ISSN 00124966, Doklady Biological Sciences, 2016, Vol. 467, pp. 65–67. © Pleiades Publishing, Ltd., 2016. Original Russian Text © A.M. Ezhkova, A.Kh. Yapparov, V.O. Ezhkov, L.M.Kh. Bikkinina, I.A. Yapparov, A.P. Gerasimov, 2016, published in Doklady Akademii Nauk, 2016, Vol. 467, No. 2, pp. 242–245.
GENERAL BIOLOGY
Development of Nanostructured Phosphorite: Study of the Safety of Application A. M. Ezhkova, A. Kh. Yapparov, V. O. Ezhkov, L. M.Kh. Bikkinina, I. A. Yapparov, and A. P. Gerasimov Presented by Academician V.G. Sychev September 9, 2015 Received September 3, 2015
Abstract—A nanostructured mineral food supplement with a particle size of 60.0–120.0 nm was manufac tured from phosphorite by ultrasonic dispersion. It was found that intragastric administration of nanostruc tures phosphorite to mice is relatively safe: clinical signs of intoxication appeared after a single administration of the preparation only at a dose of 90 mg/kg; a dose of 150 mg/kg caused death of 8% of mice, in which inju ries of organs of the gastrointestinal tract were observed. When the preparation was administered subcutane ously, intramuscularly, or intraperitoneally, small phosphorite conglomerates and inflammation of the sur rounding tissues and organs were observed at the injection site. Death of 25% of animals was observed in the group of mice which received intraperitoneal injections of nanophosphorite at a dose of 200 mg/kg. DOI: 10.1134/S0012496616020034
Biomedical Research Involving Animals [3]. The study of the potential modes of administration of nanostructured phosphorite into mice and the possible toxic properties of preparation was carried out accord ing to the Methodical Guide [4] on the Assessment of Nanomaterial Safety no. 1.2.252009 and the A Guide to Experimental (Preclinical) Trials of Pharmaceutical Agents [4, 6]. Preparation of nanostructured phosphorite and study of its properties were performed at the Nanoma terials and Nanotechnologies Research Innovation and Application Center (Kazan, Tatarstan, Russia). Nanostructured phosphorite was obtained by sonica tion of a finely ground phosphorite meal at a frequency of 18.5 kHz (±10%). The specific power of an UZU0.25 instrument (Vektor, Russia) was 80 W/L; the oscilla tion amplitude of ultrasonic waveguide, 5 µm; the duration of exposure, 20 min. Nanostructured phos phorite was stabilized in deionized water at a concen tration ratio of 1 : 20. The structure was studied by intermittentcontact atomic force microscopy (AFM) on a MultiMode V scanning probe microscope (Veeco, United States). The chemical composition of phosphorite was determined by atomic emission spectrometry with the use of an iCAP 6300 DUO spectrometer (Thermo Sci entific, United States). Not only phosphorus, but a large amount of essen tial elements necessary for the functioning of a biolog ical object, were found in studying the chemical com position of phosphorite meal. The chemical composi tion of phosphorite meal was (in percent): Р2О5, 12.0;
Nanotechnology opens up broad prospects for the development of new highly efficient pharmaceuticals and food additives. There are published data on the manufacture of dosage forms based on natural miner als [8]. Their use is caused by the ion exchange and sorption properties and the presence of a wide range of biogenic elements in their composition. One of the unique natural minerals is phosphorite. The use of phosphorite as a mineral supplement to the food of farm animals contributes to their productivity and improvement of the quality of functional foodstuff sat urated with phosphorus compounds of biological ori gin. Therefore, the purpose of this study was to obtain nanostructured phosphorite and determine its safe doses for using as a highly efficient animal food addi tive. Nanostructured phosphorite was obtained from phosphorite meal (sieve no. 0.16) made of natural phosphorite from the Syundyukovskoe deposit of Tatarstan. Experiments for estimating safety were car ried out on male outbred mice aged 4 months and weighing 23.9–26.2 g (obtained from the Tatar Inter regional Veterinary Laboratory, Kazan, Tatarstan, Russia). Laboratory animals were kept and fed accord ing to [2, 5]. Experiments were carried out in accor dance with the International Guiding Principles for
Tatar Research Institute for Agricultural Chemistry and Soil Science, Kazan, 420059 Tatarstan, Russia email: egkova
[email protected];
[email protected] 65
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CaO, 37.8; MgO, 3.4; Fe2O3, 8.0; Al2O3, 8.4; F, 1.3; CO2, 4.0; K2O, 1.8; Na2O, 1.5; SiO2, 18.0; SO2, 3.8. The mineral composition was (in percent): phosphate, 64.0; glauconite and hydromica, 22.0; quartz, 7.0; cal cite, 0.7; siderite, 2.0; pyrite, 3.5; gypsum and other sul fates, 0.7; other (feldspar and montmorillonite), 0.1. When studying phosphorite by AFM, we found that its structure was represented by capsuleshaped parti cles. The particles had a width of 320.0–400.00 nm and a length of 0.8–1.2 µm; the edges of capsules had a rounded shape. Two kinds of particles with sizes from 60.0 to 120.0 nm were observed in the structure of modified phosphorite. Particles with larger sizes had an elongated conical shape with a sharp apex; particles of smaller sizes were of an oval–spherical shape. The reduction in size and changes in the nano structured phosphorite particle shape provide grounds for assuming a change in its properties in the