Characterization of Synthetized Silver Nanoparticles using Juniperus polycarpos
Change in color of solution with Juniperus polycarpos extract was apperceived after incubation of flask containing AgNO3 at 37º C for 24 hours. The color of the extract solution altered from light yellow to dark brown after 24 hours. The alteration in color indicates the accepted synthesis of silver nanoparticles. AgNPs formation also was verified using UV-vis, XRD, FTIR, EDS, SEM and TEM.
UV-vis analysis
Figure 1 shows the recorded UV-Vis absorption spectra of the synthesized AgNPs by Juniperus polycarpos extract. For UV–vis analysis, the absorption of NPs was detected in 438 nm wavelength in UV–vis spectrum. Moreover, no additional peaks were noticed in the spectrum, which revealed great purification of synthesized AgNPs .
Characterization of size, morphology and DLS analysis of AgNPs
SEM and TEM images of AgNPs formed by Juniperus polycarpos indicate the shape, size and morphology of nanoparticles. According to microscopically investigation the green synthesized AgNPs at optimal conditions had maximum average size of 10–50 nm with mean size of 12.96 nm (Fig. 2).
According to the DLS analysis, the average size of AgNPs was estimated 133.4 nm and Polydispersity Index (PdI) value was recorded at 0.254.
The silver nanoparticles were found to be well, monodispersed from each other and spherical-shaped based on TEM and low pdl value of DLS analysis (Fig. 2c).
XRD, EDX & Zeta potential
The XRD patterns of the AgNPs synthesized by Juniperus chinensis extract, four intense XRD peaks were indicated at 2θ = 38.6, 44.4, 64.6, and 77.1correspond to the (111), (200), (220), and (311) crystallographic planes of facecentered cubic (FCC) structure.
Figure 3b shows the EDS analysis of Juniperus polycarpos mediated synthesis of silver nanoparticles. The EDS technique detects a potent signal at 3 keV indicates the presence of elemental silver. EDS analysis indicates the elemental analysis of the nanoparticles in which the percentage of silver ions was 99.15%.
In the current study, the fabricated AgNPs had a negative zeta potential of − 9.76 mV, indicating higher stability of the bio-functionalized AgNPs (Fig. 3c). The greater negative surface charge potential value indicates that the synthesized AgNPs are high dispersed in the medium with long-term stability [16].
The negative potential value may be due to the reducing and capping substances especially fatty acid in Juniperus chinensis extract also the aggregation of the nanoparticles could be prevented by electrostatic repulsion between negative charge of the nanoparticles [17, 18].
Fourier Transform Infrared Spectroscopy (FT-IR) analysis
The result of FTIR analysis of bio-synthesized AgNp revealed different stretches of bonds at different peaks. The strong and broad peak at 3411.89 cm-1 was related to the stretching vibrations of the O-H and N-H groups in the extracted. Absorption peaks located at 1619.28 cm − 1 and 1385.60 cm − 1 are assigned to C = C and C–H bend alkane groups, respectively. Peak at 1112.81 cm − 1 associated with C–O–C stretch ethers, while 621.51 cm − 1 represents = C-H group in alkenes and aromatic compounds. Peak at 2925.16 cm − 1 corresponds to aliphatic C–H group.
Cell Viability and MTT Test Results
The possible cytotoxicity results of various cisplatin concentrations and AgNPs on HEK293 cells ( obtained from National Cell Bank (NCBI) of Pasteur Institute of Iran), as a normal cell line, and A549, as a cancerous cell line, indicated that plant extract and AgNPs have cytotoxic effects against cancerous cell line while not toxic to the normal cells in lower concentrations(Fig. 5). It was also detected that the maximum cell death was belong to synthesized AgNPs, which was 35% for the cultured HEK293 cells after 24-hour exposure. This is when cisplatin resulted in 60% cytotoxicity toward the HEK293 cells. However, the cytotoxicity of AgNPs and the cisplatin against MCF-7 tumor cell line were approximately 60% and 40%, respectively (at the concentration of 12.5 µg/mL). As can be seen in Fig. 5, the toxicity of AgNPs is dose-dependent.
Figure 5A and 7C indicate viability of A549 and HEK293 cells exposed to 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 µg/ml of bio-synthesized AgNPs in cell culture. There was no significant difference between viability of HEK293 cells exposed to 0.78, 1.56, 3.125, 6.25, 12.5 and 25 µg/ml of AgNPs. However, HEK293 cells viability remarkably decreased in groups exposed to 50 and 100 µg/ml of AgNPs compared to control cells (P < 0.05 and P < 0.01, respectively). The reduction in A549 cells viability was observed when exposed to 3.125 and 6.25 µg/ml of AgNPs(P < 0.05 and P < 0.01, respectively). There was also statistically significant difference between viability of A549 cells exposed to 12.5, 25, 50 and 100 µg/ml of biosynthesized AgNPs(P < 0.001). For A549 and HEK293 cell lines the IC50 values by cultivation in the presence of silver nanoparticles were calculated 9.87 and 111.26 µg/ml, respectively.
