ISSN 1995-0780, Nanotechnologies in Russia, 2015, Vol. 10, Nos. 7–8, pp. 640–644. © Pleiades Publishing, Ltd., 2015. Original Russian Text © E.A. Titov, M.A. Novikov, L.M. Sosedova, 2015, published in Rossiiskie Nanotekhnologii, 2015, Vol. 10, Nos. 7–8.
Effect of Silver Nanoparticles Encapsulated in a Polymer Matrix on the Structure of Nervous Tissue and Expression of Caspase-3 E. A. Titov, M. A. Novikov, and L. M. Sosedova East Siberian Institute of Medical and Environmental Research, Angarsk, 665827 Russia e-mail:
[email protected] Received July 20, 2014; in final form, April 9, 2015
Abstract—Results of histological and immunohistochemical studies of the nervous tissue of mongrel albino rats exposed to the influence of a nanobiocomposite containing silver nanoparticles and a natural biopolymer such as arabinogalactan for 9 days are under discussion. It is proved that both under acute exposure and in the survival period, the use of the examined substance leads to structural and functional changes in nervous tissue such as an increase in the number of hyperchromic cells, a creduction of the total number of cells per area unit, and the development of perivascular edema. The triggering of apoptosis of neurons of the brain manifested by a statistically significant increase in the number of cells which express caspase-3 pro-apoptotic protein is found. It is determined that silver nanoparticles encapsulated in a natural arabinogalactan polymer matrix penetrate the blood–brain barrier, thus causing long-term morphological and functional disturbances of the brain tissue in rats. Keywords: histology, immunohistochemistry, arabinogalactan, nanosilver, caspase-3, rats, brain. DOI: 10.1134/S1995078015040205
INTRODUCTION Specific medical and diagnostic pharmaceuticals of direct action based on metal nanoparticles, in particular silver, commonly studied in vitro are under intensive development despite scientific data on their adverse biological effects on the whole organism. The properties of silver nanoparticles and their influence on the body were extensively described in the literature. Thus, it was found that the nanoparticles are capable of depositing in the liver and penetrating into the olfactory bulb of the brain via the axonal transport [1, 2]. Silver nanoparticles were determined to be highly stable in the environment and may retain toxic properties for a long time [3]. Nanoparticles from 5 to 50 nm in size are characterized by strong antibacterial activity and cytotoxicity towards rat hepatocytes in vitro [4–7]. It was found that the mechanism of toxicity is due to oxidative stress, mitochondrial dysfunction, and increased membrane permeability [8]. The maximum allowable concentration of silver nanoparticles in air established in the United States is equal to 2.16 × 106 particles/cm3 [1]. Silver nanoparticles are commonly encapsulated in matrices of various natures to reduce toxic effects and provide the targeted delivery of the nanoparticles [9]. Thus, arabinogalactan (AG), the natural polymer isolated from Larix sibirica L. via the known technique [10, 11], is used as a matrix for nanostabilization. AG is actually a nontoxic compound characterized by
antioxidant and immunomodulatory properties and capable of forming conjugates with various functional groups, reducing the intensity of free radical processes and activating phagocytosis. It also shows a high hepatotropic and membrane protective effect [12]. Physical and chemical properties of AG depend on the molecular weight of its macromolecules. According to literature data, the molecular weight of AG isolated from Larix sibirica L. is between 9000 and 13000 kDa. Thermal and hydrolytic stability are important characteristics of AG determining the possibility of its use. The investigations show that the properties of AG are not changed after prolonged heating at 105°С and slightly changed at 130°С. The enhancement of temperature up to 150°С results in an increase in weight loss, the average degree of polymerization of AG, and the number of high molecular weight fractions, indicating that condensation processes are occuring, for example, intermolecular dehydration [13]. The identity of the nano-Ag-AG sample is confirmed by IR spectroscopy, X-ray, microscopic, elemental, titrimetric, and HPLC analysis. The content of Ag(0) in the nano-Ag-AG preparation is equal to 16.7%. The elemental composition of the compound is determined by elemental analysis to be C, 34.26%; H, 5.39%; and Ag, 15.4%. The data of transmission electron microscopy of the studied sample shows the presence of separate Ag(0) globular particles with sizes ranging between 0 and 20 nm, with sizes from 4.0 to 8.9 nm being predominant (up to 81.5%) [11].
