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Article no: LIPI 487489
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The eradication of harmful agents such as bacteria, mold, yeast, and virus is one of the most challenging problems which human has been faced with it. Therefore, numer- ous natural and inorganic antimicrobial agents with dif- ferent properties have been used to destroy them.(1)
Silver and silver ions are known for excellent anti- bacterial activities, although the mechanism is not well-understood. Silver and its by-products have been employed in different applications such as eye drops to prevent Gonococcal ophthalmia neonatorum infection and also preparing potable water and healing fresh burns for a long time.(2) The resurgence in the use of silver-based antimicrobial agents may be linked to broad-spectrum activity and lower propensity to induce microbial resist- ance than antibiotics.(3–5) Moreover, there is an increased
demand for finding ways to formulate new types of safe and cost-effective biocidal materials. Hence, nanoscale materials (less than 100 nm) have emerged up due to their high surface area to volume ratio and exclusive chemi- cal and physical properties.(5,6) Considering their unique properties in inhibiting the growth of wide variety of microorganisms, they could be applied in different fields such as wound dressings, contraceptive devices, surgical instruments and bone prostheses likewise room spays, laundry detergents, water purificants and wall paints. As a matter of fact, nanosilver (nano-Ag) is employed widely in the referred applications. In addition, there are textiles like underwear and socks in the market which contain nano-Ag and it is even common to trace nano-Ag in washing machines nowadays.(4)
The toxicity of different sizes of silver nanoparticles on rat liver cell line (BRL 3A), keratinocytes fibroblast
Address for Correspondence: Tooba Ghazanfari, Department of Immunology, Medical Faculty, Shahed University, Tehran, Iran. E-mail: tghazanfari@yahoo.
com and [email protected]
R E S E A R C H A R T I C L E
In vitro toxicity of silver nanoparticles on murine peritoneal macrophages
Zeinab Shavandi
1,2, Tooba Ghazanfari
1,3, and kiumarz Nazari Moghaddam
41Immunoregulation Research Center, Shahed University, Tehran, Iran, 2Department of Biology, Shahed University, Tehran, Iran, 3Department of Immunology, Shahed University, Tehran, Iran, and 4Department of Endodontics, Faculty of Dentistry, Shahed University, Tehran, Iran
Abstract
Silver nanoparticles, the new generation of antimicrobial agents, are becoming one of the progressively growing products in nanotechnology. Meanwhile, the potential side effects of these nanoparticles have not been studied thoroughly yet. Macrophages, one of the most important immune cells in innate and acquired immune responses, are a key component of the clearance mechanisms in semi-open interfaces of human body against nanosilver (nano-Ag). In this experimental study, we assessed the effect of commercial col- loidal nano-Ag on murine peritoneal macrophages by MTT and nitric oxide (NO) production assay in vitro.
A significant decrease in cell viability was observed for 1 ppm (part per million) to 25 ppm of nano-Ag con- centrations compared to the control group (P < 0.01) after 24 h of cell culture. Also, a significant decrease in the cell viability was observed for 2–25 ppm of nano-Ag concentrations after 48–72 h, respectively (P < 0.05).
In our study, exposure to 0.4–25 ppm of colloidal nano-Ags brought about a considerable decline (P < 0.05) in NO production by macrophages. Nevertheless, there is no evidence of substantial difference at lower concentrations. This acute cytotoxic dose-dependent effect of nano-Ag particles on peritoneal macrophages which declines cell viability and NO production urges caution about the usage for sensitive surfaces. It is highly recommended to carry out the in vivo investigation for human to confirm its use.
Keywords: Nanoparticles; macrophages; in vitro; nitric oxide; cell viability
(Received 17 March 2010; revised 14 April 2010; accepted 19 April 2010) AQ2
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cultures, and a cell line with spermatogonial stem cell characteristics (C18-4 cell) have been studied and it was concluded that the cytotoxicity of nano-Ag to the mito- chondrial activity enhanced with the increase in the concentration of silver nanoparticles.(7,8)
As reviewed by Dobrovolskaia and McNeil, nano- materials interact with the immune system to elevate immunotoxicity.(9) Macrophages are notable for their capacity of phagocytosis which determines the fate of for- eign particles. The activation of macrophages can result in release of chemokines, cytokines, reactive oxygen spe- cies, and other mediators which initiate inflammation.
