Effect of silver nanoparticles on the reactions in the rats' tissues at endotracheal and intraperitoneal introduction

Chegodar D.V.; Kubyshkin A.V.; Fomochkina I.I.; Aliev L.L.; Pisareva O.A.; Litvinova S.V.

УДК 612.015.13+616.381+546.57

EFFECT OF SILVER NANOPARTICLES ON THE REACTIONS IN THE RATS' TISSUES AT ENDOTRACHEAL AND INTRAPERITONEAL INTRODUCTION

Chegodar D. V., Kubyshkin A. V., Fomochkina I. I., Aliev L. L., Pisareva O. A., Litvinova S. V.

Department of clinical and general pathophysiology Medical Academy named after S.I. Georgievsky of Vernadsky CFU, 295051, Lenin Boulevard, 5/7, Simferopol, Russia

For correspondence: Denis V. Chegodar, PhD, Associate Professor, Department of clinical and general pathophysiology Medical Academy named after S.I. Georgievsky of Vernadsky CFU, e-mail: dvcheg77@mail.ru

Для корреспонденции: Чегодарь Денис Владимирович, кандидат медицинских наук, доцент кафедры общей и клинической патофизиологии Медицинской академии имени С.И. Георгиевского ФГАОУ ВО «Крымский федеральный университет им. В.И. Вернадского», e-mail: dvcheg77@mail.ru

Information about authors:

Chegodar D. V., https://orcid.org/0000-0001-8067-6411 Kubyshkin A. V., https://orcid.org/0000-0002-1309-4005 Fomochkina I. I., https://orcid.org/0000-0003-3065-5748 Aliev L. L., https://orcid.org/0000-0001-9401-4398 Pisareva O. A., https://orcid.org/0000-0002-1156-4544 Litvinova S. V., http://orcid.org/0000-0001-8271-3415

SUMMARY

The effects of silver nanoparticles stabilized in the solution by sodium alginate (linear particle size is 10-20 nm) at a single-dose endotracheal and intraperitoneal introduction to intact animals have been investigated. As markers to evaluate the effect of silver nanoparticle solution, the study of reactions of proteinase inhibitory system components in blood serum, bronchoalveolar and peritoneal lavage was used. It has been found out that a single-dose endotracheal and intraperitoneal introduction of silver nanoparticle solution to intact male rats of "Wistar" line results in minimal activation of proteinase inhibitory system components, both at a systemic and local level. The microscopic examination of the lungs and tissues of the abdominal cavity showed no signs of silver nanoparticle-induced inflammation.

Key words: silver nanoparticles, sodium alginate, proteinases, proteinase inhibitors.

ВЛИЯНИЕ НАНОЧАСТИЦ СЕРЕБРА НА РЕАКЦИИ В ТКАНЯХ КРЫС ПРИ ЭНДОТРАХЕАЛЬНОМ И ИНТРАПЕРИТОНЕАЛЬНОМ ВВЕДЕНИИ

Чегодарь Д. В., Кубышкин А. В., Фомочкина И. И., Алиев Л. Л., Писарева О. А., Литвинова С. В.

Кафедра общей и клинической патофизиологии Медицинская академия имени С.И. Георгиевского ФГАОУ ВО «КФУ им. В.И. Вернадского», Симферополь, Россия

РЕЗЮМЕ

Изучены эффекты наночастиц серебра, стабилизированных в растворе альгинатом натрия (линейный размер частиц 10 - 20 нм) при однократном эндотрахеальном и интраперитонеальном введении интактным животным. В качестве маркеров оценки эффектов раствора наночастиц серебра использовали изучение реакций компонентов протеиназ-ингибиторных систем в сыворотке крови, бронхоальвеолярном и перитонеальном смыве. Установлено, что однократное эндотрахеальное и интраперитонеальное введение раствора наночастиц серебра интактным крысам-самцам линии «Wistar» приводит к минимальной активизации компонентов протеиназ - ингибиторной системы, как на системном, так и на локальном уровне. Микроскопическое исследование легких и тканей брюшной полости показало отсутствие индуцированных наночастицами серебра признаков воспалительного процесса.

Ключевые слова: наночастицы серебра, альгинат натрия, протеиназы, ингибиторы протеиназ.

the specific markers of the inflammatory process [8, 7, 1]. The purpose of the research is to study the reactions in the intact rat tissues at different ways of introduction of silver nanoparticles stabilized in sodium alginate solution based on assessment of reactions of proteinase-inhibitory system components at a systemic and local level.

MATERIAL AND METHODS

The experimental research was carried out on 52 white male rats of "Wistar" line having body mass of 180-210g in accordance with the principles

The possibility of using silver nanoparticles as a component of medicinal drugs [6, 5, 3, 14] requires further detailed investigation of different unfavorable side effects, in particular, their possible participation in the development of inflammatory reaction in organs and tissues [2, 4]. Marked antibacterial effect of silver nanoparticles and their active participation in intercellular communication suggest the involvement in the process of phagocytic cells, whose release of nonspecific proteinases against a background of the reaction of their endogenous inhibitors is one of

of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986).

