Evaluation of genotoxicity and cytotoxicity of silver nanoparticles
Anastasia Evgenievna Pecherskaya1, Daria Vladimirovna Andreeva2, Kamilla Magomedovna Abdulazizova3, Fariza Magometovna Sampieva4, Madina Beslanovna Albogachieva4, Anna Garikovna Babayan5, Yana Ruslanovna Esenova6*, Anastasia Gennadievna Lubentseva4
1 State Budgetary Healthcare Institution "City Polyclinic No. 62", Moscow, Russia. 2Department of Therapy, Faculty of Medicine, Kursk State Medical University, Kursk, Russia. 3Department of Therapy, Faculty of Medicine, Institute of Clinical Medicine named after N.V. Sklifosovsky, First Moscow State Medical University named after I. M. Sechenov, Moscow, Russia. 4Department of Therapy, Faculty of Pediatrics, Rostov State Medical University, Rostov-on-Don, Russia. 5Department of Therapy, Faculty of Medicine, Stavropol State Medical University, Stavropol, Russia. 6Department of Therapy, Faculty of Medicine, North Ossetian State Medical Academy, Vladikavkaz, Republic of North Ossetia-Alania, Russia.
Correspondence: Yana Ruslanovna Esenova, Department of Therapy, Faculty of Medicine, North Ossetian State Medical Academy, Vladikavkaz, Republic of North Ossetia-Alania, Russia. [email protected]
ABSTRACT
The toxicological and hygienic assessment of the safety of nanoscale colloidal silver produced on a large scale by the modern nano industry is an important task because of its employment medicine. This work aimed to evaluate the mutagenic and cytotoxic properties of silver nanoparticles stabilized with polyvinylpyrrolidone. Silver nanoparticles stabilized with polyvinylpyrrolidone were synthesized for the experiment. The silver particles had a size of about 300 nm, but some of them turned out to be slightly smaller, about 80 nm. The absorption spectrum of UV/Vis electromagnetic waves displayed an absorption peak of 409 nm, which is typical for silver nanoparticles. The exposure time was 10 minutes. Control cultures were placed in saline without glutamate for 10 minutes. Cultures were then returned to the original nutrient medium and placed in a CO2 incubator for 4.5 hours. The excess of β-galactosidase activity over the control in PVP was 0.9±0.03 and 1.2±0.03, and in AgNPs+PVP – 0.6±0.38 and 0.6±0.12. MCF7-test showed that in the presence of silver nanoparticles, the cells acquired a spherical shape and did not show proliferation. AgNPs+PVP activated cell proliferation, which can enhance the wound healing effect.
Keywords: Silver nanoparticle, Polyvinylpyrrolidone, Surgery, Wound healing, Toxicity
Introduction
Modern developments of new methods of treatment of infected wounds and inflammatory skin diseases are devoted to the use of silver nanoparticles, which have several advantages: multilevel antimicrobial effect (antibacterial, antifungal, antiviral), slow development of resistance, low level of toxicity [1-3]. Recent literature data indicate that the wound-healing effect of drugs with silver nanoparticles is due not only to antibacterial but also to anti-inflammatory, reparative properties (regulating the activity of several anti-inflammatory cytokines) [4-6]. At the same time, along with antibacterial and anti-inflammatory activity, silver nanoparticles can also have a toxic effect, expressed, among other things, by genotoxicity and cytotoxicity [7].
Compared to macro-sized silver, silver nanoparticles can potentially exhibit much greater toxicity. The mechanism of the toxic effect may be associated with oxidative stress, mitochondrial dysfunction and increased membrane permeability [8]. Experimental data obtained by various authors on the toxicity of silver nanoparticles are quite contradictory. There is no explanation of how, what time and what concentration of silver nanoparticles were injected into animals. Thus, some authors claim that the LD50 for mice should be 3000 mg/kg, others – 11600 mg/kg, others – 4500 mg/kg [9]. A number of researchers note that the experimental animals accumulated silver nanoparticles in the tissues of the body when receiving it with drinking water at doses of 20-50 mg/l [10]. Experiments involving mammalian test organisms, although they are able to bring us closer to understanding the effects of nanoparticles on human health, are currently shrouded in disputes about the ethics of methods. Therefore, the advantage of using primary cell cultures and continuous cell cultures of higher vertebrate organisms in vitro is obvious. Thus, when studying the cytotoxic effect of silver nanoparticles on primary fish cell cultures (hepatocytes) and continuous fish cell cultures (RTL-W1, RTH-149, RTG-2), it turned out that the latter are very sensitive indicators of their toxicity [11]. A similar effect for the same particles was demonstrated in a study on human mesenchymal stem cells and peripheral blood mononuclear cells, where silver nanoparticles showed toxicity to all test objects in a narrow concentration range from 12.5 to 50 mg/l [12]. Another study revealed cytotoxic effects of silver nanoparticles in primary gill cells and hepatocytes of rainbow trout [13]. As a result, it was noted that the indication of the level of glutathione and ROS can act as biomarkers of the toxicity of silver nanoparticles.
