3.1. Characterization of Nanoparticles
Figure (2) Show TEM images of prepared nanoparticles, the image show that Au as a dark semi spherical with average size 18nm as in Fig. 2- A, While Fig. 2-B, refer to ZnO NPs, shiny dark as a cross-linked refer to pure ZnO NPs as adherence on Au NPs with average size 25nm, as in Fig. 2-C. D, refer to the curcumin as irregular spherical size as a nanocluster, while, E) shows pure curcumin when mixing with Au/ZnO NPs, appearances the important role of curcumin played a capping of Au/ZnO NPs. The FTIR spectra were recorded in the 400–4000 cm-1 spectral range. Figure 3 shows the FTIR spectra of Au/ZnO nanocomposites. Several bands can be found in the FTIR spectra of the samples [41, 42]. The large and wide band between 3200 and 3600 cm-1 is assigned to the characteristic stretching vibration mode of the water O–H, which changes as the concentration of Au nanoparticles increases. The presence of CO2 molecules in the ambient air causes bands around 2076 cm-1. Carbon dioxide O = C = O stretching is represented by the small peak at 2356 cm-1 and 2333 cm− 1. The O–H bending vibration mode is assigned to the strong band near 1640 cm-1 for Au, ZnO, and Au/ZnO. The spectrum of pure curcumin and Cur-Au/ZnO CSNPs is shown in Fig. 5B, with the orange line referring to active groups for pure curcumin. The band seen at 1640 cm-1 is caused by the uncoordinated phen's ring vibrations. The peaks at 1137 and 3346 cm-1 are possibly due to O–H deformation and stretching due to moisture adsorbed on the NP surface, respectively . When Cur-Au/ZnO CSNPs are mixed with Au/ZnO NPs, several peaks disappear, leaving only four main peaks: the broad peak at 670 cm-1 showed the predicted Zn–O stretching vibrations, and the broad peak at 3455 cm-1 is the characteristic O–H stretch . In Fig. 4, the absorbance spectra of the Au, ZnO, Au/ZnO, and Cur-Au/ZnO CSNPs, suspensions are shown. Because of interband transitions and SPR oscillations in Au nanoparticles, the UV–visible absorption spectra of gold nanoparticles display absorptions in the ultraviolet and visible regions, respectively. Au nanoparticles have a plasmon peak at 527 nm. Figures (3) shows the UV absorption peak of ZnO nanoparticles exciton absorption at 330 nm. This finding is identical to previous PLA-prepared ZnO nanoparticles . UV absorption is observed in the Au/ZnO nanocomposites. Due to the low concentration of Au nanoparticles in colloidal solution of ZnO NPs, the strongly damped absorption becomes weak and wide. For Au/ZnO suspension, a large peak with a red shift of 540 nm was observed, which corresponds to the localized SPR of the partially shaped gold nanoparticles. Figure 3-B, show the UV-Visible Spectrophotometer for Pure Cur. and Cur-Au/ZnO CSNPs and show the peak of pure curcumin at 364nm  and Cur-Au/ZnO CSNPs appear a broad peak for mixture of nanoparticles and this refer to the pure curcumin play a role as a shell to cover on intensity of absorption for Au and ZnO nanoparticles.
3.2. Antibacterial activity of Cur-Au/ZnO NPs
The antibacterial of Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs was investigated using S.aureus. The inhibition zones after exposing the organisms to tested nanoparticles used were measured and illustrate in Fig. 4. From the result Cur-Au/ZnO NPs found to be effective than ZnO NPs, Au NPs, Cur NPs, and Au/ZnO NPs. Cur-Au/ZnO CSNPs produced an inhibition zone diameter of more than 30 mm against S.aureus. The results showed the effect of prepared nanoparticles on the growth of S.aureus, especially after 12h of treatment as shown in Fig. 5. The inhibitory effect of Cur-Au/ZnO CSNPs was observed to be more than that of ZnO NPs, Au NPs, Cur NPs as proven by the statistical analysis.
