Synthesis of Mixed Metal Oxides Nano-Colloidal Particles and Investigation of the Cytotoxicity Effects on the Human Pulmonary Cell Lines: A Prospective Approach in Anti-Tuberculosis Inhaled Nanoparticles

Alireza Jafari¹, Sharmin Kharazi², Nader Mosavari³, Farahnaz Movahedzadeh^4,5, Majid Tebyaniyan⁶, Saeedeh Jafari Nodooshan⁷, Ali Majidpour^1,8 and Tahereh Mosavi¹

¹Antimicrobial Resistance Research Center, Rasoul-e-Akram Hospital, Iran University of Medical Sciences, Tehran, Iran.

²Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

³Reference Laboratory for Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization, Tehran, Iran.

⁴Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, United States of America.

⁵Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy University of Illinois at Chicago, Chicago, Illinois, United States of America.

⁶Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.

⁷Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

⁸Department of Infection Disease, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Corresponding Author E-mail: Taherehtavakol@gmail.com

DOI : http://dx.doi.org/10.13005/ojc/330358

In today's world, Nano-science in association with synthesis of effective anti-tuberculosis nanoparticles (NPs) is expanding day by day. Also, personalized medicine in the field of Mycobacteriology; will play a significant role in the diagnosis, control, and even treatment of infectious diseases, especially tuberculosis in the next future. In this study, the colloidal Ag & ZnO NPs were synthesized with chemical reduction and deposition method. The SEM, TEM, DLS, UV-visible, and ICP-MS were used to identify of characteristics, structure, and estimate of NPS size, as well as the dispersion. Next, NPs were mixed together with 2_Ag:8_ZnO, 5_Ag:5_ZnO and 8_Ag:2_ZnO ratios. The toxicity effects of different ratios of NPs in contrast to human macrophage (THP-1) and normal human lung fibroblast (MRC-5) cell lines were evaluated using MTT method. The average size of NPs were estimated about 13±3.14 nm and 4±0.88 nm, respectively. The NPs were spherical in shape with a smooth surface morphology, as well as agglomerates. The results of UV- visible spectrums shows two peak in 420 nm and 350 nm for NPs, respectively. The intensity distribution spectrum indicate that mid-range of hydrodynamic diameter of the NPs were about 28.91 nm and 57 nm. The PDI and zeta potential of NPs were 1.00 and 0.545, also -20.7 mV and 3.13 mV, respectively. The results showed that the 5_Ag:5_ZnO ratio of mixed NPs in the dilution of 1:64 0.663 ppm), did not have any toxicity effects against THP-1 and MRC-5 cells. The ZnO NPs have had strongly toxic effects on THP-1 and MRC-5.  Also, in the dilution of 1:64 of Ag NPs ( 0.390 ppm), we did not find any evidence case of toxicity against normal lung and THP-1 cell lines. We are looking for exclusive ratio : dilution of NPs that used in the next future as a safety anti-tuberculosis agent in the personalized medicine and treatment process of respiratory disease; spatially “tuberculosis”. We discovered, if the NPs are mixed with a ratio of 5:5 in dilution of 1:64 0.663 ppm) of initial concentration, we will not see any toxic effects on the human respiratory cells.

Mixed Nano-colloidal metal oxides; Cytotoxicity; THP-1; MRC-5; Anti-tuberculosis; Personalized medicine

The Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

The morphology of Ag & ZnO NPs were analyzed using scanning electron microscopy (SEM) (Hitachi, S4169 Japon). The NPs suspensions were diluted in distilled water and dropped onto copper stubs. After air drying, particles were coated with a thin layer of gold under vacuum to make them conductive. Surface was scanned and photographs were taken at a 10 kV acceleratingvoltage^27-28. The Transmission electron microscopy(TEM)is one of the highest resolution analysis methods of materials. The three dimensional recognition of the materials is difficult by TEM because the observation data is a projection images through the materials²⁹. For morphological assessment, one drop was placed on a carbon coated copper grid and the excess amount was removed with a piece of filter paper. A drop of aqueous solution of Phosphortungistic acid (H₃PW₁₂O₄₀) (MERCK, Germany) was added onto the grid and staining was allowed for 5 min at room temperature. The prepared sample was then evaluated by TEM using a CM 120 (Philips, Germany) instrument at 200KV³⁰.

