Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo

Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo

The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. Materials and Methods: Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by lase...

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Дата:2012
Автори: Rieznichenko, L.S., Dybkova, S.M., Gruzina, T.G., Ulberg, Z.R., Todor, I.N., Lukyanova, N.Yu., Shpyleva, S.I., Chekhun, V.F.
Формат: Стаття
Мова:English
Опубліковано: Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України 2012
Назва видання:Experimental Oncology
Теми:
Original contributions
Онлайн доступ:http://dspace.nbuv.gov.ua/handle/123456789/138708
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Назва журналу:Digital Library of Periodicals of National Academy of Sciences of Ukraine
Цитувати:Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo / L.S. Rieznichenko, S.M. Dybkova, T.G. Gruzina, Z.R. Ulberg, I.N. Todor, N.Yu. Lukyanova, S.I. Shpyleva, V.F. Chekhun // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 25-28. — Бібліогр.: 34 назв. — англ.

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spelling irk-123456789-1387082018-06-20T03:06:13Z Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo Rieznichenko, L.S. Dybkova, S.M. Gruzina, T.G. Ulberg, Z.R. Todor, I.N. Lukyanova, N.Yu. Shpyleva, S.I. Chekhun, V.F. Original contributions The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. Materials and Methods: Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interaction with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard biochemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by “Comet-assay” method. Results: The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na+,K+-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. Conclusions: Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It’s necessary to take into account a size-dependent biosafety level of nanoparticles. 2012 Article Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo / L.S. Rieznichenko, S.M. Dybkova, T.G. Gruzina, Z.R. Ulberg, I.N. Todor, N.Yu. Lukyanova, S.I. Shpyleva, V.F. Chekhun // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 25-28. — Бібліогр.: 34 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138708 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language English
topic Original contributions
Original contributions
spellingShingle Original contributions
Original contributions
Rieznichenko, L.S.
Dybkova, S.M.
Gruzina, T.G.
Ulberg, Z.R.
Todor, I.N.
Lukyanova, N.Yu.
Shpyleva, S.I.
Chekhun, V.F.
Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo
Experimental Oncology
description The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. Materials and Methods: Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interaction with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard biochemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by “Comet-assay” method. Results: The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na+,K+-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. Conclusions: Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It’s necessary to take into account a size-dependent biosafety level of nanoparticles.
format Article
author Rieznichenko, L.S.
Dybkova, S.M.
Gruzina, T.G.
Ulberg, Z.R.
Todor, I.N.
Lukyanova, N.Yu.
Shpyleva, S.I.
Chekhun, V.F.
author_facet Rieznichenko, L.S.
Dybkova, S.M.
Gruzina, T.G.
Ulberg, Z.R.
Todor, I.N.
Lukyanova, N.Yu.
Shpyleva, S.I.
Chekhun, V.F.
author_sort Rieznichenko, L.S.
title Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo
title_short Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo
title_full Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo
title_fullStr Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo
title_full_unstemmed Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo
title_sort gold nanoparticles synthesis and biological activity estimation in vitro and in vivo
publisher Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
publishDate 2012
topic_facet Original contributions
url http://dspace.nbuv.gov.ua/handle/123456789/138708
citation_txt Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo / L.S. Rieznichenko, S.M. Dybkova, T.G. Gruzina, Z.R. Ulberg, I.N. Todor, N.Yu. Lukyanova, S.I. Shpyleva, V.F. Chekhun // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 25-28. — Бібліогр.: 34 назв. — англ.