In addition, according to a number of authors, silver nanoparticles may exhibit some degree of toxicity to all organisms and cell cultures. It was found that nanosilver has almost the same negative effect in the concentration range from 1 to 100 mg/l, regardless of the choice of the test object (viruses, bacteria, microalgae, fungi, animal and human cells) [14]. The noted effect is probably related to their ability to easily form ions. Nanoparticles and their ions activate cell signaling pathways, penetrate through the cell wall and membrane, change the permeability of the latter, damage membrane proteins, nucleic acids and enzymes, inducing the formation of ROS in the cell [11].
In this regard, the toxicological and hygienic assessment of the safety of nanoscale colloidal silver produced on a large scale by the modern nano industry is an important task due to its presence in a large number of consumer products (including medicines, foodstuffs, and biologically active food additives) and the possibility of contamination of various environmental objects with it [10-12].
The work aimed to evaluate the mutagenic and cytotoxic properties of silver nanoparticles stabilized with polyvinylpyrrolidone to argue in favor of the safety of its use.
Materials and Methods
Synthesis of silver nanoparticles
Silver nanoparticles were also obtained using the polyvinylpyrrolidone 10000 cationic surfactant (PVP). The synthesis was carried out in two stages. In the first stage, aqueous solutions of silver nitrate and PVP 10000 were prepared. In 25 ml of distilled water, silver nitrate was dissolved in an amount of 4.24 grams. Further, 2 grams of PVP 10000 were dissolved in 200 ml of distilled water. In the second stage, 1 ml of an aqueous solution of silver nitrate and 0.03 ml of hydrazine hydrate were dissolved in 200 ml of an aqueous solution of PVP. The prepared solution was stirred for 20 minutes at room temperature until the color changed from transparent to dark orange [15].
Characterization of silver nanoparticles
The morphology and size of the obtained nanoobjects were studied using a Sigma Zeiss scanning electron microscope (Germany) with SiO2 plates as a substrate. The optical properties of the obtained nanoobjects were studied by UV spectroscopy on a UV-2600 spectrometer (Shimadzu, Japan). Spectral range: 1400-220 nm; spectral resolution: 0.01 nm.
Strains used
Salmonella typhimurium TA98 (hisD3052, rfa, uvr-, bio-, pkm101) and Salmonella typhimurium TA1535/pSK1002 strains were used to test the mutagenicity of compounds. All bacterial strains were cultured in LB medium (trypton – 10 g/l; yeast extract – 5 g/l; NaCl – 5 g/l; pH 8.5) [16, 17].
Assessment of geno- and cytotoxicity
The mutagenicity of substances was evaluated in the Ames test. 5 ml of a night culture of Salmonella typhimurium TA98 was transferred to 20 ml LB with ampicillin (25 mcg/ml) and incubated at 37 ° C with a swing for 2 hours until the culture reached an exponential growth phase. The cells were then collected by centrifugation for 15 min at 4000 rpm. The precipitate was resuspended in a single solution of a salt base. The concentration of the silver nanoparticle solution was 1, 10, and 100 mg/ml, which exceeds the planned pharmaceutical concentration by 10 times. Sodium azide (NaN3) was used as a positive control. The tested substance was considered mutagenic if the number of revertant colonies in the experiment significantly exceeded that in the control (solvent) by more than 2 times. The DNA-damaging activity of the compounds was evaluated in an SOS chromatest on a strain of S. Typhimurium TA1535/pSK1002. The bacterial culture was diluted in LB medium 10 times. The resulting culture liquid was poured 3 ml into test tubes and grown for 4 hours in the presence of the studied compounds in the required concentrations. Then the cells were centrifuged and the activity of β-galactosidase in them was determined [18].
The cytotoxicity of the compounds was studied on the MCF7 line of human breast cancer cells. The cells were incubated in 96-well plates on DMEM medium supplemented with glutamine, FBS, penicillin, and streptomycin. Cell culture (4000 cells per well) was seeded into 96-well plastic plates (Cellstar Grenier bio-one No. 655 180) and the studied compounds were introduced. Then the cell culture was incubated for 24 hours at a temperature of 37℃ and an MTS test was performed. This test is based on the suppression of mitochondrial dehydrogenase activity in the presence of a toxic compound and the ability to oxidize the substrate MTS (3-(4,5-dimethylt-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) using phenazine methosulfate (PMS) as electron-binding reagent. MTS is reduced by cells to a colored soluble product that can be measured photometrically [19].