Furthermore, impairing the phagocytic function of mac- rophages lead to retention of particles in the organs and building up of a toxic dose.(6) Therefore, this study was carried out to determine the effect of commercial col- loidal nano-Ag (nanocid L2000®) containing 4000 ppm (part per million) nano-Ag particles with the size range of 18–34 nm on peritoneal macrophages. An attempt was made to study the cell viability of macrophages and elucidate the nitric oxide (NO) product changes in the supernatant of macrophages after exposure to different concentrations of nano-Ag.
Material and methods
Silver nanoparticles
Silver nanoparticles used in the present study were the commercial product Nanocid L2000 (supplied from Iran Nasb Niroo CO., Iranian Patent NO. 33064) containing 4000 ppm silver nanoparticles. They were used as col- loidal aqueous suspension and meanwhile, were found to retain their stability in the culture media. The N90% of cubic face centered nanoparticles was revealed to be in the size range of 18–34 nm by high resolution transmis- sion electron microscope. The data on particle size distri- bution measured by dynamic light scattering (Zetasizer, Malvern Instruments, UK) revealed the z-average size of 8.89 nm. The Zeta Potential of the nanoparticles was measured to be -33.5 mV which shows the moderate stability of nanoparticles.
Chemicals
RPMI 1640 medium (with l-glutamine) and penicillin–
streptomycin (1000 mg/mL) were procured from Sigma Chemical Company (St Louis, MO) whereas fetal bovine serum was obtained from Gibco BRL (Paisley, UK).
Moreover, 3-(4, 5-Dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) and all other analytical grade chemicals for biochemical studies were provided from Merk Chemical Company (Darnstadt, Germany).
The plasticware and glassware used for cell culturing
were also obtained from Nunc (Myriad industries, San Diego, CA).
Isolation, cell culture, and treatment of peritoneal macrophages
Six- to eight-week-old male Balb/C mice were prepared from animal laboratory of Shahed University. All ethical criteria included in Helsinki Declaration and all on the care and use of laboratory animals were observed in dif- ferent stages of the project. Mice were housed under nor- mal laboratory conditions. The mice were anesthetized with diethyl ether and the skin of chest and abdomen areas of mice were carefully dissected without opening the peritoneum. Peritoneal exudates cells were obtained from each mouse by lavage method (10) in which 5 mL of cold normal saline was injected twice intraperitoneally.
The cells were centrifuged, washed, and resuspended in RPMI medium. Cells were counted with neobar cytom- eter and dead cells were calculated using trypan blue which were less than 3% all the times. Then 4 × 105 cells/
well were cultured in 96-well microtiterplates (Falcon, San Francisco, CA) and incubated at 37°C under 5% CO2 atmosphere for 2 h. The nonadherent cells were removed by washing the plate with normal saline (37°C) and the adherent cells were treated with different concentrations of nano-Ag (25, 20, 15, 10, 4,2, 1, 0.5, 0.4, 0.2, 0.1, and 0.05 ppm). Cells were incubated for 24, 48, and 72 h. All procedures were conducted under aseptic condition.
Cell viability assay
MTT reduction assay was used for evaluating cells viabil- ity. In this assay, living cells reduce MTT tetrazolium salt into formazan crystals. At first, MTT (Merck, Germany) powder was dissolved in phosphate-buffered saline (5 mg/mL), filtered and stored at –20°C to be employed later in the process. Followed by the appropriate expo- sure of cells to nano-Ag, the cells were incubated for 4 h with MTT solution (MTT solution was added to each well at one-tenth of its volume, then the supernatants were gently removed after 4 h, and MTT formazan crys- tals were extracted in acidic isopropanol (0.04 M HCl in isopropanol)). Cell viability assessment or mitochondrial activity of living cells in all of the cultures was fulfilled by measuring the relative absorbance or optical density (OD). Measurement of OD was performed at 540 nm while using acidic isopropanol as blank reference.