series Experimental Oncology
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fulltext GOLD NANOPARTICLES SYNTHESIS AND BIOLOGICAL ACTIVITY ESTIMATION IN VITRO AND IN VIVO L.S. Rieznichenko1,*, S.M. Dybkova1, T.G. Gruzina1, Z.R. Ulberg1, I.N. Todor2, N.Yu. Lukyanova2, S.I. Shpyleva2, V.F. Chekhun2 1F.D. Ovcharenko Institute of Biocolloidal Chemistry, NAS of Ukraine, Kyiv 03142, Ukraine 2R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. Materials and Methods: Water dispersions of gold nanoparticles of different sizes have been synthesized by Da- vis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interac- tion with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard bio- chemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by “Comet-assay” method. Results: The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na+,K+-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. Conclusions: Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It’s nec- essary to take into account a size-dependent biosafety level of nanoparticles . Key Words: gold nanoparticles, U937 cells, Guerin carcinoma, affinity, biological activity. Metal nanoparticles are a principally new class of compounds that possess significant biological activity and are potentially perspective for diagnos­ tics and treatment of diseases of different ethiology, especially cancer [1–5]. Gold nanoparticles, in comparison with other me­ tals, are characterized by unique physical, chemical, biological properties and functional activity [6–10]. The nanoparticle size and shape substantially define their properties [11–15]. High affinity to tumor cells, surface modification ability and special optical properties create the basis for effective usage of GNP as vectors for target antitumor drug delivery [16, 17], in cancer phototermal therapy [18–20], as contrasting agents in magnetic resonance and computer tomography [21, 22]. The aim of this work was the synthesis of gold nanoparticles with different sizes and estimation of their biological activity in vitro and in vivo. MATERIALS AND METHODS GNP have been synthesized by Davis’ method from the tetrachloroauric (III) acid (HAuCl4 · 3H2O) (≥99.9% trace metals basis, Sigma­Aldrich) [23] and have been characterized by their size using laser­correlation spectroscopy (Zetasizer­3, Malvern Instruments Ltd, UK) and transmission electron microscopy (JEM­1230, JEOL, Japan) methods. The concentrat ion of obtained GNP was 38.6 μg/ml by metal for each size of preparations. For in vitro experiments U937 (human leukemic monocyte lymphoma) cell line as model of tumor cells has been used. The cell line was obtained from the Bank of Cell Lines from Human and Animal Tissues, R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine. The cell’ viability has been estimated by trypan blue exclu­ sion test. The quantity of alive cells was > 90 % in all experiments. The GNP interaction with tumor cells has been visualized by confocal microscopy method (LSM 510 META, Carl Zeiss, Germany). U937 cell total membrane fraction isolation has been carried out by the method [24]. Protein content in membrane preparations has been evaluated by the method of Lowry [25]. Na+,K+­АТР­ase activity (E.C. 3.6.1.3) of U937 cell membrane fraction has been measured by method [26] at 37 °С in 1 ml incubation medium (50 мМ Tris­ HCl, 5 мМ MgCl2, 100 мМ NaCl, 20 мМ KCl, 3 мМ АТР (рН=7.5)). Membrane aliquot (15–20 μg of protein) was added into incubation medium, incubated for 10 min and stopped by addition of 1 ml 10% trichlo­ racetic acid. The phosphorus content has been mea­ sured by Fiske­Subbarow method [27]. For estimation of GNP influence on the enzyme activity the membrane fraction protein (150–200 μg) was mixed with GNP 3 min before the incubation (at concentration range of 0.11–1.1 μg/ml by metal). 20 мМ Тris­HCl buffer has been added in control sample instead of GNP. For in vivo experiments white inbred rats (males and females) with average weight of 180–230 g from vivarium of R.E. Kavetsky Institute of Experimental Pa­ thology, Oncology and Radiobiology, have been used. All experiments with laboratory animals using have been carried out in compliance with “Guide for the Care and Use of Laboratory Animals”. The transplantation of Guerin carcinoma to the laboratory rats has been carried out subcutaneously Received: September 29, 2011. *Correspondence: Fax: +38(044)424-80-78 E-mail: Reznichenko_LS@mail.ru Abbreviation used: GNP — gold nanoparticles. Exp Oncol 2012 34, 1, 25–28 26 Experimental Oncology 34, 25–28, 2012 (March) on the back using 23% suspension of tumor tissue in physiological solution. The study of GNP influence on the tumor growth in vivo has been initiated when tumor size reached ~ 30 mm × 40 mm × 20 mm. Experimental tumor­bearing animals have been kept at the standard regimen and housed in four groups: control group — 3 animals without treatment; and 3 experimental groups (8 animals per group) treated with single intravenous GNP injection (1 ml of GNP, 38.6 μg/ ml by metal) on the basis of 20, 30 and 45 nm