Silver is the sixty third most common element in the Earth’s crust and silver compounds can be found on every continent. It is hard to say when people first discovered silver, but it is clear that it was known and used by prehistoric people. Silver alloys were actively used for producing coins, ornaments and various household items. The medical properties of the metal, especially its powerful antibacterial effects, were known to people at the time of Hippocrates. Later, there were attempts to use silver for treatment of epilepsy, neonatal ophthalmic diseases, cholera, dysentery, sexually transmitted diseases, traumatic and other types of infections. One should also mention surgical instruments, bone prostheses and catheters made of silver alloys [1
]. Developments in biotechnology have allowed incorporation of ionizable silver into fabrics for clinical use to reduce the risk of nosocomial infections, and for personal hygiene [3
Discovery of colloidal silver at the end of the 19th century, when the German surgeon and the son of C. Credé (a famous gynecologist who introduced silver nitrate eyedrops as an antiseptic for the prevention of ophthalmia neonatorum in newborns)—B. Credé (1847–1929) together with several chemists suggested using nonionized silver in the form of colloidal solutions of metallic silver (“Unguentum Credé”, collargol) or silver oxide sol, which can be considered a breakthrough in the medical and pharmaceutical application of the metal [5
]. Outstanding antimicrobial activity together with the lack of resistance of most pathogens make silver a very promising material for medical applications especially taking into account the new life-threatening antibiotic-resistant bacterial strains [4
Development of nanoindustry gave rise to vast research of silver nanoparticles (AgNPs). Active studies are being conducted in order to receive a better understanding of both existing and potential application of AgNPs for water purification and disinfection [6
], clothing and footwear manufacturing [8
], cosmetics [9
], household appliances and cleaning products, etc [10
]. The exceptional potential of AgNPs for application in medicine and hygiene practices can be explained through their prominent antibacterial [12
], antiviral [14
] and antifungal [15
] properties. Currently, AgNPs-based wound dressing materials and water purifying agents are also being actively developed [4
Though silver has been actively used throughout most of human history, the first data on its potential toxic effects only appeared in the 20th century [4
]. AgNPs are the least studied silver-containing substances, and the data on their toxicity to mammals and humans are inconclusive and often contradictory. Upon oral AgNPs administration, the first pathological changes are observed in the digestive tract and liver [18
]. It is a well-known fact that a healthy human gastrointestinal tract is colonized by about one trillion commensal bacteria comprising over 3000 species that form intestinal microbiota [20
]. The intestinal microbiome is of utmost importance to human health while any imbalance can affect the host’s metabolism and immunity which are closely connected with chronic inflammatory diseases [21
]. It is reasonable to assume that when administered orally, AgNPs might damage the intestinal microbiota balance due to their powerful antibacterial effects. The results of a study where AgNPs stabilized with polyvinylpyrrolidone (PVP) with a diameter of 10–80 nm were orally administered to rats for 62 days in doses of 0.1 mg/kg, 1.0 mg/kg and 10 mg/kg of body weight per day indicated that AgNPs produced no significant impact on the composition of the main components of normal rat cecum microbiota. The total amount of aerobe and anaerobe bacteria, the volume of lactobacilli and enterobacteria remained unchanged in all studied groups. At the same time, the bifidobacteria population inhabiting the bodies of rats receiving the 0.1 mg/kg dose of AgNPs was reduced compared to the control group, although the absolute value of the decrease was not significant (less than half of the initial quantity). Besides, for streptococci and staphylococci in the group receiving the 10 mg/kg dose of AgNPs, the total values were below those of the control group by more than an order of magnitude [22
]. Alkaline Comet Assay and Micronucleus Assay were used to study the effects of orally administered 20 nm AgNPs at a dose of 50 mg/kg, 150 mg/kg and 300 mg/kg on male and female mice. Comet assay was performed on liver, spleen, blood, duodenum and kidney, and Micronucleus assay was performed on spleen lymphocytes, to evaluate the genotoxic potential. The result displayed that AgNPs first accumulated in duodenum from where they migrated to kidney, liver and spleen; in liver and duodenum cells, the nanoparticles (NPs) were discovered both in cytoplasm and in organelles, except the cell nucleus. However, the authors discovered neither genotoxicity, nor tissue damage in all the studied organs [23
]. Jeong et al. [24
] showed that the rats treated for 28 days with 60 nm AgNPs at a dose of 30 mg/kg, 300 mg/kg and 1000 mg/kg exhibited higher numbers of goblet cells, resulting in more mucus materials in the intestinal canal.