NO production measurement
NO was assessed by measuring the nitrite concentration in supernatant of macrophages (Griess method) 16-h after culture. Briefly, 100 μL of supernatant from each well of microplate was transferred to a 96-well flat-bottom
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microtiter plate and 50 μL of 1% sulfanilamide (Fluka) solution and 50 μL of 0.1% N-(1-Naphtyl) ethylendi- amindihydrochloride (Merck, Germany) solution (both in 5% phosphoric acid) were added to each sample and all standards and were incubated together at room tem- perature for 10 min. The absorbencies were read at 540 nm and the amount of nitrite was calculated as µM.
Statistical analyses
Data were analyzed by Student’s unpaired T-test Differences which were considered significant at P <
0.05
Result
Macrophages cell viability 24 h after nano-Ag exposure Peritoneal macrophages were exposed to various concen- trations of nano-Ag and the cell viability was measured after 24 h. As showed in Figure 1, a significant decrease in cell viability was observed by doses of 25–1 ppm of nano-Ag concentrations compared to the control group (P < 0.01) as 25 ppm indicated 77.26% decrease in cell viability and 4, 2, 1 ppm sequentially indicated 50.92, 39.96, 38.44% decrease in cell viability. In addition, inverted microscopy images showed no MTT formazan crystals in macrophage cultures exposed to 25–4 ppm of nano-Ag concentrations. However, a few MTT formazan crystals were observed at 2 and 1 ppm (Figures 2 and 3).
The concentrations lower than 1ppm did not show any significant differences compared to the control group.
Macrophages cell viability 48 h after nano-Ag exposure The same experiment was carried out and the cell viabil- ity was measured 48 h after nano-Ag exposure. A sig- nificant decrease in cell viability was observed by doses of 25–2 ppm of nano-Ag concentrations compared to the control group (P < 0.05) (Figure 4) as 25 ppm indi- cated 72.9% decrease in cell viability and 10, 4, 2 ppm
sequentially indicated 73.11, 72.69, 30.07% decrease in cell viability. Furthermore, inverted microscopy images displayed no MTT formazan crystals in macrophage cul- tures exposed to 25–4 ppm of nano-Ag concentrations.
Nevertheless, a few MTT formazan crystals were observed at 2 ppm (images aren’t shown here). The concentrations lower than 2 ppm did not indicate any considerable dif- ferences compared to the control group.
140
** ** ** ** ** ** **
120 100 80
Cellular viability (%)
60 40 20
0 C 25 20 15 10 2
Nano-Ag concentrations (ppm)
1 0.5 0.4 0.2 0.10.05 4
Figure 1. Cell viability of macrophages 24 h after exposure to various nano-Ag concentrations. A significant decrease in cell viability was observed by doses of 25–1 ppm of nano-Ag concentrations compared to the control group as 25 ppm indicated 77.26% decrease in cell viabil- ity and 4, 2, 1 ppm sequentially indicated 50.92, 39.96, 38.44 decrease in cell viability.. The concentrations lower than 1 ppm has not shown any significant differences in cell viability compared to the control group. **Significant differences of P < 0.01 with control group. ppm, part per million.
A B
Figure 2. Inverted microscopy images of macrophage culture after exposure to (A) nano-Ag (25 ppm), (B) nano-Ag (4 ppm). There were not MTT formazan crystals in macrophage culture exposed to nano-Ag from 4 to 25 ppm. ppm, part per million
A B C D
Figure 3. Inverted microscopy images of macrophage culture after exposure to: (A) no nano-Ag (control), (B) nano-Ag (2 ppm), (C) nano-Ag (1 ppm), (D) nano-Ag (0.5 ppm). Few MTT formazan crystals were observed at 1 ppm and 2 ppm, but in lower concentrations MTT formazan crystals as same as control group. ppm, part per million.
