ISSN 10623590, Biology Bulletin, 2014, Vol. 41, No. 3, pp. 255–263. © Pleiades Publishing, Inc., 2014. Original Russian Text © Yu.P. Buzulukov, E.A. Arianova, V.F. Demin, I.V. Safenkova, I.V. Gmoshinski, V.A. Tutelyan, 2014, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2014, No. 3, pp. 286–295.
ANIMAL AND HUMAN PHYSIOLOGY
Bioaccumulation of Silver and Gold Nanoparticles in Organs and Tissues of Rats Studied by Neutron Activation Analysis Yu. P. Buzulukova, E. A. Arianovab, V. F. Demina, I. V. Safenkovac, I. V. Gmoshinskib, and V. A. Tutelyanb a National
b
Research Center Kurchatov Institute, pl. Kurchatova 1, Moscow, 123182 Russia Institute of Nutrition, Russian Academy of Medical Sciences, Ust’inskii proezd 2/14, Moscow, 109240 Russia c Bakh Institute of Biochemistry, Russian Academy of Science, Leninskii pr. 33/2, Moscow, 119071 Russia email:
[email protected] Received March 6, 2013
Abstract—Bioaccumulation of silver (Ag) and gold (Au) nanoparticles (NPs) with mean sizes of 35 nm and 6 nm, respectively, has been studied after their intragastric administration to rats at a dose of 100 µg/kg of body weight for 28 or 14 days. The organs and tissues (liver, kidney, spleen, heart, gonads, brain, and blood) were subjected to thermal neutron activation, and, then, the activity of the 110mAg and 198Au isotopes gener ated was measured. The NPs of both metals were detected in all biological samples studied, the highest spe cific weight and content of Ag NP being found in the liver, and those of Au being found in kidneys of animals. The content of Ag NPs detected in the brain was 66.4 ± 5.6 ng (36 ng/g tissue), no more than 7% of these NPs being localized in the lumen of brain blood vessels. The content of Ag and Au NPs found in organs and tissues of rats could be regarded as nonhazardous (nontoxic) in accordance with the known literature data. DOI: 10.1134/S1062359014030042
INTRODUCTION Nanoparticles (NP) and nanomaterials are consid ered nowadays as a factor that can have various, including toxic, effects on the human organism (Onishchenko and Tutelyan, 2007; Onishchenko et al., 2007; Khotimchenko et al., 2009; Gmoshinski et al., 2010). NPs in the composition of foodstuff can be present either as contaminants (components migrating from packing materials) or as food additives introduced intentionally and dietary supplements (DS) (Chaudhry et al., 2008; Vernikov et al., 2009; Nevzorova et al., 2009). Among the diversity of engi neered nanomaterials, silver (Ag) and gold (Au) NPs are some of the most popular products of nanoindus try. Their application has spread rapidly in the field of antimicrobial agents, cosmetics, packaging materials, and food supplements (Ag NP), as well as in medicine for diagnostics and delivering drugs to damaged organs and tissues (Au NP). Thus, safety evaluation of these materials and investigation of their absorption, biodis tribution, and affinity to different organs and tissues is an important task. Methods of detection and identification of NPs in biological objects such as transmission electron microscopy (TEM) (Tiede et al., 2008; Raspopov et al., 2012) are not always capable of detecting the NPs introduced by oral intake since their content in organs and tissues might be extremely low. In the case of the NPs of noble metals, which are not character ized by biotransformation in the organism (Abdelhaim
and Jarrar, 2012; Levard et al., 2012; Walczak et al., 2013), their absorption and distribution in organs and tissues could be studied by elemental analysis. It is sig nificant that the content of elements such as Ag and Au in the normal state in the organism is extremely low, so the presence of NPs of these metals entering an organism from the environment could easily be detected. One of the highly sensitive methods to deter mine the elemental composition of abiotic and biolog ical objects is neutron activation analysis (NAA), which consists of the treatment of samples with inten sive flux of thermal neutrons in a nuclear research reactor (Kuznetsov, 1974). As a result, the atoms of chemical elements in the composition of NPs absorb ing neutrons become radioactive; i.e., they function as labels marking NPs. As a rule, herewith β– and β+ active nucleotides are formed, which are also sources of characteristic γradiation in a narrow spectral energy range. Their activity proportional to the con tent of the element studied in a sample is recorded using a sensitive lowbackground γspectrometer. The advantages of the methods are the very high sensitivity, no need for complicated sample preparation (mineral ization), and the possibility to monitor simultaneously a number of elements entering into the composition of several nanomaterials introduced into an animal at the same time and present in an organism endogenously. The purpose of this work is to study the absorption and accumulation of Ag and Au NPs by NAA upon their repeated intragastric administration to adult rats.