3.3. Bacterial morphology
The effect of Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs on the structure of S. aureus under treatment was assessed using a SEM technique. The images demonstrated that there were differences in the bacteria cell morphology between treated bacterial strains and the untreated samples (control). Untreated control bacterial strain confirmed the cluster-form colonies as in Fig. 7-A. Since S. aureus is Gram-positive bacteria and thus exists in clusters, SEM images demonstrated that they were destroyed after they were treated with Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs as shown in Fig. 7- B, C, D, E, and F. The Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs was observed to have huge activities on bacterial strains as demonstrated in the bacterial cell structural changes as in Fig. 7. The Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs had effect on tested microorganisms’ outer membrane, as it was observed that the bacterial strain cell membrane had more pores and damage. The damage occurred as a result of osmotic imbalance leading to a leak of bacterial cells and it resulted to changes in morphology, osmotic balance, and cells’ structural integrity after it was treated with Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs. It was observed that in the bacterial strains treated with Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs there was aggregation and membrane rupture compared to the untreated strains. Additionally, the bacteria membrane surface potential became neutralized and this led to higher surface tension, abnormal structure, rapturing and damage on bacterial cells membrane.
3.4. Nanoparticles induces production of reactive oxygen species
The AO/EtBr staining technique was used to detect generation of ROS after the bacterial strains were treated with Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs. The indicators that show the presence of ROS are nitric oxide and hydrogen peroxide. When AO/EtBr dye comes into contact with reactive oxygen species, produced when an organism is under stress, it undergoes oxidation. The EtBr component will only pervade cells whose membrane integrity has been damaged and reacts with cells nucleic acid. The dead cells are stained in red while the viable cells are stained green. The bacterial strains that treated with Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs showed generation in ROS compared to the untreated cells. The Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs treated S.aureus resulted in more structural deformities as well as higher levels of ROS production as in Fig. 8, as demonstrated by a high number of bacteria strains that are reddish. Overall, the results showed that Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs were suitable as antibacterial agents that can be applied in biomedical and biological fields.
3.5. Nanoparticles attenuated invasion of bacterial strain to REF cells
REF cells were pretreated with Au, ZnO, Cur, Au/ZnO NPs and Cur-Au/ZnO CSNPs for 1 h and then infected with bacterial strains at MOI (200:1) for 1 h. The results shows that the Au, ZnO, Cur, Au/ZnO NPs and Cur-Au/ZnO CSNPs are attenuated the binding of bacterial strains to REF cells as indicated in Fig. 9. To determine whether Au, ZnO, Cur, Au/ZnO NPs and Cur-Au/ZnO also inhibit the invasion of bacterial strains, REF cells were pretreated with Au, ZnO, Cur, Au/ZnO NPs and Cur-Au/ZnO for 1 h and then infected with bacterial strains for 3 h. The cell invasion of bacterial strains in the presence and absence of Au, ZnO, Cur, Au/ZnO NPs and Cur-Au/ZnO CSNPs was significantly decreased as shown in Fig. 10. Taken together, these results of present study demonstrated that the Au, ZnO, Cur, Au/ZnO NPs and Cur-Au/ZnO mediate the adherence and invasion of bacterial strain in REF cells.
3.6. Nanoparticles blocks S. aureus α-Hemolysin production
As shown in Fig. 11, treatment with Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs attenuated the α-Hemolysin activity. Western blot analysis was performed to verify whether the decreased hemolytic activities of S. aureus. The results showed the ability of prepared Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs in reduction of production of α-Hemolysin.
3.7. Nanoparticles prevents S. aureus mediated alveolar epithelial cell injury
The ability of prepared Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs to prevent α-Hemolysin-mediated alveolar epithelial cell injury was tested using co-culture system. As in Fig. 12, upon co-culturing with S. aureus, cell death was evident, as indicated by an increase in the number of orange-red fluorescent dead cells and a transform in the cellular morphology of the live cells. Remarkably, after added of Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs in the co-culture system conferred a robust protection against S. aureus-mediated cell injury. Furthermore, to measure the ability of Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs to inhibits the effect of S. aureus on A549 cells. LDH release from A459 was tested using a LDH release assay, and the results are showed as percentage of cell death Fig. 13. The results showed the ability of Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs to reduce the percentage of LDH release when added to the co cultures of A549 cells and S. aureus.
3.8. Cur-Au/ZnO CSNPs improves lung injury in S. aureus
In the present study, we investigated the in vivo effects on S. aureus in a mouse model, the lung tissue of mice that infected with S.aureus then received PBS was kermesinus and had a firm texture as in shown in Fig. 14-B, the mice that were treated with PBS typically revealed that significant alveolar destruction had occurred along with infiltration of large numbers of inflammatory cells. Notably, the alveoli were open and contained no large areas of inflammation, although occasionally small areas of congestion were observed in Au NPs, ZnO NPs, Cur NPs, Au/ZnO NPs, and Cur-Au/ZnO CSNPs -treated mice as shown in Fig. 14 (C, D, E, F, and G).