phase of nanodispersion, which leads to the necessity of study ing its toxicity. It is known that the toxicity of nanopar ticles is determined not only by their sizes, but also by their shape. Nanoparticles of a dendritic and spindle shape exhibit greater cytotoxicity as compared with spherical particles [7, 9, 10]. Testing of the way of administration of a substance in the body that will be used in daily practice is man datory in the study of the potential modes of adminis tration of new substances [6]. We propose the use of nanostructured phosphorite as a food additive; there fore, the study of delivery into the animal body by means of an intragastric flexible atraumatic probe has become a priority. We also tested other modes of administration of nanostructured phosphorite, such as intramuscular, intraperitoneal, and subcutaneous injections. In the experiment, we used an aqueous sus pension of nanostructured phosphorite that was administered at a single dose of 20 mg/kg for all modes of administration under investigation. The choice of this dose is based on the results of previous studies of nanosized bentonite manufactured in a similar man ner from a natural mineral [8]. We did not detect any signs of intoxication in the mice receiving the preparation by intragastric admin istration. The mice were alive throughout the entire period of observation. An increase in the signs of intoxication was observed during the first three days of intramuscular and subcutaneous injections; the ani mals exhibited weak water and food excitability. Com plete refusal of food and water was observed in mice injected intraperitoneally. Death of three of 12 indi viduals was observed in this group of animals that received the preparation at a dose of 20 mg/kg. Total depletion and peritonitis were observed at a postmor tem autopsy; nonresorbed nanostructured phospho rite was present in the abdominal cavity in the form of a punctulate cheesy mass.
On the fifth day after administration of nanostruc tured phosphorite, we conducted a diagnostic autopsy of mice in all groups. In mice injected intraperito neally, intramuscular, and subcutaneously, we observed unresolved phosphorite in the subcutaneous space, muscle tissue, and the abdominal cavity, which caused inflammatory responses in the tissues and organs adjacent to the injection site. The mineral was not found in the gastrointestinal tract of mice receiv ing intragastric phosphorite injection; there were no changes in internal organs and tissues. Thus, oral administration of nanostructured phos phorite is a relatively safe route of its delivery to the animal body. The optimum doses of native minerals for animal feeding are 150–500 mg/kg body weight [5]. At the next step of the study, in order to determine the safe doses of nanostructured phosphorite for oral adminis tration, we used the lowest dose from this range as an initial dose, which was further decreased by 20, 40, 60, and 80%. The preparation was administered intragas trically at a single dose; mice were observed for 14 days. Control mice received native phosphorite and deionized water. The scheme of the experiment and the indicators of animal death are presented in the table. General depression, low mobility, and indifferent attitude to food and water were observed in mice of all groups in the first minutes after the introduction of an atraumatic probe with the tested preparation. The clinical signs of intoxication appeared within the first hour after administration of phosphorite to mice in groups I–III (the dose range 90–150 mg/kg): animals gathered in groups, huddled close to each other, and did not respond to external stimuli; visible mucous membranes and tails were cyanotic. At the same time, responses of different intensities to external stimuli appeared in mice of groups IV–VII. By the end of the fourth hour after administration of nanostructured phosphorite, we observed diarrhea in mice in groups I and II (doses of 150 and 120 mg/kg); visible mucous membranes were cyanotic, low mobility and indiffer ent attitude to food and water persisted. In mice of group III (90 mg/kg), behavioral reactions were restored and interest in water appeared. Diarrhea was observed in four animals from this group. Deviations in behavior of mice in groups IV, V (doses of 60 and 30 mg/kg), and VI (control, native phosphorite at a dose of 150 mg/kg) were not observed: the animals responded to external stimuli, were active; water and food excitabilities were retained. On the second day, behavior responses of mice in groups III–VI were identical to those of control ani mals in group VII. Diarrhea and cyanotic mucous membranes were retained in mice of group I and II (doses of 150 and 120 mg/kg); responses to external stimuli appeared; excitabilities to water and food were DOKLADY BIOLOGICAL SCIENCES
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DEVELOPMENT OF NANOSTRUCTURED PHOSPHORITE
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The scheme of the experiment, doses, and amounts of components administered intragastrically to mice Group of animals
Phosphorite dose, mg/kg
Phosphorite amount, mg per mouse
Water amount, mL
Number of dead mice
150 120 90 60 30 150 –
3.7 3.0 2.2 1.5 0.7 3.7 –
0.5 0.5 0.5 0.5 0.5 0.5 0.5
1 0 0 0 0 0 0
I II III IV V VI (control, native phosphorite) VII (control, deionized water)
Mice in groups I–V received nanostructured phosphorite (n = 12 in each group).