We used 0.1% solution of silver nanoparticles with a linear size of 10-20 nm in a matrix of 0.6% sodium alginate in water medium (99.3%). The composition has been developed at Taurida National University (Simferopol) with participation of staff members of the Institute of Biology of the Southern Seas (Sevastopol) [11]

The experimental research included two series of experiments. In the first series of experiments the effect of silver nanoparticle solution with alginate stabilizer on tissues of bronchoalveolar tree was investigated. The laboratory animals (n = 25) were divided into three groups. In the test group (n=9) the base solution of silver nanoparticles, previously diluted with 0.9% sodium chloride solution in ratio 1:1 was given as a single dose endotracheally in amounts of 0.2 ml. Two other groups of laboratory animals were used as control groups: the first one (n=8) with a single-dose endotracheal introduction of 0.2 ml 0.9% sodium chloride solution and the second one (n=8) with a 0.3% sodium alginate solution.

In the second series of experiments the evaluation of effect of silver nanoparticle solution on the abdominal tissues was conducted. Laboratory animals (n=27) were divided into three groups. In the test group (n=9) 0.05% solution of silver nanoparticles with sodium alginate was introduced as a single dose endotracheally in a volume of 2 ml. Similarly to the first series of experiments, two groups of rats were used for control, with introduction of 0.9% sodium chloride solution (n=9) and 0.3% sodium alginate solution in a volume of 2 ml (n=9) intraperitoneally as a single dose. Euthanasia of animals was performed 24 hours after the beginning of the experiment under ether anesthesia by decapitation with the following material sampling.

The material for the research was blood serum, bronchoalveolar and peritoneal lavage. Blood sampling was performed from the jugular vein. To obtain serum, blood was centrifuged at 5000g for 15 minutes. Bronchoalveolar lavage was obtained by isolation of cardiopulmonary complex by 6-8-time washing of the lungs through the trachea with 10 ml of physiological salt solution. After washing, 7-8 ml of bronchoalveolar lavage was obtained. Peritoneal lavage was obtained by 5-time washing of the abdominal cavity with 10 ml of isotonic NaCl solution during a minute with the following aspiration by a syringe.

The activity of components of proteinase-inhibitory system was determined by enzymatic methods [9] with the help of spectrophotometer "Biomat 5" (UK). Method for determination

of trypsin-like activity (TLA) is based on spectrophotometric measurement of elimination rate of N-benzoyl-L-arginine (BA) from the synthetic substrate of N-benzoyl-L-arginine ethyl ester (BAEE). Determination of elastase-like activity (ELA) was based on the study of hydrolysis rate of the synthetic substrate N-t-BOC-alanine-p-nitrophenil ester (BANPE). Determination of concentration of alpha-1-proteinase inhibitor (antitryptic activity, ATA) and acid-stable inhibitors (ASI) was carried out by inhibiting BAEE decomposition by trypsin. Protein in all samples was determined by Lowry method.

To assess the pathological changes in the tissues, serial sections 4-5 ^m thick were prepared which were stained with hematoxylin-eosin. The research of micro slides was carried out by light microscopy using a light microscope "Olympus CX-41" with magnification 100 and 400.

The statistical processing of the obtained data was carried out by methods of variation statistics with calculation of average values (M), assessing the probability of discrepancies (m), assessing the significance of changes using the Student t-test. Difference in the mean values at p<0.05 was taken as reliable. Distribution of signs corresponded to normal according to Shapiro-Wilk statistics.

RESULTS

The performed research of the first series of experiments showed that endotracheal introduction of silver nanoparticle solution to intact animals did not cause any changes of investigated parameters in blood serum as compared to both control groups. The study of changes in bronchoalveolar lavage in a day after the first endotracheal introduction of silver nanoparticle solution and sodium alginate solution did not reveal any authentic changes of the investigated parameters either. Elastase-like and antitryptic activity underwent the most significant changes, which tended to increase in comparison with the control groups (Fig. 1A).

Morphological studies of lung tissues showed that in the group with endotracheal introduction of silver nanoparticles and in control groups with introduction of saline solution and sodium alginate a similar morphological pattern of the lung tissue with absence of marked signs of inflammation were observed.