After entering blood vessels, AgNPs are mostly accumulated in the liver [25
], often leading to pathological changes in its tissues and cells. In order to study the pathological potential of 8.7 nm AgNPs, the NPs were administered abdominally to female rats for 28 days at a dose of 1 mg/kg, 2 mg/kg and 4 mg/kg. Identification of the oxidative stress signs in the liver tissue, determination of the silver nanoparticles concentration in tissues, description of hepatic histopathological alterations and detection of possible chromosomal aberrations in the bone marrow were carried out. Results revealed various dose-dependent hepatic histopathological lesions. The effect of AgNPs on the hepatic malondialdehyde and glutathione levels varied in different treated groups compared to the control. Residues of AgNPs were found in the hepatic tissue and their amount depended on the initial treatment dose. In addition, AgNPs induced variable chromosomal aberrations that were dose dependent. The reported results display the hepato- and genotoxic properties of AgNPs [27
]. A single intravenous exposure of rats to 20.1 ± 2.8 nm AgNPs (5 mg/kg) produced no toxic effects on the liver cells despite considerable AgNPs accumulation in the liver (60% of the introduced NPs within the first 24 h) according to the histopathological and biochemical studies [25
]. Park et al. [28
] studied the effects of the exposure of mice 42 nm AgNPs at a dose of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 28 days. The authors observed a dose-dependent increase of the alanine transaminase (ALT) and aspartate transaminase (AST) levels in all treated groups. Although the histological studies of the liver and intestine tissues revealed no pathologies, the adverse impact on liver and kidney was observed in the high dose-treated group (1.00 mg/kg). In the longer lasting experiment where the doses of 0.1 mg/kg, 1 mg/kg and 10 mg/kg of PVP-stabilized AgNPs were administered to rats for 92 days, N.V. Zaytseva et al. [29
] observed histological changes in the liver tissue even in the case of the lowest studied dose. Eosinophilic infiltration of the portal tracts was also observed, accompanied by the emergence of medium and large-drop fat vacuoles in the cytoplasm of hepatocytes, swelling and lympho-macrophage infiltration of the portal tracts at the doses of 1.0 and 10.0 mg/kg. These changes can be treated as symptoms of inflammation of hepatocytes.
Interestingly, in some cases, the higher doses of AgNPs produce a less pronounced toxic effect, as shown, for example, in the paper by Kim et al. [30
]. Their research revealed that when exposed to orally administered 60 nm AgNPs at the doses of 30 mg/kg, 300 mg/kg and 1000 mg/kg, rats did not show any significant changes in the body weight that could be related to the doses of AgNPs during the 28-day experiment. In the case of the maximal dose, biochemical blood examination revealed some significant changes in the alkaline phosphatase and cholesterol levels related to bile ducts hyperplasia revealed during histological examinations.