recovered. One individual died in group I upon administration of preparation at a dose of 150 mg/kg (LD8). Punctulate hyperemia of mucosa of the esoph agus, stomach, and duodenum was observed at a post mortem autopsy. Isolated petechial hemorrhages were observed under the liver capsule. The kidneys on the cut had a strained capsule; there was no clear bound ary between the cortical and medullary layers. In the 14day observation period, the general con dition of the mice of the experimental groups was sat isfactory; water and food excitabilities were preserved; behavioral reactions, condition of hair, visible mucous membranes, and skin were identical to those of the control animals. Thus, intragastric administration of nanostruc tured phosphorite at single doses of 30 and 60 mg/kg did not affect the general state of animals; clinical signs of intoxication appeared at a dose of 90 mg/kg, the lethal dose (LD8) was 150 mg/kg. ACKNOWLEDGMENTS This study was performed in accordance with the plan of research work of the Tatar Research Institute of Agricultural Chemistry and Soil Science under the State Contract “Basic and Applied Scientific Research in the Framework of Implementation of the Program of Basic Scientific Research of the State Academies of Sciences for 2013–2020,” item 19, research project no. 074620140012 “Determining the Biological Safety of Nanosized Minerals for Their Use in Feeding of Farm Animals.” REFERENCES 1. Agromineral’nye resursy Tatarstana i perspektivy ikh ispol’zovaniya (Agromineral Resources of Tatarstan and Prospects of Their Exploitation) Yakimov, A.V., Eds., Kazan: Fen, 2002. 2. Veterinarnaya laboratornaya praktika (Veterinarian Laboratory Practice), Moscow: Sel’khozizdat, 1963, vol. 2. DOKLADY BIOLOGICAL SCIENCES
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3. Mezhdunarodnye rekomendatsii (eticheskii kodeks) po provedeniyu medikobiologicheskikh issledovanii s ispol’zovaniem zhivotnykh (International Guiding Prin ciples for Biomedical Research Involving Animals), Moscow: Sovet Mezhdunar. Nauch. Organizatsii, 1985. 4. MU 1.2.252009. Gigiena, toksikologiya, sanitariya. Toksikologogigienicheskaya otsenka bezopasnosti nano materialov. Metodicheskie ukazaniya (MU 1.2.252009. Hygiene, Toxicology, and Sanitary: Toxicological– Hygienic Assessment of Safety of Nanomaterials: A Methodical Guide), Moscow: Federal’nyi Tsentr Gigieny i Epidemiologii Rospotrebnadzora, 2009. 5. Ob utverzdenii pravil laboratornoi praktiki (On Authori zation of the Rules of Laboratory Practice) (Order no. 708n of the Ministry of Health and Social Development of the Russian Federation of August 23, 2010). http://www.consultpharma.ru/index.php/ru/documents/ drugs/299708232010. 6. Rukovodstvo po eksperimental’nomu (doklinicheskomu) izucheniyu novykh farmakologicheskikh veshchestv (A Guide to Experimental (Preclinical) Trials of Phar maceutical Agents) Khabriev, R.U, Eds., Moscow: Meditsina, 2005. 7. Yapparov, A.Kh., Aliev, Sh.A., Yapparov, I.A., Ezh kova, A.M., et al., Nanotekhnologii v sel’skom kho zyaistve: nauchnoe obosnovanie polucheniya i tekhnologii ispol’zovaniya nanostrukturnykh i nanokompozitnykh materialov (Nanotechnologies in Agriculture: The Sci entific Basis of Fabrication and Technologies of the Use of Nanostructured and Nanocomposite Materials), Yapparov, A.Kh, Ed., Kazan: Tsentr Innovats. Tekh nologii, 2013. 8. Ezhkova, A.M., Yapparov, A.Kh., Ezhkov, V.O., Yappa rov, I.A., Sharonova, N.L., Degtyareva, I.A., Khisam utdinov, N.Sh., and Bikkinina, L.M.Kh., Nanotech nol. Russia, 2015, vol. 10, no. 1/2, pp. 120–127. 9. Jiang, J., Oberdrster, G., Elder, A., Gelein, R., Mercer, P., and Biswas, P., Nanotoxicology, 2008, vol. 2, no. 1, pp. 33–42. 10. Wang, J., Zhou, G., Chan, C., et al., J. Phys. Chem. Toxicol. Lett., 2007, vol. 168, no. 2, pp. 176–185.
Translated by G. Levit