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MATERIALS AND METHODS A preparation of Ag NPs, Argovit (VektorVita, Russia), was used in this study, which represents an aqueous dispersion of Ag NPs containing from 1.0 to 1.4% by weight of Ag and from 18.6 to 19.0% of a non toxic biocompatible polymer polyvinylpyrrolidone (PVP) as an authorized food additive, E1201. The principle of the production of the preparation mainly coincides with that described earlier (Lee et al., 2008). Ag preparations of this type are widely used in Russia and abroad as components of DS, cosmetics, disinfec tant agents, and other consumption goods. According to the TEM data (Fig. 1a), the mean size of the NP was 34.9 ± 14.8 (M ± SD); the minimal size was 8.4, and the maximal, 80.9 nm. The shape of the particles was close to spherical (Fig. 1c). Using dynamic light scat tering (DLS), it was found that more than 89% of Ag particles in the preparation have a diameter less than 100 nm (Fig. 1e). The significantly higher value of the mean diameter of the particles (64.2 ± 50.3 nm, M ± SD) determined by this method might be explained by the presence on the surface of NPs of extended solvation shells of PVP, which are not detectable using TEM, but affect the assessment of the hydrodynamic diame ter of the particles (Tiede et al., 2008). Au NPs were obtained according to the Safenkova method (Safenkova et al., 2012) in the presence of sodium citrate. The choice of the Au NP preparation mentioned obtained using the conventional technique is due to the fact that the NPs of this type are most widely used in the composition of medical diagnostic preparations including those that are introduced in vivo (Day et al., 2010). According to the TEM data (Fig. 1b), the mean diameter of the particles was 5.8 ± 0.9 (M ± SD), the maximal one was 8.5 nm, and the shape of the particles was close to spherical (Fig. 1d). According to the DLS data, more than 100% of the par ticles had a diameter less than 60 nm, the mean size of the particles being 9.74 ± 4.33 nm (M ± SD) (Fig. 1f). The carrier to introduce the NP preparations into the animal stomach was represented by ultrapure (ISO 3696 (1987) and NCCLS (1988)) deionized water obtained using a MilliQ Advantage А10 facility (Millipore SAS, France). The study was conducted on 15 male Wistar rats with an initial weight from 100 to 120 g. The animals were given ad libitum access to a balanced standard semisynthetic diet (Reeves et al., 1993) providing all the major required macronutrients and micronutrients and drinking water ( Russian Federation sanitary reg ulation no. 2.1.4.1074901). The NP preparations tested were intragastrically administered every day using a probe for 28 or 14 days of the experiment. Three groups of animals participated in the experi ment, each of them containing five rats. The first group is the control with the administration of deion ized water; the animals of the second and the third groups were administered Ag and Au NPs at a dose of
100 µg/kg of body weight for 28 or 14 days, respec tively. Throughout the feeding period, the animals were weighed daily using an electronic balance with preci sion up to ±1 g and changes of their appearance and behavior were registered. After the end of the experi ment, the animals were subjected to deep ether anes thesia and exsanguinated using the inferior vena cava. The abdominal cavity was aseptically opened, and the liver, kidney, gonads, spleen, heart, and brain were sequentially taken and put with sterile instruments into disposable polyethylene containers. The samples were stored at –20°C until analysis. Sampling and transfer of the samples in the National Research Cen ter Kurchatov Institute for the NAA were performed under cold chain conditions. The neutron activation of the isotopes entering into the composition of the Ag and Au NPs was carried out by irradiation with the flux of thermal neutrons (0.005 eV < En < 0.4 eV) in a VEK9 vertical experimental channel of