The Preparing of Different Ratios of Colloidal Ag & ZnO NPs

To prepare the mixture of 2_Ag:8_ZnO, 5_Ag:5_ZnOand 8_Ag:2_ZnO NPs, Ag & ZnO NPs, dispersed in a cold ultrasonic bath to 25^oC for 30s,before used. For the preparation of 10 ml of8_ZnO: 2_AgNPs ratios, 2 ml of Ag NPs with a value of 8 ml of ZnO NPs were mixed in a sterile Falcon tube. Also, to prepare 10 ml mixture 5_ZnO:5_Ag of Ag & ZnO NPs, 5 ml of Ag NPs with 5 ml of ZnO NPs were mixed. The 2_ZnO: 8_Agratios of NPs was performed also according to the table I. Also, The approximate concentration of Ag NPs, ZnO NPs and 8_ZnO:2_Ag, 5_ZnO:5_Ag and 2_ZnO:8_Ag mixed NPs in different dilution in a final volume of 1 ml calculated in table II.

Table 1: The approximate concentration of Ag NPs, ZnO NPs and 8_ZnO:2_Ag, 5_ZnO:5_Ag and 2_ZnO:8_Ag mixed NPs in a final volume of 10 ml.

Table 2: The approximate concentration of Ag NPs, ZnO NPs and 2Ag:8ZnO, 5Ag:5ZnO and 8Ag:2ZnO mixed NPs in different dilution in a final volume of 1 ml. Click here to View table

The Preparation of Different Ratios: Dilution of Mixed Nps in Cell Culture Medium

Firstly, about 11 Falcon tubes were prepared containing 10 ml sterile Gibco RPMI 1640 medium for serial dilution of the NPs in cell culture medium. Subsequently, all of the falcon tubes were numbered and was floated in cold ultrasonic baths at room temperature for 30s. Next, 10 ml (about 200 ppm) of the Ag NPs was added to the tube (number 1). At this time, the concentration of silver NPs in first falcon tubes (1) was reduced to the half of initial concentration. After Pipetting, about 10 ml of number 1 falcon tube was added to the second falcon tube (number 2). Continuously, serial dilution of NPs was continued until the last falcon tube (number 9) and also, variety of obtained concentrations for each dilution was approximately calculated. Separately, the serial dilution of the ZnO NPs, 2_Ag:8_ZnO, 5_Ag:5_ZnO, and 8_Ag:2_ZnO of mixed NPs were performed. The last two Falcon tubes (number 10 & 11) did not have any NPs. They were containing only RPMI and were selected as controls. Also, Dilution for different ratios of nanoparticles in a medium (DMEM-F12) medium (Sigma-Aldrich) for MRC-5 cell line was carried out with the same method.

The THP-1 and MRC-5 Cell Cultures

The THP-1and MRC-5 cell lines that had been obtained from cell bank of the Pasteur Institute of Iran (IPI). The THP-1had been derived from the peripheral blood of a 1-year-old male with acute monocyte leukemia. These cells also were phagocytic. Also, the MRC-5 cell line had been derived from normal lung tissue of a 14-week-old male fetus. The THP-1 cells were seeded in GibcoRPMI-1640 medium with Gibco FBS 10% and the penicillin 100 UNIT.ml-1 (Sigma-Aldrich) and the streptomycin 100 mg.ml-1 (Sigma-Aldrich), at 37°C and CO2 5%. When the cell density reached to 80%, about 104cell/well of these cells were seeded in 96-well plates. In addition, the MRC-5 cells were seeded in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) medium (Sigma-Aldrich) and Gibco FBS 10%.