preparations. The euthanasia of animals from control and experi­ mental groups has been performed 1 h after injection (4 animals from each experimental group) and 24 h after administration. The GNP distribution in tissues (thymus, brain, spleen, liver, kidneys, adrenal glands, lungs, heart and tumor) has been studied using the atomic­absorption spectrometry method [28] on the complex “Graphit ­2” (Ukraine). The sensitivity of the method is 6 ng/ml. The GNP DNA­damaging activity in vivo has been estimated by “Comet­assay” method (alkaline gel­elec­ trophoresis of isolated eukaryotic cells) [29]. Cell isolation from liver, kidneys, spleen, bone marrow, intestine and tu­ mor tissue has been performed using standard protocols. The statistical analysis of the obtained data has been carried out using Student’s t­criterion [30]. The differences p < 0.05 were considered as significant. RESULTS AND DISCUSSION Water dispersions of coagulation­resistant gold nanoparticles have been synthesized for analysis of their activity in vitro and in vivo. Average sizes of synthesized GNP preparations were 10; 20; 30 and 45 nm according to the data of laser­correlation spec­ troscopy and transmission electron microscopy. The electron­microscopic image of synthesized 10 nm gold nanoparticles is presented on Fig. 1. Fig. 1. Electron­microscopic image of synthesized gold nanopar­ ticles with average size of 10 nm (JEM­1230, JEOL, Japan) The method of synthesis used for the GNP production is based on the conversion of dissolved gold (water solu­ tion of tetrachloroauric acid) into insoluble condition with subsequent aggregation and crystallization of insoluble particles which form dispersed phase. The peculiarities of gold nanoparticle structure play an important role in conditions of their contact with different types of bio­ logical objects. The different quantity of constituent at­ oms, depending on the size of nanoparticles, bind to the surface: the percentage of surface atoms is higher for smaller particles. The increase of active surface area per mass, changes in interatomic distance and crystal lattice period affect the nanoparticle ability to penetrate into the cell, their biological activity as well as chemical, physical and pharmacological properties [31, 32]. High level of the GNP accumulation in U937 tumor cells has been determined by confocal microscopy by layer­by­layer scanning (Fig. 2). Fig. 2. Confocal­microscopic image of U937 tumor cells (cell concentration is 106 cells/ml) after 3 min incubation with 30 nm gold nanoparticles at the concentration of 12.7 μg/ml by metal. Z­scanning with 1 μm step; GNP maximum accumulation is pre­ sented as red staining (LSM 510 META «Carl Zeiss», Germany) The most effective GNP accumulation by U937 tu­ mor cells has been observed for 20 and 30 nm gold nanoparticles. In vitro biological activity of synthesized gold nanoparticles is estimated through measuring the values of Na+,К+­АТР­ase and Mg2+­АТР­ase activi­ ties of membrane fraction of U937 cells treated with GNP. The obtained results have demonstrated the dependence of such activity from nanoparticles size (Fig. 3, curves 1–4). GNP with average size of 10 nm in all studied concentrations (0.11–1.1μg Au/ml) inhibited the enzyme activity by 70% in comparison with control cells (curve 1), GNP with 20 nm diameter — by 20 % (curve 2). At the same time GNP with average size 30 nm and 45 nm in concentration range 0.11–1.10μg Au/ml stimu­ lated Na+,K+­АТР­ase activity by 30–40 % (curve 3), and 20–40% (curve 4), respectively. Na +/ K+ -A TP -а se a ct ivi ty А /А 0 , % 0 20 40 60 80 100 120 140 160 180 0 0,2 0,4 0,6 0,8 1 1,2 Gold nanoparticles concentration, µg/ml by metal 1 2 3 4 Fig. 3. Changes of U937 tumor cell membrane fraction Na+,К+­ АТР­ase activity value (А/А0, %) upon the influence of gold nanoparticles with average sizes: 1–10 nm, 2–20 nm, 3–30 nm, 4–45 nm. (M±m; n=5, p < 0.05 compared to control — А0). Native Na+,К+­АТР­ase activity value is considered as 100 % (control) Experimental Oncology 34, 25–28, 2012 (March)34, 25–28, 2012 (March) (March) 27 T h u s , i n G N P c o n c e n t r a t i o n r a n g e of 0.11–0.28 μg Au/ml (45nm) we have registered 20% elevation of the enzyme activity, while in concentration range 0.28–0.55 μg Au/ml GNP this value increased from 20% to 40% and was equal to 40% in concentra­ tion range of 0.55–1.10 μg Au/ml. However, treatment of U937 cells with GNP (in all in­ vestigated sizes) has no significant influence on Mg2+­ АТР­ase activity of cell membrane fraction. In vivo study of GNP biological activity after their intravenous injection is important for estimation of GNP perspective usage in cancer diagnostics and therapy. That’s why we have estimated the patterns of GNP distribution and accumulation in organs and tissues of experimental animals. Via atomic absorption spectrometry the GNP distribution and accumulation in organs and tissues of experimental normal animals and Guerin carcinoma bearing animals have been studied. For gold content analysis thymus, brain, spleen, liver, kidneys, adrenal glands, lungs, heart and tumor tissue of experimental animals have been examined 1 and 24 h after single GNP i.v. injection. It has been revealed that the gold is not present in studied organs of normal experimental animals either in 1 h nor 24 h after GNP injection. It may be suggested that tumor influences gold nanopar­ ticles distribution in tumor­bearing organism resulting in their accumulation in non tumor tissue in contrast to the non­tumor­bearing host. However, in studied organs of tumor­bearing animals the picture was different. 