In the 14-day experiment on BALB/C mice receiving 20 and 50 ppm of 40 nm AgNPs orally, the researchers observed significantly increased levels of ALT and AST. During histological examination hepatotoxic effects in the form of necrosis, hepatocytic inflammation and total lymphocyte aggregation were observed [31
]. Similar results were obtained when 30–50 nm AgNPs were orally delivered to rats for 21 days at a dose of 50 mg/kg. The treatment resulted in severe hepatotoxicity and histological changes, increased DNA fragmentation and down regulation of the antioxidant gene expression in the liver [32
Intraperitoneal administration of 33 nm AgNPs at a dose of 78 mg/kg to mice during the period of 14 days resulted in the pronounced increase in the ALT (+126.6%) and AST (+53.5%) levels, while the histological inspection of the liver revealed vascular hyperemia as well as parenchymatous and perivascular aggregations of mononuclear cells [33
]. Another 30-day study where rats were injected intraperitoneally three times a week with 54.9 nm AgNPs revealed a severe hepatotoxic effect. AgNP exposure enhanced the hepatic lipid peroxidation (+281.7%) along with a decline in the reduced glutathione (−58.3%) levels. The apparent hepatic oxidative damage was associated with the obvious hepatic dysfunction as shown by the alteration of the serum liver enzymatic biomarkers, the lipid profile and the pathological hepatic lesions. Variously located cases of focal liver necrosis with inflammatory cell infiltration, hydropic cell degeneration and bile duct epithelium degeneration were observed [34
Various methods of the AgNPs’ toxicity reduction are currently being studied, such as green synthesis [35
] or NPs stabilization and surface functionalization [37
]. In the paper [37
], the authors investigated the hepatotoxicity of citrate-coated AgNPs (cAgNPs) after single intravenous administration to rabbits at the doses of 0.5 mg/kg and 5 mg/kg. The results showed that the structure and function of the liver tissue were disrupted. The authors confirmed the development of dose-dependent oxidative stress in the liver tissue significantly exceeding the control group levels. It should be noted that the levels of oxidative stress markers remained abnormal 28 days after the single exposure to cAgNPs, suggesting a potential genotoxic effect of the studied nanoparticles. Toxicity of cAgNPs was evaluated in a longer lasting (42 days) experiment where rats received the 62.5 mg/kg, 125 mg/kg and 250 mg/kg doses of the material orally. Hematologic study, biochemical blood serum analysis and histopathological examination revealed no significant differences between the treated and the control groups. Toxicity endpoints of the reproduction screening test were measured and revealed no evidence of the cAgNPs toxicity [38
Oral administration of the doses of 50 mg/kg, 100 mg/kgand 200 mg/kg of PVP-covered AgNPs to rats for 90 days led to a dramatic increase in reactive oxygen intermediate production that, as a result, stimulated a dose-dependent increase in the hepatotoxicity markers, intensified autophagy and depleted the insulin signaling pathways. Such effects can influence apoptotic, necrotic and autophagic molecular processes [39
Thus, finding stabilizers for AgNPs that would increase their biocompatibility remains an important problem. The goal of our research was to modify the surface of AgNPs in a way that would reduce their potential toxicity to organs and cells of mammals without compromising the antibacterial effectiveness of nanosilver.
Previously we have studied the in vitro activity of AgNPs stabilized with benzyldimethyl[3-myristoylamine)-propyl]ammonium chloride monohydrate (BAC) against gram-positive and gram-negative bacteria, yeast and fungi. BAC is a well-known active ingredient used in many antiseptic medicines. It was assumed that modification of the AgNPs surface with a cationic surfactant displaying no toxicity against mammal cells [40
], and possessing antibacterial properties [42
] would produce a highly effective antibacterial agent containing small-diameter AgNPs with the positive surface charge that would require no additional surface modification or purification [46
]. During in vitro experiments we confirmed high effectiveness of AgNPs-BAC. In particular, its antibacterial activity against E. coli was twice higher than that of unmodified AgNPs and twenty times higher than the activity displayed by BAC, making this combination a powerful antibacterial agent [50
]. This effectiveness was confirmed in in vivo experiments when it was used for treating enteritis of dogs [51
]. Stabilization with BAC may reduce the toxic effects of unmodified AgNPs. Thus, the present work is aimed at evaluating the entero- and hepatotoxicity of the AgNPs-BAC active complex by means of acute and sub-acute in vivo experiments on laboratory mice.
2. Materials and Methods
AgNPs were synthesized according to the method described in our previous work [52
]. Benzyldimethyl[3-myristoylamine)-propyl]ammonium chloride monohydrate (BAC)—trade name Miramistin®
(Infamed K, LLC, Kaliningrad Oblast, Russia)—a commercially manufactured antiseptic medicine, belongs to the chemical group of quaternary ammonium compounds efficient against pathogenic microorganisms including fungi, bacteria and protozoa. In order to study the effects of BAC on animals, the substance was administered at a dose of 0.5 mg/kg.