On microscopic examination of the rats' lungs of the first control group, whom a saline solution was introduced endotracheally, a slightly marked edema and hyperemia of interalveolar septa were detected; in the lumen of the alveoli the isolated alveolar macrophages were found (arrow). In the terminal bronchioles the morphological pattern was characterized by focal desquamation of epithelial

Figure 1. А. Changes of nonspecific proteases and their inhibitors in the bronchoalveolar lavage of intact animals when endotracheally administered silver nanoparticles solution. B. Changes of nonspecific proteases and their inhibitors in the peritoneal lavage of intact animals when intraperitoneally

administered silver nanoparticles solution.

cells (Fig. 2A). In the group with introduction of sodium alginate to intact rats and in the group with introduction of silver nanoparticles a more substantial increase of alveolar macrophage number in the alveoli lumen was marked (arrow), as well as the occurrence of focal lympholeukocytal infiltrates in the interstitium, in combination with congestion and edema (Fig. 2B). It should be noted that alveolar macrophages which are the components of the cellular immunity of the alveolar wall have the whole set of proteolytic enzymes, which release may be connected with the formation of inflammation of the lung tissue [10, 12].

The results of the second series of experiments showed that in the blood serum on intraperitoneal introduction of sodium alginate to intact animals a significant increase in trypsin-like activity by 60% occurred as compared to the control group. On intraperitoneal introduction of a silver nanoparticle

solution a statistically significant increase in trypsin-like activity to more than double as related to the control group was also observed. At the same time a significant increase of antitryptic activity by 20% as compared to the values of the control group and by 14% as to the group of animals with introduction of sodium alginate testifies to compensatory reaction of antiproteinase potential at a systemic level. In peritoneal secretions of the control group and groups with introduction of sodium alginate and solution of silver nanoparticles a substantial activation of nonspecific proteases was not observed, while the increase in the level of antitryptic activity and acid-stable inhibitors testifies to activation of local antiproteinase potential (Fig. 1B). On histologic study of rats' peritoneum in the control group with intraperitoneal introduction of 0.9% sodium chloride solution the morphological pattern was characterized by slightly marked tissue reaction with

Figure 2. А. Respiratory bronchioles of rat in the control group. Magnification 400. Hematoxylin and Eosin Stain. B. Lung of rats when endotracheally administered silver nanoparticles solution. Magnification 400.

Hematoxylin and Eosin Stain.

moderate tissue edema and loosening interstitial spaces. On intraperitoneal introduction of sodium alginate to intact animals the development of a more expressed tissue reaction was observed with the presence of focal accumulations of lymphocytes with isolated neutrophils (arrow). Thickening of the peritoneum due to edema, separation of collagen fibers in subperitoneal space, focal congestion of vascular microcirculation and minor erythrodiapedesis were marked (Fig. 3A). The morphological picture of the visceral peritoneum covering omentum of the rats of the experimental group with a single intraperitoneal introduction of silver nanoparticle solution was characterized by the absence of signs of acute inflammatory process and was similar to the microscopic structure of the peritoneum of rats in the control group. In a number of observations congestion of vessels (arrow) with the phenomena of margination and focal leucodiapedesis was noted (Fig. 3B).

contact with the external environment. Changes in proteolysis system and microscopic examinations testify to absence of proinflammatory effects of silver nanoparticles at their action on the peritoneum and abdominal organs or these effects are minimal [13].

CONCLUSION

1. A single endotracheal and intraperitoneal introduction of silver nanoparticle solution with sodium alginate to intact animals is characterized by minimal changes of morphological picture of bronchoalveolar tree tissues and parameters of proteinase-inhibitory system components, both at a systemic and local level.

2. Slight changes of parameters of proteinase-inhibitory system components and absence of morphological signs of acute inflammatory process indicate the absence of expressed pro-inflammatory effects in the investigated nanocomposition which allows to recommend

Figure 3. А. Histological structure of the peritoneum and omentum of rat when intraperitoneally administered sodium alginate. Magnification 400. Hematoxylin and Eosin Stain. B. Histological structure of the peritoneum and omentum of rat when intraperitoneally administered silver nanoparticles solution.

Magnification 100. Hematoxylin and Eosin Stain.

DISCUSSION

Thus, as a result of the research it was found out that introduction of silver nanoparticles stabilized in sodium alginate solution to intact animals leads to slight changes of components of proteinase-inhibitory system, both at a systemic and local level. Comparative characteristics of changes in the bronchoalveolar and peritoneal lavage show that changes in the lung tissue are more expressed than in the abdominal cavity. The above-mentioned differences in reactions to the introduction of solutions can be explained by the peculiarities of participation of bronchoalveolar and peritoneal system in the protective reactions of the organism. Bronchopulmonary tissue is an open system which is constantly in contact with the external environment, as a result of which the reaction to environmental effect may be more dynamic, as opposed to the peritoneal cavity, which is not in

further research to study the possibilities of using silver nanoparticles as components of drugs.

Conflict of interest. The authors have no conflict of interests to declare.

Конфликт интересов. Авторы заявляют об отсутствии конфликтов интересов.

The work was performed as part of a research project supported by the Ministry of Education and Science of the Russian Federation No 3884.

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