MTT Assay

The effect of Ag NPs, ZnO NPs and 8_ZnO:2_Ag, 5_ZnO:5_Ag and 2_ZnO:8_Agofmixed NPs on the viability of a Human Monocyte (THP-1) and Normal Lung (MRC-5) cell lines in 24h were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The cells (10⁴_cell/well) were seeded in GibcoRPMI-1640 medium(200 ml per well) in 96-well plates and incubated at 37 ^oC in 5% CO₂for 24 h, to induce cell adherence. Next, dilution of 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64 of each of the NPs were prepared. Separately, the cells were treated with 100 ml of freshly prepared NPs (Ag, ZnO and 2_Ag:8_ZnO, 5_Ag:5_ZnO, and 8_Ag:2_ZnO mixed NPs) and further incubated at 37 ^oC in 5% CO₂ for 48 h. For the MTT assay, 20ml of MTT solution (5 mg.mL^-1 in incomplete medium) was added and incubated for 4–6 h. Next, 100 ml DMSO was added and the plates put on a shaker for 5 minutes. Absorbance was measured at 560 nm using a microtiter plate reader (Biotech, USA). The absorbance of the test group (treated cells) and control group (untreated cells) was used for the determination of cell viability percentage³¹.Cell survival in control cells was assumed to be100%. The percentage of cells viability were calculated by using the following equation:

Statistical Analysis

Statistical analyses were done using non-parametric form of variance, kruskalwall is tests and non-parametric form of t-test, Mann whitney u test (u-test) as appropriate. Longitudinal comparisons between the groups during the intervention were performed. Results were expressed as mean ± standard deviation of the mean (SD). The statistical significance threshold was defined as P-value≤ 0.05.The amount of the IC₅₀ and EC₅₀ was estimated  through Graph pad prisim (version 6).All of charts were done by Microsoft office Excel software (Version 2010).

Results

Characterization of NPs

The UV-Visible Spectroscopic

In order to prove the existence of Ag & ZnO NPs, UV-visible spectrum was prepared. One of the interesting characteristics of metal NPs is their optical properties, which varies according to the size and shape of NPs. Surface Plasmon resonance of metallic NPs is responsible for the unique optical properties of them. These characteristics will vary with the size, shape, spacing from each other and the refractive index of the surrounding NPs³². Figure I shows that absorption of Ag & ZnO NPs were between 200 nm and 800 nm. Characteristic absorption of surface Plasmon resonance band was occurred at 420 nm and 350 nm, respectively.

Figure 1: The UV-visible absorbance spectrums of Ag (Blue) & ZnO (Red) NPs from 200 nm to 800 nm. Inset shows the derivative of the absorbance spectrum. Click here to View figure

The Dynamic Light Scattering (DLS), Polydispersity Index(PDI), Zeta Potential

The Dynamic Light Scattering (DLS) was done using Stereoscope-IN (A-ONE Enc., Korea). The results of intensity distribution spectrum indicate that mid-range of hydrodynamic diameter of the Ag & ZnO NPs colloids were about 24.4 nm and 257 nm, respectively²³.

The Polydispersity Index (PDI) is a measure of the size distribution of NPs. Accordingly, the PDI of Ag & ZnO NPs were estimated about 1.00 and 0.545, respectively. Results showed that the scattering of particles was mono-dispersed. In other words, indicated that the NPs population was homogeneous. Also, Zeta potential of NPs were -20.7 mV and 3.13 mV, respectively. A high zeta potential helps the NPs to repel each other, which ensures long-term stability and inhibitsparticleaggregation³¹.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

The ICP-MS with different properties such as simultaneous multi-element analysis, low detection limit, and linear of the range most appropriate method for the measurement of inorganic nanoparticles such as NPs. In this test, the concentrations of 53 elements were analyzed. According to the recorded results, the amount of Ag concentration in colloidal solution was about 25 ppm and also the concentration of ZnO NPs was shown around 55 ppm^33-34.