Figure 5B and 5D also shows viability of A549 and HEK293 cells exposed to 3.125, 6.25, 12.5, 25, 50 and 100 µg/ml of cisplatin in cell culture. A549 cells viability significantly declined in groups exposed to 50 and 100 µg/ml of cisplatin compared to control cells (P < 0.001). Exposure of A549 cells to 6.25 (P < 0.05), 12.5 and 25 (P < 0.01) µg/ml of cisplatin also led to significant decrease in viability of A549 cells. However, there was no significant difference between viability of A549 cells exposed to 3.125 µg/ml of cisplatin compared to control cells. HEK293 Cell viability decreased greatly after exposure to 12.5 (P < 0.05), 25, 50 (P < 0.01) and 100 (P < 0.001) µg/ml of cisplatin. However, no significant differences were observed in the HEK293 cells viability at low doses (3.125 and 6.25 µg/ml) of cisplatin. The IC50 values of cisplatin determined for A549 and HEK293 cell lines were 24.67 and 43.35 µg/ml, respectively.
Gene expression
Figure 6 show the induction expression levels of caspase-3 and caspase-9 genes treated by AgNPs. According to Fig. 8a, the expression levels of caspase-3 and caspase-9 in cells exposed to synthetic AgNPs were higher than cisplatin treated cells. The results also revealed that AgNPs can down-regulation the expression levels of MMP2 and MMP9 in A549 cells which higher than cisplatin effect. Our result also was shown in Fig. 8c indicates the up-regulation of p53 and reduction of cyclin D1 in both AgNp and cisplatin treated A549 cells.
Flow cytometric Apoptosis analysis
To subsequent characterize apoptosis induction of AgNPs, A549 cells were stained with Annexin-V/PI assay, followed by flow cytometry test. The determination finding of flow cytometry assay was demonstrated in Fig. 10. The flow cytometry data obviously indicated that biosynthesized AgNPs can stimulate cell death procedure in A549 cells. Based on the Annexin V-FITC/PI staining, 96.1% of control cells were detected viable with early apoptotic value of 0.84%, late apoptotic value of 1.1%, and necrotic value of 1.9% of cells, which are common process for cells can be proliferated in culture mediums. The A549 cells exposed to AgNPs dramatically induced the late apoptotic and necrotic cells as compared with untreated control cells. An increase in the percentage of apoptotic (early and late, Q2 + Q3) cells was detected with the value of 34.4 and 16.08% for ic50 of synthetic AgNPs and cisplatin, respectively. AgNPs significantly (p < 0.001) has lower necrotic effects (Annexin V -/PI+) (4.0%, Q1) in A549 cells at ic50 concentrations than cisplatin (Annexin V -/PI+) (13.4%, Q4), indicative of apoptotic cell death and side effects of cisplatin drug (Fig. 7).
Cell cycle analysis
To indicate the distribution of Juniperus polycarpos leaf extract AgNps and cisplatin-treated A549 cells in different phases of the cell cycle, the DNA content in cells was detected by PI staining and analyzed by flowcytometry. The results indicated that the treatment with both AgNPs and cisplatin led to an increase in the population of cells in the G0/G1 phase (Fig. 8). The results showed that 15.58% and 7.58% of the cells treated with nanoparticles and cisplatin respectively were recorded in the sub-G1 phase.
Inhibition of migration and invasion of A549 cells
Scratch wound healing assay
To examine the anti-metastatic properties of each drug, a scratch wound assay was performed in all three cell lines. A wound healing assay was performed to exclude the growth inhibitory effect of AgNPs and cisplatin on migration. The effect of AgNPs and cisplatin on A549 cells was detected, and it was identified that both AgNPs and cisplatin have great effects on cells migration. The results of the wound healing assays are presented in Fig. 9. The area that the cells had migrated (toward the initially scratched midline, from the border line) was measured. The cells incubated with AgNPs migrated across an area that was less than that of the cells incubated with the cisplatin and control medium, indicating that both AgNPs and cisplatin weaked the migration of the A549 cells.
Figure 9A Indicates the migration rate in control cells, B, shows the migration rate in A549 cells were treated with AgNPs and C, revealed the migration rate A549 cells were treated with cisplatin for 24 h. Migration ability was determined by the migration rate of migrating cells at 24 h.
Invasion assay
We accessed the effects of both AgNPs and cisplatin on invasion ability of A549 cells. As shown in Figs. 13, both AgNPs and cisplatin inhibited the migration dramatically compared with the control group. AgNPs and cisplatin decreased the migration ability from 85–30% and from 85–50%, respectively (Fig. 10).
Induction activation of caspases and ROS
Caspases are the cysteine-aspartate proteases that play an important role in apoptosis. Caspase-3 is the initiator caspase and caspase-9 is the executor caspase. The executor caspase expression level was increased in A549 lung cancer cell lines treated with both AgNPs and cisplatin but the caspase-3 expression level had significant increased only in cells treated with AgNPs (Fig. 11A). The intracellular ROS content of control, cisplatin and AgNPs treated A549 cells is depicted in Fig. 11B. Forty percent and 90% increase in ROS content were observed in cisplatin and AgNPs-treated cells, respectively.
Determination of Apoptotic Effects in A-549 Cells
Apoptotic pathway or programmed cell death includes several changes in cells which contains morphology of cells, chromatin condensation, DNA, and nuclear fragmentation [35, 37]. In order to comparison the effect of AgNPs and cisplatin on cell death in A549 cells, nuclei of the cells were stain by DAPI stain. As shown in Fig. 12, higher levels of nuclear fragmentation, disintegration and condensation of chromatin at the boundary of the nuclear membrane and cell death were seen in AgNPs treated cells, compared to cells treated with cisplatin[19].