3.1. IR spectra
The IR spectrum of the synthesized composite (Fig. 1) showed absence of the characteristic bands assigned to M-O or M-C bonds in the range 500–650 cm− 1 which indicates the absence of oxides or carbides materials, respectively, in the chemical structure of the synthesized composite [26]. Also, the spectrum showed no bands in the ranges 1550–1680 cm− 1 and 3300–3450 cm− 1 assigned to bending and stretching vibrations of H2O molecules, respectively, indicating that no chemical bonded or physical adsorbed water molecules which confirmed the anhydrous structure of the metallic composite [27]. The multiple weak bands observed at 420–480 cm− 1 are due to metal-metal interaction [28].
3.2. XRD
XRD was used to confirm the structure of the trimetallic nanocomposite (Fig. 2). The positions of high intensity peaks refer to the formation of pure Ru/Ag/Pd trimetallic NPs. Peaks appeared at 2θ = 40.11, 46.15, and 68.31 which correspond to (111), (200), and (220), respectively, and are consistent with a conventional Pd-NPs phase pattern [29, 30]. Ag NPs showed typical peaks corresponding to the (111), (200), (220), and (311) planes at 2θ values of 38.14, 44.35, 65.00, and 77.5, respectively [31]. For the hexagonal structure of Ru-NPs, five different diffraction peaks were found and indexed with the planes (100), (002), (101), (102), and (110) at 38.42°, 42.12°, 43.98°, 58.32°, and 69.42°, respectively [32]. The size of the crystals was determined using the Scherrer equation [33] as follows.
D=\(\frac{0.9. \lambda }{\beta . Cos\theta } \left(2\right)\)
Where λ = 1.5418 Å is wavelength of X-ray (for Cu Kα1), θ is XRD angle. β is half maximum width. The crystal size of the nanocomposite was calculated to be 15.67 nm.
3.3. TGA
The TGA curve of the synthesized composite revealed a straight line with no thermal decomposition steps. These findings confirmed the complete degradation of nitrate and acetate groups in the precursors during the preparation of nanocomposite and the thermal stability of the product.
3.4. Morphological study
The morphology of the synthesized composite was examined using SEM at different magnifications. As depicted in Fig. 4, the synthesized NPs exhibited highly homogenous structure.
3.5. In vitro anti-cancer activity of (Ru/Ag/Pd)-NPs before and after UV exposure
The cytotoxic activity of (Ru/Ag/Pd)-NPs before and after UV exposure was evaluated using the Caco-2, HepG2 and K562 cells. Our data revealed that (Ru/Ag/Pd)-NPs treatment before UV exposure exhibited cell cytotoxicity with IC50 of 47.35 ± 2.7 µg/ml, 68.8 ± 3.9 µg/ml and 35.87 ± 2.0 µg/ml against Caco-2, HepG2 and K562 cells, respectively. Interestingly, after UV exposure for 20 min, (Ru/Ag/Pd)-NPs treatment results in cytotoxic activity with IC50 of 9.32 ± 0.52 µg/ml, 46.77 ± 2.6 µg/ml and 28.32 ± 1.6 µg/ml toward Caco-2, HepG2 and K562 cells, respectively (Fig. 5). These data proved that photoactivation markedly enhanced the anticancer efficacy of (Ru/Ag/Pd)-NPs.
Cell cycle arrest is a crucial mechanism through which anticancer drugs produce their antiproliferative effects [34, 35]. As a result, we investigated how (Ru/Ag/Pd)-NPs before and after UV exposure affected the distribution of Caco-2 cells throughout the cell cycle. The purpose of the current study was to examine the cell cycle distribution and proliferation potential Caco-2 cells following treatment with (Ru/Ag/Pd)-NPs before and after UV exposure. To ascertain the total population distribution in the various phases (G0/G1, S, and G2/M), asynchronously growing Caco-2 cells were exposed to the (Ru/Ag/Pd)-NPs before and after UV exposure for 24 h. The cells were then stained with PI and subjected to flow cytometry analysis.
Herein, Caco-2 cells treated with the IC50 of (Ru/Ag/Pd)-NPs before photoactivation (47.35 µg/ml) and (Ru/Ag/Pd)-NPs after photoactivation (9.32 µg/ml). (Ru/Ag/Pd)-NPs before photoactivation and after photoactivation induced apoptosis as indicated by an increase in G2/M phase by 19.3% and 31.06%, respectively, in comparison with that of control Caco-2 cells (5.87%). Moreover, in Pre-G1 phase, (Ru/Ag/Pd)-NPs before photoactivation resulted in apoptosis induction by 17.03%, while the photoactivated (Ru/Ag/Pd)-NPs produced marked cells apoptosis with 32.41%, in comparison with that of control Caco-2 cells (2.23%) as represented in Fig. 6. As a result, treatment with (Ru/Ag/Pd)-NPs before and after UV exposure can encourage the transition of colon cancer cells from the G1 to the S phase and subsequently induce cycle arrest in the S phase, thereby weakening their ability to proliferate and decreasing their viability.