The Scanning Electron Microscopy (SEM) and Transmission electron microscopy (TEM)

The shapes and surface morphologies of these NPs were evaluated by SEM. The particles were spherical with a smooth exterior, as shown in Figure II. The use of this technique is not very accurate, there are some deficiencies. It seems that the particle size is larger than the actual size. Also, the SEM just takes photo from adhering NPs on the sample surface. Due to the extra small size of NPs on the surface, they are agglomerated. Figure II shows the SEM image of Ag & ZnO NPs. Distribution of particle size was done with a 60 K magnification on a scale of 500 nm (a) and 750 nm (b). The TEM images of the prepared Ag and ZnO NPs are shown in Figure III. The NPs were spherical in shape with a smooth surface morphology, as well as agglomerates. Also, TEM image also shows that the produced NPs are more or less uniform in size and shape³⁴.The average size of Ag & ZnO NPs were estimated with Digimizer software (v4.1.1.0) at about 13±3.14 nm and 4±0.88 nm, respectively.

Figure 2: The SEM images of Ag (a) & ZnO (b) NPs ,distribution of particle size was done with a 60 K magnification on a scale of 500 nm (a) and 750 nm (b). Click here to View figure

Figure 3: The aggregated Ag NPs (a) 460 K and ZnO NPs (b) 200 K magnification TEM images, on a scale of 20 nm and 50 nm, respectively. Click here to View figure

MTT Assay

Cytotoxicity tests on 10 experimental groups, as well as a control group and a total of 60 tests in six different concentrations, each in three separate wells was performed(Figure IV, V and Table III, IV). Based on results mentioned, more than 50 % of THP-1 cells at a dilution of 1:8 of Ag NPs ( 3.125 ppm) in compared to the control group, were alive. When the concentration of NPs was halved, we found that cell viability increased too. So that, after dilution of 1:32 ( 0.781 ppm), about 100% of human macrophage cells were still alive. This despite the fact that, the statistical results showed that the Ag NPs at all dilutions were toxic against THP-1 cells (P-value ≤ 0.05). Interestingly, the ZnO NPs showed different behavior in front of THP-1cells and at all dilutions had toxicity effects against macrophage cells (P-value ≤ 0.05). Mixing of Ag & ZnO NPs with a ratio of 2_Ag:8_ZnO did not change circumstances. For this reason until dilution of 1:16 ( 3.312 ppm), less than 50% of THP-1 cells were alive. Anyway, after dilution of 1:32 ( 1.656 ppm), almost all of THP-1cells were alive. However, the P-value rang in both dilutions of 1:32 and 1:64 ( 0.828 ppm) were non-significant. This means that in these two dilutions, mixed NPs were non-toxic against macrophage cells (P-value ≥ 0.05). Mixing Ag & ZnO nanoparticles with a ratio of 2_Ag:8_ZnO, only at 1:32 and 1:64 dilutions showed the lowest percentage of cell viability (Up to 50%). The statistical results showed too that in 1:32 and 1:64 we have found insignificant value for P-value (P-value ≥ 0.05) that firmly say we do not have any toxicity against THP-1 cell lines. In the ratio of 5_ZnO:5_Ag , we had more chance. This means that at all dilutions more than 50% of cells survived. So that at dilutions of 1:16 ( 2.656 ppm), 1:32 ( 1.327 ppm), and 1:64 ( 0.663 ppm), more than 80 % of THP-1 cells compared to control group were survived (Figure IV). The statistical results showed too that at 1:32 and 1:64 dilutions of 5_Ag:5_ZnO we have found an insignificant value in P-value (P-value ≥ 0.05) that clearly says that we do not have any toxicity against THP-1 cell line, this could be optimistic. In other experiments, Ag NPs & ZnO NPs with the ratio of 8_Ag:2_ZnO were tested. In this case, the percentage of cell viability after dilution of 1:8 ( 4 ppm) increased, gradually. Besides, percentage of living cells at 1:32 (1 ppm) dilution reach to 100 %. Apparently, normal human lung cells (MRC-5) showed more sensitive to NPs (Figure V). About 70% of MRC-5 cells at 1:16 ( 1.562 ppm) dilutions of Ag NPs, were alive. This amount following of the reducing the concentration of Ag NPs is increased, gradually. Until the 1:64 ( 0.390 ppm) dilution, the percentage of cell viability compared to the control group reach to 97 %.The investigation shows that, cell viability of MRC-5 in contrast to ZnO NPs and the ratio of 8_ZnO:2_AgNPs were less than 50% in the all of dilutions. The statistical results showed too that in all of dilutions of 5_Ag:5_ZnO we have found a significant value in P-value (P-value ≥ 0.05).Undoubtedly, we can say that they have toxicity against THP-1 cell line, in all over the dilution. In addition, only at 1:64dilution of ratios 5_Ag:5_ZnONPs ( 0.663 ppm) and 8_Ag:2_ZnO NPs ( 0.5 ppm), the percentage of cell viability reach to more than 50%. Also, statistical data showed that does not exist a significant relationship between MTT results of THP-1 and MRC-5 cells after 24 hours and 48 hours. The MRC-F cells shows the most senility to the nanoparticles comparison to THP-1. So that, almost the all of the ratios: dilutions of NPs in statistic tars in spas indicated a significant value in compare to control.  Apart from Ag dilutions 1:64 ( 0.390 ppm),the ratio of 5_Ag:5_ZnO that were significand and safe, it means that all of this ratios:dilutions are toxic against MRC-5. (P-value ≤ 0.05).