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In-depth studies of biological effects of silver nanoparticles encapsulated in a polymer matrix are required for the widespread use of nanobiocomposites, with importance of the investigations being determined by possible risks to human health due to direct contact with these substances. A significant number of researches assessed biological effects of silver nanoparticles in preclinical studies mainly via acute or subchronic experiments [14], while the possibility that long-term effects of nanoparticles will appear was not examined. The methods for estimating the safety of nanopreparations are based on a morphological analysis of structural changes in internal organs and tissues of the body and molecular diagnostics of the expression of intracellular proteins, thus making it possible to prevent the production and use of drugs and diagnostic pharmaceuticals leading to longterm effects. There are no standard approaches to the biomedical assessment of exposure to nanobiocomposites, which hinders the development of effective preventative health-care programs. The aim of the present work was to assess structural changes in nervous tissue and the expression of proapoptotic protein in nerve cells of the brain of albino rats in both the early period of exposure to silver nanoparticles encapsulated in a polymer matrix and the long-term survival period. EXPERIMENTAL Seventy-two 3-month-old mongrel albino male rats weighing between 240 and 280 g were received for the studies from a vivarium of the East Siberian Institute of Medical and Environmental Research. The animals were kept in a special room with a 12 : 12 h light/dark cycle at controlled temperature (22 ± 3°С) and humidity with free access to clean tap water and food containing all necessary vitamins and minerals. All investigations with animals were performed according to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Purposes (Strasbourg, 1986) and Rules of Laboratory Practice approved by Order no. 708n of the Ministry of Healthcare and Social Development of the Russian Federation dated August 23, 2010. Groups of animals were chosen according to the Safety Assessment of Nanomaterials guideline approved by Order no. 280 dated October 12, 2007 (Moscow, Russia). Animals of the first experimental group (Ie, 12 animals) were intragastrically (as a gavage) administered with an aqueous solution of AG containing silver nanoparticles (nano-Ag-AG) in 0.5 mL of distilled water (100 μg of silver per 1 kg of weight) for 9 days, followed by decapitation on the next day after the end of exposure (the early period of observation). Animals of the second experimental group (IIe, 12 animals) received the same solution and were left alive for 6 months (which corresponds to approximately 15 years NANOTECHNOLOGIES IN RUSSIA
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of human life) after exposure (the long-term survival period). Animals of the third experimental group (IIIe) were intragastrically administered with the equivalent dose of pure AG and were decapitated like those of Ie group. IVe group received pure AG and was left alive after the experiment. The animals of Ic and IIc control groups (each of 12 animals) were intragastrically administered with 0.5 mL of distilled water for 9 days and decapitated like animals of Ie and IIe experimental groups, respectively. After decapitation, the brain of each test animal was removed and fixed in neutral buffered formalin (10%), dehydrated with ethanol of increasing concentrations (70, 80, 90, 95, and 100%), and placed into a HistoMix homogenized paraffin medium for histological studies (BioVitrum, Russia). Sections of 4–5 μm thickness were prepared with the use of a HM 400 microtome (Microm, Germany) and stained with hematoxylin and eosin on simple histology slides to perform microscopic analysis. Nissl staining was also used for the visualization of nerve cells. The sections were examined using an Olympus BX 51 research light optical microscope (Japan) with the possibility of transferring microimages to a personal computer with the use of an Olympus camera. The immunohistochemical method for determining the pro-apoptotic activity of caspase-3 was used to investigate the biological response of the organism at the subcellular level. Sections prepared with a microtome were placed on polylysine slides (Menzel, Germany), stained to visualize antibodies to caspase-3 protein according to the protocol of the manufacturer (Monosan, Netherlands), fixed with polystyrene, and covered with a cover glass. After the drying of polystyrene, the micropreparations were examined with the use of a research light optical microscope, followed by an analysis of predetermined parameters of micrographs, such as the total number of neurons per area unit, the number of hyperchromic neurons, and that of normal neurons which express or do not express caspase-3 pro-apoptotic protein, by means of an ImageScope S microscopy system. A statistical analysis of results was performed using Statistica 6.0 software (Statsoft Inc., United States). The numerical data are given as a percentage of the total number of neurons per area unit. Statistically significant differences between independent samples were determined via the Mann–Whitney method, with the significance level being set at p < 0.01. RESULTS AND DISCUSSION An analysis of prepared samples of the brain of albino rats exposed to silver nanoparticles shows that the total number of neurons per area unit is not statistically significantly different from the control value of the first experimental group, while in the cases of the survival groups the total numbers of neurons are significantly lower for both the control group and the 2015
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Expression of caspase-3 protein after exposure to AG and nAG100 in various periods of observation (percentage of the total number of cells per 0.2 mm2). Me (Q25–Q75) Period of observation Studied parameter
nano-Ag-AG (the early period) Ie
Hyperchromic neurons with caspase-3
2.7
Hyperchromic neurons without caspase-3 Normal neurons with caspase-3 Normal neurons without caspase-3 Total number of cells (pcs.)
