1. MORPHOLOGICAL CHANGES IN ORGANS OF IMMUNOGENESIS AND INDICATORS OF PERIPHERAL BLOOD UNDER THE INFLUENCE OF GOLD NANOPARTICLES IN THE EXPERIMENT Alla B. Bucharskaya, Saratov State Medical University, Russia Svetlana S. Pakhomy, Saratov State Medical University, Russia Olga V. Zlobina, Saratov State Medical University, Russia Galina N. Maslyakova, Saratov State Medical University, Russia Olga V. Matveeva, Saratov State Medical University, Russia Irina O. Bugaeva, Saratov State Medical University, Russia Nikita A. Navolokin, Saratov State Medical University, Russia Boris N. Khlebtsov, Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, Russia Vladimir A. Bogatyrev, Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, Russia Nikolai G. Khlebtsov, Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, Saratov State University, Russia Valery V. Tuchin, Saratov State University, Russia

2. The aim of the work was to study in vivo the effects of gold nanoparticles (GN) of different sizes on the morphological structure of immunogenesis organs and the peripheral blood indicators in laboratory animals PURPOSE OF THE RESEARCH Colors of various sized monodispersed gold nanoparticles http://www.mi2g.com/cgi/mi2g/frameset.php?pageid=http%3A//www.mi2g.com/cgi/mi2g/press/110210.php

3. 2 nm GN, peroral administration during 15 days (n=6) 15 nm GN, peroral administration during 15 days(n=6) 50 nm GN, peroral administration during 15 days (n=6) Сontrol group, peroral administration of 0,9% saline solution during 15 days (n=6) Design of experiment The experiments were conducted accordingly “International guiding principles for biomedical researches involving animals” (CIOMS, ICLAS, 2012).

4. 1.Morphological method: Hematoxylin and eosin staining 2.Hematological test 0f peripherical blood 3.Morphological method of bone marrow examination: May- Grunwald staining 4.Morphometric analysis of the histological samples was conducted using the digital image analysis system Mikrovizor medical μVizo-103 (LOMO, Russia). Materials and methods

5. Results At the analysis of peripheral blood indicators of laboratory animals the decrease of leukocytes number was found. After administration of 15 nm gold nanoparticles the significant reduction of leukocytes and lymphocytes number were noted, the reducing of number of all types of leukocytes were detected in the group with administration of 50 nm gold nanoparticles.

6. Results The bone marrow examination after administration of nanoparticles the signs of irritation of leukocyte germ hematopoiesis were noted in all groups – the increasing of number of myelocytes, metamyelocytes and stab leukocytes. The most pronounced changes were observed in the groups with 15 nm and 50 nm nanoparticles administration, the decrease of leuco-erythroblastic ratio was noted, probably due to the decrease of segmented neutrophils. Fig.1 Bone marrow. May-Grunwald staining The indicators of bone marrow Control group Experimental groups with GN administration 1-3 nm15 nm50 nm Myeloblasts1,0±0,671.33±0.670.75±0.471.66±0.57 Neutrophil myelocytes1.16±0.286±2.16.75±1.5*7±1.73* Neutrophil metamyelocytes 3±0.67*13.3±2.88*10.75±2.36*11.3±3.3* Neutrophil stab leycocytes 7.5±1.817±1*17.5±1.73*13±1.73* Neutrophil segments 32.8±8.3122±6.0823.25±3.8623±2.645 Eosinophils1.66±0.671.66±0.571.25±0.9570.66±0.57 Basophils0,25±0,250,75±0,320,33±0,170,25±0,25 Monocytes 0.66±02±1.731.75±1.2581.33±1.15 Lymphocytes16±014.33±3.0516.5±4.0414.3±3.21 erythroblasts000.5±10.33±0.57 Basophilic normocytes 1±02±01.75±1.252.66±0.57 Polychromatic normocytes 1217.66±4.1613.5±6.420±4.35 Oxyphilic normocytes3±02.66±1.153.25±0.54.66±1.52 Leukocytic- erythroblastic ratio 2.6±01.7±0.732.05±0.37*1.19±0.32*

7. Results After administration of gold nanoparticles the morphokinetics of cellular elements of mesenteric lymph nodes had size-dependent character and was characterized by lymphocytopoiesis activation. The most striking changes occurred after administration of 15 and 50 nm gold nanoparticles compared with the control group. The number of test objects (on 6400 square) Zones of lymphatic nodes Control group Experimental groups with GN administration 1-3 nm15 nm50 nm Small lymphocytes Lymph follicules 64.2±2.162.3±1.671.2±1.2*83.3±1.7* Paracortical60.1±1.261.2±1.266.2±1.1*72.4±1.1* Brain cords20.2±1.318.3±1.012.6±0.120.8±0.6 Middle lymphocytes Lymph follicules 18.2±0.416.6±1.230.1±2.1*35.3±1.0* Paracortical12.1±1.114.3±0.819.1±0.826.6±0.8 Brain cords10.2±1.710.4±1.012.7±1.122.3±1.2 Large lymphocytes Lymph follicules 00.5±0.012.2±0.79.61±1.2 Paracortical0.75±0.10.67±0.11.2±0.31.2±0.23 Brain cords10.2±1.70.3±0.10.5±0.13.6±1.2 Immunoblasts Lymph follicules 2.5±0.32.9±0.14.8±0.3*10.4±0.3* Paracortical000.7±0.1*2.9±0.1* Brain cords2.3±0.32.0±0.33.5±0.45.2±1.0 Plasmocytes Lymph follicules 00.1±0.0200.35±0.5 Paracortical0.71±0.101.2±0.081.75±0.4 Brain cords15.3±0.617.9±1.226.4±3.4*38.4±3.4* Mastocytes Lymph follicules 000.6±0.40.8±0.7 Paracortical000.3±0.012.0±0.6 Brain cords0.3±0.010.1±0.071.4±0.033.4±0.5 Mitotic figures Lymph follicules 0.2±0.010.7±0.14.0±0.3*4.6±0.07* Paracortical0.8±0.160.9±0.21.2±0.062.1±0.16 Brain cords0.7±0.10.9±0.163.5±0.14.2±0.2 Conglomerates of 50-nm GNPs in the cytoplasm of macrophages. G.-E. x1500

8. Results After GN administration the morphological changes in spleen were presented in the form of circulatory disorders of varying severity and activation of proliferative processes. Normal blood supply of the spleen was observed only in 25% animals after 15 nm GN administration, in other cases the lack of blood supply was noted. The lymphoid follicles contain bright germinative centers represented by large blast cells, which indicates activation of proliferative processes. Spleen. Increasing of lymphoid follicles at 16-day administration of 50 nm GN. Hematoxilin-eosin.

9. Conclusions At study of peripheral blood indicators the size-dependent decrease in the number of neutrophils and lymphocytes was noted, especially pronounced in rats after administration of 50 nm GN. At bone marrow examination the stimulation of lymphocytic and myelocytic germs of hematopoiesis was recorded after GN administration. The signs of strengthening of cell maturation processes was noted in mesenteric lymph nodes, which was reflected in increased amounts of immunoblast and large lymphocytes. The quantitative changes of cellular components in immunogenesis organs indicate the activation of differentiation and proliferation processes. We suggest the presence of immunomodulating effect of 15 nm and 50 GN on on lymphocyte subpopulations in spleen and lymph nodes.