Apoptosis can be induced by arresting cell cycle. The anticancer activities of NPs synthesized by green synthesis has been proved in several investigations [36] showing that the cytotoxic activity was mediated by different mechanisms, including blocking cell cycle in G0/G1 [37] or in G2/M [38] phases. In this study, we explored the impact of (Ru/Ag/Pd)-NPs on the ability of Caco-2 cells to undergo apoptosis before and after exposure to UV light. The presence of phosphatidylserine (PS) residues on the surface of the cell which are typically concealed by the plasma membrane is used for the identification and quantification of apoptosis. One of the distinctive cues for macrophages to recognize and remove apoptotic cells is the presence of PS on the cell surface. Annexin V has demonstrated a high affinity for binding to PS and hence could be used to check integrity of the cell membrane which is compromised as the apoptotic process develops. It is feasible to discriminate between early and late apoptotic cells as well as dead cells using DNA-specific viability dyes such as PI [39].
Cell cycle analysis of Caco-2 after treatment with (Ru/Ag/Pd)-NPs either before or after photoactivation showed preG1 peak that proved apoptosis (Fig. 7). Consequently, to confirm the effect of both (Ru/Ag/Pd)-NPs on apoptosis induction and the impact of photoactivation, Caco-2 cells were stained with Annexin V/PI, incubated for 24 h, and analyzed. The results proved that both (Ru/Ag/Pd)-NPs and its photoactivation potently induced apoptosis in Caco-2 cells from to 17.03% and 32.41%, respectively, in comparison with that of Caco-2 control cells (2.23%). These results indicated that photoactivation of the (Ru/Ag/Pd)-NPs resulted in 1.9-fold increase in its ability to induce apoptosis.
3.6. Antimicrobial activity
Aspergillus flavus (MT550030), Aspergillus niger (MW596373), Candida albicans (MW534712), Candida glabrata (MW865705), Escherichia coli (MW534699), and Bacillus cereus (MW830387) were chosen to evaluate the antimicrobial activity of the trimetallic NPs composite. The minimum inhibitory concentration (MIC) was determined using four different dosages of nanomaterials (0.0125, 0.025, 0.05, and 0.1 mg/mL) (Fig. 8 and Table) and was found to be 0.0125 mg/mL.
For comparison, antimicrobial experiments were carried out for each single metal as depicted in Table 2 and Fig. 9. The findings indicated that all tested materials demonstrated high antimicrobial activity and followed the sequence (Ru/Ag/Pd)-NPs > Pd-NPs = Ag-NPs = Ru-NPs > garlic extract in comparison to different isolate species of bacteria and fungi.
Table 1
MIC values in mg/ mL of composite (Ru/Ag/Pd)-NPs against different microbes.
Conc. (mg/mL) | Inhibition % |
Aspergillus flavus | Aspergillus niger | Candida albicans | Candida glabrata | Escherichia coli | Bacillus cereus |
0.0125 | 37.1 | 39.4 | 34.2 | 38.5 | 32.9 | 31.6 |
0.025 | 55.3 | 59.4 | 50 | 52.8 | 59.2 | 54.8 |
0.05 | 60.7 | 62.8 | 65.7 | 69.3 | 60.2 | 60.4 |
0.1 | 94.8 | 91.6 | 89.3 | 91.6 | 88.8 | 87.1 |
Table 2
The antimicrobial activity of different NPs.
| Inhibition % |
Aspergillus flavus | Aspergillus niger | Candida albicans | Candida glabrata | Escherichia coli | Bacillus cereus |
Miconizol | 32.7 | - | - |
Amikacin 30 | - | - | - | - | 30.6 |
Garlic extract | 19.7 | 15.5 | 20 | 14 | 20 | 19 |
Ag-Np | 26.2 | 31.4 | 25 | 30 | 32.9 | 29.8 |
Pd-Np | 24.9 | 29.7 | 32.4 | 29.5 | 25 | 27.4 |
Ru-Np | 28.7 | 26.4 | 21.5 | 26.3 | 28.5 | 29.3 |
(Ru/Ag/Pd)-Np | 37.1 | 39.4 | 34.2 | 38.5 | 32.9 | 31.6 |