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LIPI 487489
Macrophages cell viability 72 h after nano-Ag exposure The same experiment was carried out and the cell viability was measured 72 h after nano-Ag exposure. A substantial decline in the cell viability was observed by doses of 25–2 ppms of nano-Ag concentrations compared to the control group (P < 0.05) (Figure 5) as 25 ppm indicated 68.855%
decrease in cell viability and 10, 4, 2 ppm sequentially indicated 69.33, 61.3, 69.66 decrease in cell viability.
Moreover, inverted microscopy images showed no MTT formazan crystals in macrophage cultures exposed to 25–2 ppm of nano-Ag concentrations (images aren’t shown here). The concentrations lower than 2 ppm did not illustrate any significant differences compared to the control group.
NO production of macrophages
The effect of various nano-Ag concentrations on macro- phages NO production was also assessed in this study.
As shown in Figure 6, a significant decrease (P < 0.05) in macrophages NO production occurred following expo- sure to nano-Ag doses from 25 to 0.4 ppm as 25 ppm indicated 43.79% decrease in NO production and 1, 0.5, 0.4 ppm sequentially indicated 83.33, 75.82,77.78%
decrease in NO production. Although there was no sub- stantial difference at lower concentrations compared to the control.
Discussion
The results suggest that high doses of nano-Ag (more than 4 ppm) indicate extreme cytotoxicity effect on macrophages in all three periodic times. But nano-Ag medium doses (4–2 ppm) show moderate cytotoxicity
on macrophages 24 and 48 h after nano-Ag exposure and extreme cytotoxicity effect after 72 h nano-Ag exposure in vitro. The results also showed nano-Ag low doses (less than 2 ppm) do not bring about any cytotoxicity effect on macrophages. It is also shown that nano-Ag cytotoxicity effects on macrophages are dose and time-dependent.
This result is in agreement with the other similar nano- Ag-toxicity studies on different major cell types (3T3 fibroblast cells, BRL 3A rat liver cells, keratinocytes, pri- mary liver cells isolated from Swiss albino mice, neuro- nendocrine cell line (PC-12 cells), spermatogonial stem cell (C18-4 cell) and mast cells).(8,11–19) In all cases authors reported nano-Ag toxicity at high doses including cell viability decrease, cell morphology alteration, reduction of mitochondrial function, increased membrane leakage,
140
** ** ** ** **
* 120
100 80
Cellular viability (%)
60 40 20
0 C 25 20 15 10 2
Nano-Ag concentrations (ppm)
1 0.5 0.4 0.2 0.10.05 4
Figure 4. Cell viability of macrophages 48 h after exposure to various nano-Ag concentrations. A significant decrease in cell viability was observed by doses of 25–2 ppm of nano-Ag concentrations compared to the control group as 25 ppm indicated 72.9% decrease in cell viability and 10, 4, 2 ppm sequentially indicated 73.11, 72.69, 30.07% decrease in cell viability. The concentrations lower than 2 ppm do not show any significant differences in cell viability compared to the control group.
*Significant differences of P < 0.05 with control group. **Significant differences of P < 0.01 with control group. ppm, part per million.
140
** ** ** ** ** **
120 100 80
Cellular viability (%)
60 40 20
0 C 25 20 15 10 2
Nano-Ag concentrations (ppm)
1 0.5 0.4 0.2 0.10.05 4
Figure 5. Cell viability of macrophages 72 h after exposure to various nano-Ag concentrations. A significant decrease in cell viability was observed by doses of 25–2 ppm of nano-Ag concentrations compared to the control group) as 25 ppm indicated 68.855% decrease in cell viability and 10, 4, 2 ppm sequentially indicated 69.33, 61.3, 69.66%
decrease in cell viability. The concentrations lower than 2 ppm do not show any significant differences in cell viability compared to the con- trol group. **Significant differences with control group with P < 0.01.
ppm, part per million.