Figure 4: Percentage of Survival fraction (SF) of THP-1 cell lines in contraction with different ratio : dilution of Nanoparticles   Click here to View figure

Table 3: The mean, standard deviation, percentage of Survival fraction (SF) & Growth inhibitory (GI) of different (ratios:dilution) of NPs in contraction with THP-1 cells after 24 hours follow-up time. (**P.value≤0.05 are representative of significant values and ***P.value≥0.05 are indicative of non-significant values). Click here to View table

Table 4: The mean, standard deviation, percentage of Survival fraction (SF) & Growth inhibitory (GI) of different (ratios / dilution) of NPs in contraction with MRC-5 cells after 24 hours follow-up time. (**P.value ≤ 0.05 are representative of significant values and ***P.value ≥ 0.05 are indicative of non-significant values).  Click here to View table

Figure 5: Percentage of Survival fraction (SF) of MRC-5 cell lines in contraction with different ratio / dilution of Nanoparticles Click here to View figure

Figure 6: Normal lung fibroblasts cell line (MRC-5) treated with a dilution of 1:32 (a) and 1:64 (b) of mixture 5Ag:5ZnO NPs and control group (c) after 24h . (labomed TCM 400 invert microscopy, U.S, magnification 100×). Click here to View figure

Human macrophage cell line (THP-1) treated with a dilution 1:32 (d) and 1:64 (f) of mixture 5_Ag:5_ZnO NPs and control group (g) after 24h. (labomed TCM 400 invert microscopy,

U.S, magnification 200×)

Table 5: The IC₅₀ and EC₅₀ values of Ag, ZnO, 2_Ag8_ZnO, 5_Ag5_ZnO and 8_Ag2_ZnO NPs in contraction with THP-1 and MRC-5 cell lines after 24 hours follow-up time. The IC₅₀ is the concentration of an inhibitor where response (or binding is reduced by half) and The EC₅₀ is the concentration of drug that gives half-maximal response.

Discussion

At the 65th World Health Assembly in 2012, Member States, called upon WHO to develop a new post- 2015 TB strategy and targets. It is estimated that there are more than 8.7 million cases of TB in the world. More than 1.4 million of them, lose the chance to live and die. Besides, up to 0.5 million people suffer from drug resistant Mycobacterium tuberculosis (MDR) and also 150,000 of them lose their lives³⁵. Hence, governments and organizations are always looking for drugs, novel anti-mycobacterial agents and new strategies to eliminate TB from human communities³⁶.So it sounds as if combination of nanotechnology and personalized medicine can play a vital role in achieving this goal^37-38. It was not long ago that we understand some metal oxide nanoparticles show amazing antibacterial properties against various bacteria, but there has always been concern about cytotoxicity effects of nanoparticles against human cells^39-40.