AgNPs-BAC were obtained via the Tollens’ technique by reducing the ammonia silver complex with glucose during gentle heating. The process consisted of three stages:
Obtaining an ammonia complex of silver oxide:
150 µL of 0.01% sodium hydroxide solution (3.8 × 10−5 mol) were added to 50 mL of aqueous solution of 0.017 g (1 × 10−4 mol) of silver nitrate. Then, the mixture containing unreacted silver nitrate and precipitated silver oxide was mixed with a 25% ammonia solution (approx. 50 μL) until full dissolution of Ag2O was achieved.
Reducing diamminesilver(I) complex with glucose in the presence of BAC as a capping agent:
50 mL of an aqueous solution of 0.011 g of BAC were added dropwise to the obtained diamminesilver(I) complex. After 15 min of stirring, 0.35 g of glucose were added. Reduction process took place at 49 °C.
Purification of the AgNPs’ dispersion:
Dispersion of AgNPs with the remains of reagents like glucose, sodium hydroxide and ammonia was purified by means of dialysis. Dialysis of dispersion of AgNPs was carried out by soaking of dialysis bags SERVAPOR® (SERVA Electrophoresis GmbH, Heidelberg, Germany) with pore diameters of 2.5 nm containing dispersions of AgNPs-BAC into a 0.01% solution of pure BAC in double distilled water with the ratio of volumes of dispersion of silver and solution of BAC equal to 1:10. Dialysis was carried out twice in the same conditions. The obtained purified dispersions were diluted with the 0.01% solution of BAC until the content of colloidal silver in them comprised 10 and 50 ppm, respectively.
2.2. Transmission Electron Microscopy (TEM)
Micrographics of AgNPs were obtained using an electronic microscope (Leo 912 AB Omega, Leo Ltd., Neu-Isenburg, Germany) with the operating accelerating potential of 100 kV. In order to prepare the samples, 1–2 μL of the solution were spread onto the copper mesh coated with Formvar™ (d = 3.05 mm), which was then dried in the open air for 5–10 min. Size distribution of AgNPs was calculated on the basis of the obtained micrographs using the software Femtoscan Online v. 2.2.91 (Center of Perspective Technologies, Moscow, Russia).
2.3. Electron Spectroscopy
To register the absorption spectra in the visible region, a spectrophotometer (JENWAY 6310 Bibby Scientific Ltd., Stone, UK) and quartz cells with the 10 mm optical path length were used.
The aqueous solution was prepared in distilled water with the AgNPs content equivalent to the tested dosage; the AgNPs were stabilized with BAC in the ratio 1:10.
2.4. Zeta Potential
Zeta potential was measured by applying an electric field across the dispersion of silver NPs using the technique of laser Doppler anemometry that involved the ZetasizerNano ZS analyzer (Malvern Instruments Ltd., Malvern, UK).
2.5. Animals and Conditions
Six-week-old male CBF1 mice were used during the experiment (body weight 25 ± 2 g). All manipulations with the animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (ILAR, DELS), the Principles of good laboratory practice [53
]. The Saint Petersburg State University of Veterinary Medicine Institutional Animal Care and Use Committee approved the animal related procedures in this study (#011–05–19).
The animals were housed according to National Standard [54
] in 440 × 270 × 140 mm3
polycarbonate boxes, 10 animals per box. Sterilized wood shavings were used as bedding. The following ambient conditions were kept under strict control: air temperature 20–22 °C, relative humidity 50 ± 5% and a 12:12 light–dark cycle. Throughout the experiment, the animals had unlimited access to food and water. Prior to the experiment, the animals underwent a 14-day quarantine period.
Acute experiment. In order to evaluate the AgNPs-BAC acute toxicity, the mice were divided into 9 groups, 10 animals in each group. AgNPs-BAC were administered using an intragastric gavage as a single dose of 0.8; 1; 1.2; 1.5; 4; 5; 7.5 mg/kg. The AgNPs-BAC doses were selected according to the previously obtained results for non-stabilized and non-functionalized AgNPs [27
]. The BAC group received the pure BAC solution in a dose of 5 mg/kg. The dose was selected based on the results of the previous research defining the optimal dose for AgNPs stabilization. The control group received the same volume of distilled water. The animals were closely observed for 14 days after the treatment, the functional status of the body was regularly evaluated and the animals were weighed once a week. At the end of the observation period, all animals were euthanized for further morphological, biochemical and histological examinations.
