM concentration of GT/AgNP reduced the viability of A549 cells more efficiently in comparison to other concentrations. It was apparent from the result that neither chemically synthesized nor green tea-mediated synthesis affected the viability of L132 cells at different concentrations used. The comparative data is given in the graph below (Figure 1).

Live cells will appear green and dead cells red when visualized under a fluorescent microscope after staining with EtBr and AO. The typical snapshots of the cells viewed under a fluorescence microscope at 10 X magnification, 24 h after treatment with 0.2 nM AgNPs (Figure 2 (b)) and GT/AgNPs are shown in Figure 2(c). Control cells are shown in Figure 2(a). Exploring the live dead cell assay, the A549 cell killing efficiency of GT/AgNPs was found to be 16.4 % and for AgNPs, it was 2 %, respectively. The results were obtained by counting the live cells’ and dead cells’ numbers for the snapshots captured at different locations of the coverslip. The cell counting results showed the percentage of killed cells after treatment with the nanoparticles and are depicted in Figure 2(d). The comparative data infers that GT/AgNPs killed the cancer cells at a higher rate compared to the AgNPs.

To gain further insights into the size of the nanoparticles and characteristics of cancer cells after exposure to AGNPs and GT/AgNPs, SEM micrographs were taken. Figure 3 (a) and (b) show the SEM images of the A549 cells treated with GT/AgNPs (Fig 3 (a)) and chemically synthesized AgNPs (Fig 3 (b)). The typical cell shrinkage, which indicates apoptosis, was observed for GT/ AgNPs treated cancer cells, whereas chemically synthesized AgNPs treated cells did not show any toxicity to the cancer cells.

The zebrafish embryos’ morphology was observed for control and after treatment with AgNPs and GT/AgNPs at different concentrations (0.05, 0.1, 0.2, 0.3 and 0.5 nM) (Figure 4). Depending on the embryo’s hatchability at different concentrations, cumulative hatchability was calculated and depicted in Figure 5. For 0.05 nM concentration, the AgNPs-treated embryos showed 70 % hatchability, whereas GT/AgNP-treated embryos showed 80 % hatchability. Abnormalities such as body bends were observed in only AgNPs-treated embryos. For 0.1 nM concentration, the hatchability percentage was 63 % for AgNPs and for GT/AgNP-treated embryos, it was 77 %, respectively. No delayed hatching was observed in these concentrations (0.05 and 0.1 nM).

For 0.2 nM concentration, in chemical-AgNPs treated embryos, only 23 % of embryos hatched at 77 hpf with abnormalities like tail bend and 78 % at GT/AgNPs treated embryos, without showing any abnormality. The hatchability percentage decreased with increasing concentrations of AgNPs-treated embryos. For 0.3 nM concentration, all the AgNPs treated embryos were dead, whereas GT/AgNPs treated embryos showed 83 % hatchability. 0.3 nM concentration of GT/AgNP showed an increased hatchability percentage in comparison to other concentrations. For 0.5 nM concentration, all embryos died in AgNPs-treated embryos and in GT/AgNPs-treated embryos, it showed 78 % of hatching and there was no delayed hatching found. The highest hatchability of 83 % was observed in GT/AgNP of 0.3 nM concentration. On the other hand, all the embryos treated with higher concentrations of AgNPs, such as 0.3 and 0.5 nM, died. Abnormalities like body bend, abnormal growth, and tail bend were seen in embryos treated with AgNP of 0.05 nM, 0.1 nM, and 0.2 nM alone, whereas GT/AgNP-treated embryos showed no developmental defects microscopically (Figure 6).

This study confirmed that biosynthesized silver nanoparticles using green tea (GT/AgNP) have reduced toxicity in zebrafish embryos compared to AgNP-treated embryos. The microscopic images of chemically synthesized treated embryos with developmental disabilities are shown in Figure 6.

The gene expression study of ZHE1 and ZHE2 in zebrafish embryos was done with reverse transcriptase PCR (RT-PCR) (Figures 7a and 7b). Beta-actin was used as a housekeeping gene where the ZHE1 was upregulated and the ZHE2 gene was downregulated in the control sample. Thus, from the results, the ZHE2 gene is relatively highly expressed in AgNP and GT/AgNP-treated embryos. ZHE1 is downregulated in AgNP and GT/AgNP-treated zebrafish embryos. Figure 7 shows the differential expression of genes. Figure 7c shows that the GT/AgNP expressed higher levels of ZHE1 and ZHE2 compared to AgNP-treated embryos.