In this study, we synthesized colloidal Ag & ZnO NPs by chemical deposition and chemical reduction, also compared the cytotoxicity properties against normal pulmonary cell lines at different ratios/dilutions or even individually. In fact, we have acquired the lowest concentration of ratios: dilutions nanoparticles whose they had the lowest cytotoxicity against both THP-1 and MRC-5 cell lines.

The results show that the physicochemical properties of nanoparticles were consistent with other researches. So that the UV-visible peaks for Ag & ZnO NPs were 420 nm and 350 nm, respectably^41-43. UV-Visible spectroscopy is significant method which gives the preliminary confirmation of nanoparticles. Drzewiecka and their colleaqes synthesized colloidal Ag NPs in 2014 and showed the absorbance peak of Ag NPs were at 420 nm, which is characteristic feature of Ag NPs⁴⁴.In addition, to determine the stability of synthesized Ag NPs, they were measured the zeta potential that was -25.2 mV.These results consistent with our zeta potential values (-20.7 mV) in this study too, which indicates that the synthesized NPs were moderately stable⁴⁴.They believed that negative zeta potential value of the particles might be due to adsorption of OH^– ions. Since the adsorption of OH^–ions on Ag NPs increase the zeta value of NPs, there is an increase in stability of NPs due to electrostatic repulsion among the negative charges. Gurunathan also argues that an OH^– ion helps in preventing the aggregate formation and maintains the smaller size of Ag NPs⁴⁵. Also, the images of TEM and SEM revealed that the Ag & ZnO NPs were poly-dispersed and spherical in shape. A large number of studies give insights regarding the cytotoxicity effects excited by metal oxide NPs⁴⁶.The cytotoxicity of Ag NPs has been intercommunicate with generation of reactive oxygen species. It is undeniable that several studies suggest that Ag NPs is only toxic when oxidized to silver ions, Ag⁺⁴⁷.Asha Rani investigated the cytotoxic effect of Ag NPs on human cells. Their results illustrate that mitochondrial function as well as induction of reactive oxygen species (ROS) by Ag NPs which result in DNA damage and chromosomal aberrations^48-49.The P-value in concentration of 1:32 (0.781 ppm) and 1:64 (0.390 ppm) was no significant. Thus,  we can say that in these concentrations the Ag NPs do not have devastating impact  on human macrophage cells. Whereas only at dilution 1:64 of Ag NPs, normal lung fibroblasts cells exhibited this behavior. The P-value of statistical data, in concentration 1:64 was more than 0.05 and showed no a significant relationship. The results demonstrated that these nanoparticles were non-toxic to MRC-5 cells. Recent studies have shown that some NP made of metal oxides, such as ZnO NPs, have selective toxicity to bacteria but exhibit minimal effect on human cells⁵⁰. Even Reddy in the studies on both prokaryotic and eukaryotic cells indicated that zinc oxide nanoparticles have selective toxicity. Although, zinc oxide nanoparticles kill E. coli bacteria, are ineffective on human T lymphocytes⁵¹. Also, Sophie Lanone and their colleagues in 2009 at the University of Paris examined the cytotoxic effects of ZnO NPs in human alveolar epithelial and macrophage cell lines. The use of MTT assay on THP-1 cells indicated that the ZnO NPs is toxic⁵².Our research showed that zinc oxide nanoparticles have considerable toxicity to human macrophage cells and lung fibroblasts cells. So that the statistical data illustrated that, in all measured dilutions, the P-value was less than 0.05 and the toxicity seen. Generally speaking, the cell viability of THP-1 and MRC-5 cell lines, after 24 hours of incubation with ZnO NPs, measured by MTT assay designated more than 44.56% and 25.35%, survive in dilution of 1:16, respectively. This amount was amplified by decreasing of the concentration of NPs in dilution of 1:64. In other hand, it increased to 62.5% and 42.85%, respectively. In fact, the percentage of living cells, at a dilution of 1: 64, increased to 62.5% and 42.85% by reducing the concentration of nanoparticles.