Subacute experiment. In order to evaluate the AgNPs-BAC subacute toxicity, 6 groups were formed, 15 animals in each group. 0.5 mL of the dispersion were administered daily for 14 days using an intragastric gavage. AgNPs-BAC were administered in the doses of 0.05; 0.25; 0.45; and 2.25 mg/kg. The BAC group received pure BAC solution at a dose of 0.5 mg/kg. The control group received the same volume of distilled water. Daily examinations together with evaluation of the functional status of the body were carried out. The animals were weighed once a week. The following parameters were observed: skin and coat health, body position, food and water consumption, weight gain dynamics. At the end of the observation period, all animals were euthanized for further morphological, biochemical and histological examinations.
2.7. Functional Status of the Animals
Activity—low, normal, increased.
Movements—loss of coordination, muscle tonus, tremor.
Body position—natural, lying on one side, hunched posture, hiding in the corner.
Body condition—adipose, well-conditioned, seriously underweight, cachectic.
Coat—shiny, dull, smooth, rough, abnormal shedding.
Eyes—watery eyes, inflammation, corneal clouding, adhesion.
Ears—color (normal, pale, reddened), inflammation, discharge, crusting, twitching.
Teeth—color, broken teeth, tooth loss.
Feet and limbs—color, swelling.
Breathing—rapid, normal, bradypnea, wheezing, coarse breathing, panting.
Salivation—insufficient or excessive.
Saliva—watery or sticky.
Food and water consumption—normal, insufficient or excessive.
The animals were weighed once a week in order to determine the weight gain dynamics during the experiment.
2.8. Hematological Examinations
The blood for the analysis was collected using 1.5 mL polyethylene tubes, incubated for 4 h at 37 °C until clotted, then centrifuged for 10 min at 3000× g. The harvested blood serum was stored at −20 °C until tested. The biochemical analysis was performed using a MindreyBA88A spectrophotometer with Olvex kits (Russia).
For red blood cell count, the full blood aliquot was diluted 1:200 with normal saline. The assay was performed within 2–3 h of dilution. For white blood cell count, full blood was diluted with 4% acetic acid using methylene blue for cell nuclei staining. The leukogram was calculated in blood smears stained by the Romanovsky-Gimza dye.
Macroscopic examinations and internal organs morphometry.
Following the experiment the animals were euthanized and the autopsy was performed for macroscopic examination of the internal organs. The organs were weighed using a digital weighing balance AND EK-6100i. For each organ, the mass coefficient km
was calculated according to the formula:
is the individual organ mass and mb
is the body mass.
2.9. Internal Organs Histology
The liver and intestine were fixated in a 10% buffered formalin solution, prepared and embedded in Histomix after the standard procedures [55
]. The embedded material was sectioned into slices of 4 μm; the slides were stained with haematoxylin and eosin.
For the intestine histological examination, a section of the jejunum 10–11 cm from the stomach was taken. Each fragment yielded 10–15 slices from three different areas. Three randomly selected samples from each animal were photographed. In each Section, 10 fields of 130,834 μm2 (418 μm × 313 μm) were randomly selected and photographed. The fields included all jejunum wall layers.
In the intestinal crypts, the percentage of cells with mitotic figures was calculated. The experimental and control groups were compared on the basis of this parameter. The cells in mitotic prophase were excluded from the total of cells with mitotic figures. The non-dividing cells were calculated by their nuclei. We counted the cells in the middle part of each crypt (about 50 μm from the bottom and from the opening of the crypt). Over 1000 crypt cells were counted for each animal.
For the histological liver examination, two sections from the median lobe were taken. From each sample, at least 10 sections were prepared. Three randomly selected samples from each animal were microphotographed. The fields were randomly selected. The microphotograph area corresponded to the area of the section (130,834 μm2, 418 μm × 313 μm).
The number of Kupffer cells was counted for each microphotograph, then the number of cells per 1000 μm2 of the liver parenchyma was calculated. The cells were determined by the nuclei. The quantities for the experimental and control groups were then compared.
2.10. Statistical Analysis
The statistical significance of differences between the mean values was tested using Student’s t-test, Mann-Whitney U-test and ANOVA. p values < 0.05 were considered statistically significant.