Next, 100 µl of PMS solution was added in 1 ml of MTS immediately before addition to the culturally liquid-containing cells. Carefully mixed and 10 µl of the combined solution of MTS and PMS was added to each well of a 96-well analytical tablet containing 50 µl of cells in a culture medium. The tablet was incubated for 1-4 hours at 37℃ in a thermostat with a content of 5% CO2 until a brown color was formed in the control wells.
Results and Discussion
Characterization of silver nanoparticles
The silver particles had a size of about 300 nm, but some of them turned out to be slightly smaller, about 80 nm (Figure 1a). The absorption spectrum of UV/Vis electromagnetic waves displays an absorption peak of 409 nm (Figure 1b), which is typical for silver nanoparticles [20-22]. The shape of the silver particles tends to be spherical.
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a) |
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b) |
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Figure 1. Characterization of silver nanoparticles: SEM micrographs (a) and absorption spectrum of UV-visible electromagnetic waves |
Thus, the synthesis of silver particles in water allowed us to obtain nanoparticles whose size is less than 10 nm, while the composition of the particles strongly depends on the purity of the reagents used. The described process does not lead to the formation of one-dimensional particles [23].
Evaluation of the genotoxicity of silver nanoparticles
It was found that in the Ames test, the excess of the number of revertants over the control at concentrations of 10 and 100 µg/ml in PVP was 0.5±0.03 and 0.4±0.06, and in AgNPs+PVP – 0.9±0.12 and 1.1±0.09, which indicates the absence of mutagenic action. None of the compounds showed an increase in the SOS response in the DNA-damaging test. The excess of β-galactosidase activity over the control in PVP was 0.9±0.03 and 1.2±0.03, and in AgNPs+PVP – 0.6±0.38 and 0.6±0.12.
Evaluation of cytotoxicity of silver nanoparticles
The results of the MTS test are shown in Table 1. None of the compounds reduced the activity of mitochondrial dehydrogenase, which indicates the absence of their cytotoxicity. The data obtained are consistent with the results of Alsaleh et al. [24] and Tiwari [25].
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Table 1. Results of cytotoxicity study |
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Substance |
Cytotoxicity, % |
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1 mcg/ml |
10 mcg/ml |
100 mcg/ml |
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PVP |
105±14 |
131±14 |
127±19 |
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AgNPs+PVP |
135±15 |
97±14 |
119±21 |
In addition to the MTS test, we investigated the effect of PVP and AgNPs+PVP on the morphology of MCF7 cells. The cells were examined by phase contrast microscopy on the 1st and 2nd days of incubation with the test substances (Figure 2).
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Figure 2. Morphology of MCF 7 cells in the absence of the studied compounds (control) and after proliferation in the presence of sodium azide, PVP, and AgNPs+PVP (10 mg/ml) |
Sodium azide, a highly toxic substance, was used as a control (Figure 2). As can be seen from the figure, in his presence, the cells acquired a spherical shape and did not show proliferation. AgNPs+PVP activated cell proliferation (Figure 2), which can enhance the wound healing effect [21, 26-28].
Conclusion
It was found that in the Ames test, the excess of the number of revertants over the control at concentrations of 10 and 100 µg/ml in PVP was 0.5±0.03 and 0.4±0.06, and in AgNPs+PVP – 0.9±0.12 and 1.1±0.09, which indicates the absence of mutagenic action. None of the compounds showed an increase in the SOS response in the DNA-damaging test. The excess of β-galactosidase activity over the control in PVP was 0.9±0.03 and 1.2±0.03, and in AgNPs+PVP – 0.6±0.38 and 0.6±0.12. The results of the MTS test are shown in Table 1. None of the compounds reduced the activity of mitochondrial dehydrogenase, which indicates the absence of their cytotoxicity. MCF7-test showed that in the presence of silver nanoparticles, the cells acquired a spherical shape and did not show proliferation. AgNPs+PVP activated cell proliferation, which can enhance the wound healing effect. Thus, the assessment of the geno- and cytotoxicity of AgNPs+PVP did not reveal their toxicity and indicates the need for further research in connection with the possibility of application in the pharmaceutical industry, veterinary medicine, and medicine.
Acknowledgments: All the authors were involved in the conceptualization, methodology, formal analysis, writing, and editing of the manuscript.
Conflict of interest: None
Financial support: None
Ethics statement: The protocol for experiments with laboratory animals complied with the requirements of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes.
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