According to Olesja Bondarenko researches, the reason for candidating the Ag & ZnO NPs, in this study is the metallic elemental composition of these two particles. Also, all the NPs are practical to fight the unfavorable growth of pathogenic microorganisms. Although among the two nanomaterials, Ag NPs are used most widely as antimicrobials, also ZnO NPs have been successfully used as biocides⁵⁰.The next case is their negative surface charge, which results from oxygen atoms in ZnO⁵³.They proclaimed that Ag NPs do not contain oxygen, the surface of metallic Ag NPs is oxidized under most environmental conditions (aerobic) and negatively charged hydroxo and Oxo groups cause the negative surface charge of the particle⁵⁴.In terms of toxicologically, perhaps the most important share property is that all the two NPs are soluble to some extent in aqueous media. They have previously shown that the solubility of Ag & ZnO NPs is the interestingly enough issue in the toxicity of metal-containing nanoparticles⁵⁵.Nonetheless, Casals and their colleagues firmly believe that solubility of NPs and the behavior of released metal ions, the proportion of intact particles, metal ions and metal complexes, depend greatly on the properties of the test environment⁵⁶.It has been also emphasized that when Ag & ZnO NPs were mixed together, the antibacterial properties of NPs incredibly increased, ^57-58 and represent the synergistic properties.

In current study, Salah and colleagues at 2013,researched on the inhibitory growth effects of lung fibroblasts (MRC-5) and THP-1cells in exposure on chitin, chitosan and low molecular weight chitin. The results suggest that low molecular chitin was highly suppressive. The lowest IC₅₀ value was 1 µg cm⁻³ for low molecular weight chitin⁵⁹.By mixing Ag & ZnO with different ratios: dilution together, their cellular cytotoxicity properties against lung cells are also changed.

Also, lung fibroblast cells compared to THP-1 cells represented more susceptible to metal oxide NPs. When Ag:ZnO NPs at a ratio of 2: 8 were mixed together, to achieve a 1:64 dilution, only 27.69% of MRC-5 cells were alive. Which was a sign of the toxicity effects of NPs on these cells. But THP-1 cells showed more differentiated behavior. In fact, dilutions of 1:32 and 1:64 of the NPs on human macrophage cells were non-toxic. But, the data which has obtained in this study, rejected the theory of using the ratio of 2:8 of  Ag:ZnO NPs in body. The results of mixing of Ag:ZnO NPs at a ratio of 8:2 was also significant . Almost at dilutions of 1:32 ( 1 ppm) and 1:64 ( 0.5 ppm) More than 90% of THP-1 cells were alive. While the MRC-5 cells showed stricter  behavior, in concentration of 1 ppm , about 60% of cells had survived in culture medium. As well as statistical data significantly analyzed, all the studied concentrations.

The statistical data indicates that mixed of 8: 2 NPs, is not yet an appropriate option for the treatment of pulmonary infectious diseases (P-value ≥0.05). By mixing ratio of 5_Ag:5_ZnONPs, less cellular cytotoxicity was also reported. So that the statistical data showed that almost 80% of lung fibroblast cells  which were treated with the NPs were alive at dilution of 1:64  (P-value ≤ 0.05).

In contrast,  about 100% of the human macrophage cells at dilutions of 1:32 and 1:64, were quite alive and active. (P-value ≤ 0.05). This represented, using a dilution of  1:64 to concentration of 0.663 ppm mixture of Ag:ZnO NPs at a ratio of 5:5 can be a proper option for future research on the effectiveness of non-toxic metal oxide NPs on infectious diseases which involving the lungs and respiratory system. However, Stebounova verdict that inhalation is considered the most important route of exposure for nanoparticles with bacteria ⁴⁷.

Conclusion

By mixing metal nanoparticles together, can reduce the risks of cellular toxicity. By following of these studies, may imagine  the treatment of pulmonary infectious diseases of the human body by Nanotechnologies for each person in the near future.

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