20 nm GNP distribution and ac­ cumulation in different organs of experimental animals are demonstrated in Table 1. In tumor bearing animals 1 h after GNP injection gold content values were as fol­ lows: in brain — 26.09±3.15 ng Au/g tissue, kidneys — 51.40±6.23 ng Au/g tissue, spleen — 60.00±5.78 ng Au/g tis­ sue, and liver — 37.60±4.07 ng Au/g tissue. Table 1. Accumulation of GNP with average size of 20 nm in organs and tissues of Guerin carcinoma bearing animals after intravenous injection of nanoparticles (1 ml) Target organ Concentration of gold, ngAu/g tissue Control group 1 h after GNP in- jection 24 h after GNP injection Tumor - 78.90±5.27 31.70±2.69 Thymus - - - Brain - 26.09±3.15 - Lungs - - - Heart - - - Adrenal glands - - - Kidneys - 51.40±6.23 - Spleen - 60.00±5.78 - Liver - 37.60±4.07 12.70±1.92 Note: “-” — gold content below detection limits (M ± m; n = 4, p < 0.05). In 24 h after GNP injection the level of gold accumu­ lation in liver, spleen and kidneys has been substantial­ ly decreased and amounted to 12.7±1.92 ng Au/g tis­ sue for liver and was absent in spleen and kidneys. These data demonstrate the principal role of these organs in detoxication and clearance of GNP from the organism. The peculiarities of GNP accumulation in brain of tumor­bearing animals have shown their ability to penetrate through hematoencephalic barrier what can be used in diagnostics and target therapy. The highest level of the gold accumulation (78.90±5.27 ng Au/g tissue), comparatively with other organs, has been registered in tumors in 1 h after GNP injection, while in 24 h it amounted to 31.7±2.69 ng Au/g tissue, what was twice higher than its residual concentration in liver. Thus, the GNP high clearance rate in the conditions of their i.v. injection to normal animals has been demo­ nstrated while as GNP predominant accumulation has been determined in tumor tissue at both time points (1 and 24 h after intravenous injection). GNP usage in diagnostics or therapy should be validated through their biosafety marker tests in vitro and in vivo. One of most sensitive marker tests of biosafety is genotoxicity test, which reveals agent DNA­damaging influence. For this purpose we have used “Comet assay” method. The GNP genotoxicity, depending on their size, has been analyzed earlier [33, 34] in vitro and in vivo. The 20 nm GNP genotoxic influence on kidney cells of tumor­bearing rats in 1 h after single GNP intrave­ nous injection has resulted in 24.04% of DNA in comet tail versus 0.24% for negative control (Table 2, Fig. 4). These data evidence on potential risk of 20 nm GNP injection for normal organs. Table 2. GNP genotoxicity evaluation in organs and tissues of Guerin car- cinoma-bearing animals 1 h after intravenous injection of nanoparticles Target organ % of DNA in co- met tail, negative control % of DNA in comet tail in GNP-adminis- tered animals 20 nm 30 nm 45 nm Liver 0.27 0.44 0.32 0.35 Kidneys 0.24 24.04 0.27 0.33 Spleen 0.23 0.27 0.27 0.27 Tumor 0.92 apoptosis 0.91 0.93 Fig. 4. Electrophoretic image of kidney cell damaged DNA (DNA­comet) 1 h after i.v. injection of 20 nm gold nanoparticles to tumor­bearing animals 1 h after 20 nm GNP injection the apoptosis rate of tumor cells yielded up to 80%. It is the evidence of total tumor cells death. The 30 and 45 nm GNP injection didn’t cause DNA damage in organs and tumor tissue of experimental animals in 1 h after i.v. injection. In other words, 30 and 45 nm nanoparticles revealed biosafety in such test. In 24 h after i.v. injection of 20, 30 and 45 nm GNP there has been registered no genotoxic influence (Table 3). 28 Experimental Oncology 34, 25–28, 2012 (March) Table 3. GNP genotoxicity evaluation in organs and tissues of Guerin car- cinoma-bearing animals 24 h after intravenous injection of nanoparticles Target organ % of DNA in co- met tail, ne gative control % of DNA in comet tail in GNP-adminis- tered animals 20 nm 30 nm 45 nm Liver 0.31 0.41 0.43 0.42 Kidneys 0.29 0.57 0.52 0.56 Spleen 0.25 0.25 0.27 0.32 Tumor 0.71 0.83 0.77 0.71 So, the high specific size­dependent biological activity in vitro and in vivo of synthesized water disper­ sions of GNP has been revealed. In vitro data show that synthesized gold nanoparticles possessed by ex­ pressed size­dependent modulation of membrane Na+,K+­АТР­ase activity in U937 tumor cells. In vivo results indicate that gold nanoparticles possess high affinity to tumor cells after their i.v. injection to ex­ perimental animals. 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