(1.49–3.4)a 4.2 (3.76–5.2)a,b 5.9 (3.34–12.8)a,b 87.21
pure AG (the early period) IIIe 0.33 (0–0.67) 1.83 (1.66–2.4) 1.92 (1.66 –2.1) 96.11
control (the early period) Ic 0.68
nano-Ag-AG pure AG control (the long-term (the long-term (the long-term period) IIe period) IVe period) IIc 3.82
(0.52–0.96) (2.78–5.05)a,b 1.68
3.03
(1.49–2.05) (0.85–4.8)a,b 1.93
8.89
(1.76–2.09) (6.25–31.24)a,b 95.52
84.02
0
1.24
(0–0.34)a
(0.97–1.74)
1.69
1.33
(0–2.21)
(1.16–1.45)
0
1.45
(0–2.03)a
(0.89–1.75)
95.53
95.49
(80.85–92.05)a,b (95.2‒96.5) (94.61–95.67) (59.7–86.35)a,b (94.83–95.72) (94.93–96.75) 204 (181–219)
294
266
(194–307)
(187–323)
134 (111–177)a,b
284
290
(203–302)
(281–295)
a Periods of observation being the same, the differences are statistically significant in comparison to the corresponding control group at p < 0.01; b periods of observation being the same, the differences are statistically significant in comparison to the corresponding group
administered with pure AG at p < 0.01. Statistical significance was calculated via the Mann–Whitney method.
first experimental group (table). Pure AG being used, the total numbers of cells per area unit are similar to the control values in both periods of observation. Hyperchromic neurons were estimated taking into account the expression of caspase-3 in these cells, with the compacted neurons containing dark cytoplasm and no distinct nucleus being considered hyperchromic. It was found that, in the first experimental group, the percentage of hyperchromic neurons which do not express caspase-3 is 2.9-fold higher than that of hyperchromic neurons which express the protein. In the long-term period of observation (in the second experimental group) the opposite pattern is observed, namely, the percentage of hyperchromic neurons which express caspase-3 is higher than that of hyperchromic neurons without the expression (table). The numbers of hyperchromic neurons which express caspase-3 in both experimental groups (in the early and long-term periods of observation) are significantly higher than the corresponding control values. As to the IIIe group administered with pure AG, the percentage of hyperchromic neurons is equal to 0.33 (0–0.67) and not statistically significantly different from the control value; however, it is significantly lower than the same parameter of Ie group. An examination of prepared samples of the brain of animals after the long-term survival period shows a significant increase in the percentage of hyperchromic neurons which express caspase-3 over the corresponding control group (table). The exposure to pure AG is
not observed to result in the appearance of hyperchromic cells with the expression of caspase-3 in the longterm period. In this period, the number of hyperchromic cells which express caspase-3 is significantly higher than that obtained in the early period of observation. This circumstance seems to be due to the worsening of adverse effects of silver nanoparticles along with the functional aging and depletion of brain cells. The percentage of normal neurons which express caspase-3 is also significantly higher in Ie and IIe groups than in the corresponding control groups in both periods of observation (table). Meanwhile, in the cases of IIIe and IVe groups, this parameter is no different from the control values and equal to zero, respectively. It should be noted that, in normal unmodified neurons, caspase-3 is localized strictly at the cell periphery or as compact groups, whereas in hyperchromic neurons the protein is distributed throughout the cell without any pronounced localization (Fig. 1). The numbers of normal cells without expression of caspase-3 in Ie, IIIe, and Ic groups have no statistically significant differences. In the long-term period of observation, this parameter is significantly lower in the IIe group than in the corresponding control value (p ≤ 0.01) and the value in IVe group (pure AG), with the latter, in turn, being no different from the control values (table).
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(b)
(c)
(d)
Fig. 1. The expression of caspase 3 in neurons of the brain after administration with silver AG (a) in the first period of observation and (b) in the long-term period of observation or with pure AG (c) in the first period of observation and (d) in the long-term period of observation. Thionine staining was used as an immunohistochemical reaction for caspase-3; 400-fold magnification.