4
** ** ** ** ** ** ** ** **
3.5 3 2.5
Amount of nitrite (µM)
2 1.5 1 0.5
0 C 25 20 15 10 2
Colloidal Nano-A concentrations (ppm)
1 0.5 0.4 0.2 0.10.05 4
Figure 6. Nitric oxide (NO) production of macrophages exposed to various nano-Ag concentrations. A significant decrease in macro- phages NO production occurred following exposure to nano-Ag doses from 25–0.4 ppm as 25 ppm indicated 43.79% decrease in NO produc- tion and 1, 0.5, 0.4 ppm sequentially indicated 83.33, 75.82,77.78%
decrease in NO production. Although there were no significant dif- ferences at lower concentration compared to the control. **Significant differences with control group (P < 0.01). ppm, part per million.
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necrosis and induction of apoptosis, abnormal size of mitochondrial cells, cellular shrinkage and irregular shape. However, different techniques have been used for assessment of nano-Ag toxicity but in all, like this study, the most important challenge with this type of particles is the dependence of dose and the potential of high toxi- cological effect caused by heavy metal dissociation.(20) In addition, other nano-studies on macrophages demon- strate the same results.(4,21–28)
In this study a significant decrease (P < 0.05) in mac- rophages NO production occurred following exposure to nano-Ag doses more than 0.4 ppm but there was no significant difference at lower concentrations compared to the control group. This conclusion recommends that high doses lead to extreme macrophage death and sub- sequently, reduction in nano-Ag macrophage phagocy- tosis. Therefore, a decrease in NO production is occurred but at low doses, nano-Ag doesn’t have cytotoxic effect.
Consequently, NO production as well as cell viability does not show any difference compared to the control group. The comparable experiments revealed that, in part of them, NO production rises after exposure to nanoparticles whereas nanoparticles induce NO release and result in inflammation.(29–31) However, it seems that addition of mitogen such as linked polymer solution or polymer conjugated to nanoparticles causes macro- phage activation.(29,32) Nevertheless, in some studies the results show that nanoparticles are not cytotoxic and do not arouse macrophage NO production in the range of concentrations studied.(33) These results demonstrate that nano-Ag low doses do not impose any restriction while consumption of nano-Ag in high doses could be challenging.
Similar to this study Soto et al.(25,34) used MTT assay for cell viability measurement. Their investigation was mainly concentrated on the effect of constant nanoparticulate Ag (5 µg/mL) on murine alveolar macrophage cell line (RAW 264.7) and human macrophage (THB-1) as comparators in only one periodic time of 48 h. Considering the results of this study, the nanoparticulate Ag brings about cytox- icity effect on macrophages and cytotoxicity level was the same for both cell lines.
These results suggest that representative cytotoxic responses for humans might be gained by nano-Ag exposures to simple murine macrophage cell line assays.
Hence, our results could be generalized (extended) to human and subsequently can be discussed in human health field and due to the fact that macrophages can determine the foreign particles fate in every semi-opened interface of human body, these results, although per- formed in vitro, would provide a preliminary understand- ing of immune response overview for potential health effects of nano-Ag.
It has to be noted that in this study, most experiments have been carried out in vitro in which cells may behave
differently in comparison to in vivo conditions because in in vivo condition, nano-Ag can have interactions with complex mixture of compounds such as enzymes, microbial flora and other compounds which might change reactivity and toxicity of the particles(4) and can also protect single cells against hazard effect of nano-Ag which leads to a decline in their toxicity. Therefore, although this study has reported the commercial colloi- dal nano-Ag toxicity effect on peritoneal macrophages in vitro at high concentrations but there are no impli- cations of the health effects in humans. Undoubtedly, cautious measurements must be taken against usage of large concentrations of nano-Ag. Approaches to truths pertaining to complex nanosystems necessitate: (1) Founding the mechanism of nano-Ag toxicity exactly and comparison to silver in bulk. (2) Accomplishment of in vivo experiments and comparison their result with that of in vitro.
Acknowledgement
This work was supported by Shahed University research grant. Authors wish to thank Dr. Roya Yaraee for her help- ful discussion during the work.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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