Thus, an increase in the number of hyperchromic neurons which express the pro-apoptotic protein and a sharp reduction in the number of normal neurons in the long-term period of observation indicates the dynamic development of pathological process in white rats. The appearance of the long-term effects induced by the administration of silver nanobiocomposites in rats and the absence of the effects in the case of pure AG may be due to physical and chemical properties of silver nanoparticles such as the long-term persistence in the body and the ability of accumulating and forming conglomerates within cell structures and intercellular space. Meanwhile, the prolonged residence and low rate of elimination of silver nanoparticles from the body is likely to contribute to the development of accumulated adverse effects. The optical microscopy of prepared samples of nervous tissue of the brain of albino rats of all experimental groups shows the development of perivascular edema around blood vessels of the brain in the early period of observation (Fig. 2a). This result indicates the hemodynamic disturbances leading, in turn, to a decrease in the trophic function of the tissue and disruption of cell metabolism. In the long-term period, the edema is not observed; however, the full recovery NANOTECHNOLOGIES IN RUSSIA
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does not occur and perivascular spaces remain enlarged (Fig. 2b). The obtained experimental results indicate the ability of silver nanoparticles encapsulated in a polymer matrix to penetrate the blood–brain barrier after their administration in laboratory animals. The disruption of the structure of nervous tissue and functional properties of neurons is an indirect confirmation of this assumption. The ability of silver molecules to block the thiol groups of proteins and inactivate ATP activity of proteins results in dysfunctions of the latter and may trigger apoptosis. In the case of disruption of metabolism in the body, a cell may undergo apoptosis in various ways, with caspase-3 being one of the end points of the cascade of activation of proteolytic enzymes leading to programmed cell death. Taking into account that the mechanism of development of toxic effects of silver is due to oxidative stress, membrane damage, and mitochondrial dysfunction, the mitochondrial pathway of apoptosis may occur, leading to activation of caspase-3. It should be noted that in recent years the triggering of apoptosis in tissues and organs of the body, including nervous tissue, induced by various nanoparticles has been reported [6]. These data, along with results obtained in the 2015
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(a)
(b)
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Fig. 2. (a) Multiple perivascular edema of blood vessels of the brain tissue induced by administration with silver AG in the first period of observation, (b) enlarged perivascular spaces induced by administration with silver AG in the long-term period of observation, (c) enlarged perivascular spaces and development of perivascular edema induced by administration with pure AG in the first period of observation, and (d) retention of enlarged perivascular spaces induced by administration with pure AG in the longterm period of observation. Staining with hematoxylin and eosin was used; 400-fold magnification.
present work, contribute to the development of a universal technology for the safe use of nanopreparations and the medical and biological assessment of exposure to nanopreparations for effective health care. CONCLUSIONS Exposure to silver nanoparticles encapsulated in a polymer matrix leads to not only structural but also functional changes in the brain tissue due to the hemodynamic disturbance of the tissue and the activation of pro-apoptotic proteins, resulting in the triggering of apoptosis in cells. REFERENCES 1. J. H. Ji, Inhalat. Toxicol. 19 (10), 857–71 (2007). 2. M. C. Stensberg et al., Nanomedicine (London), No. 6, 879–898 (2011). 3. S. K. Sahoo, S. Parveen, and J. J. Panda, Nanomed.: Nanotechnol., Biol. Med., No. 3, 20 (2007). 4. N. Lewinski, V. Colvin, and R. Drezek, Small J. 4, 26 (2008).
5. A. B. G. Lansdown, Adv. Pharmacol. Sci. 10, 910686 (2010). 6. M. G. Shurygin et al., Nanomed.: Nanotechnol., Biol. Med. 7, 827–833 (2011). 7. K. Unfried, Nanotoxicology 1, 52–71 (2007). 8. M. Allsopp, A. Walters, and D. Santino, Preprint of Greenpeace Research Laboratories (2007). 9. G. F. Prozorova, A. S. Pozdnyakov, N. P. Kuznetsova, et al., Int. J. Nanomed., No. 9, 1883–1889 (2014). 10. V. I. Dubrovina and E. P. Golubinskii, et al., “Studing of ferrogale effect on protective properties of Yersinia pestis EV,” Sibir’-Vostok, No. 3, 8–9 (2002). 11. Patent RF No. 2256668 (2005); RZh Khim., 05/2419F.22P (2005). 12. V. I. Dubrovina, S. A. Medvedeva, et al., Immunomodulating Action of Arabinogalactan of Siberian Larch (Farmatsiya, Moscow, 2001), pp. 26–27 [in Russian]. 13. E. N. Medvedeva, V. A. Babkin, and L. A. Ostroukhova, Khim. Rastit. Syr’ya, No. 1, 27 (2003). 14. M. A. Abdelhalim and B. M. Jarrar, J. Nanobiotechnol. 10, 5 (2012).
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