an IR8 nuclear reactor (Russia). The mean density of the neutron flux during irradiation was 4.7 × 1012 cm–2 s–1. The biological samples with Ag NPs were irradiated for 24 h. The biological samples with Au NPs were divided into two groups according to their mass, the samples with a larger mass (liver, spleen, kidneys) being irradiated for 3 h, and the sam ples with a smaller mass being irradiated for 22 h. This approach was taken, above all, due to the necessity for the activity of individual samples not to exceed the radiation safety standards. At the same time with the samples studied, activa tion was performed of the reference samples of Ag and Au NPs represented by aqueous dispersions in the containers with the geometry close to that of contain ers for biological samples. After the end of the irradia tion cycle and withdrawal of the preparations from the reactor core, they were kept within a biological shield no less than 10 days to decrease the γradiation back ground of shortlived isotopes (mainly, 24Na, 31Si, and 29 Al with halflives T1/2 = 14.8, 2.6, and 0.11 h, respec tively) formed during the activation of the nuclei of the container and samples to the safe level. After that, γspectrometric analysis of the samples was carried out. The activity of the samples studied was measured by a γspectrometer (Canberra, United States) con sisting of a GC 4018 germanium semiconductor detector, a DSA 1000 analyzer, and Genie 2000 (Genie S501, Genie S502) program software. γIrra diation was recorded in the energy regions corre sponding to the most intense spectral lines of the ele ments analyzed (Table 1). For the samples with Au NPs, having measured the count rate of γquanta, the correction coefficients were used to reduce all the activities measured to the time corresponding to the moment when the samples were extracted from the reactor (the correction for radioactive decay) accord ing to the data of Table 1 using the conventional BIOLOGY BULLETIN
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100 nm (b)
(а)
50 nm
(c)
(d)
30
20
10
0
20
40
60
80
102
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3
4
5
(e)
6 (f)
7
8
9
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16 12 8 4
10–1
100
101
10–1
100
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Fig. 1. The results of physical and chemical characteristics of Ag (a, c, e) and Au (b, d, f). (a) and (b) are microphotographs of the particles obtained by TEM using a JEM100CX/SEG microscope (Jeol, Japan), accelerating voltage being 80 kV and mag nification being 30000 and 100000, respectively; (c) and (d) are the particle diameter distribution according to the TEM data: xaxis is the particle diameter, nm, and yaxis is the number of particles; (e) and (f) are particle distribution with respect to the hydrodynamic diameter according to the DLS: xaxis is hydrodynamic diameter, nm, and yaxis is the part of the total number of particles, %.
method (Kuznetsov, 1974). In measurement of the samples with Ag NPs, the radioactive decay did not significantly change the activity of the samples, so there was no need for corrections. BIOLOGY BULLETIN
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The content of Ag and Au in the NPs was determined using the reference method according to the formula mx = me(Aγ, x/Aγ, e),
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Table 1. Characteristics of the initial and activated isotopes of the elements involved in neutron capture reactions during activation of biological samples containing NPs Target isotope Element isotope Ag* Au* Fe** Na*** Zn***
109
Ag
197Au 58
Fe Na
23
64
Zn
Radioactive isotope
isotope content in the natural mixture, %
atomic mass, g/mol
σ, 10–24 cm2
48.2 100 0.282 100
108.905 196.97 57.93 22.99
4.4 98.7 1.3 0.53
48.6
63.93
0.6
isotope 110m
Ag
198Au 59
Fe Na
24
65
Zn
T1/2, days
irradiation type
Eγ, keV
nγ, decay–1
249.8 2.7 44.5 0.625
β–, γ β–, γ β–, γ β–, γ
244.3
β+, γ
657 412 1099 1369 2754 1115.5
0.95 0.95 0.56 1 1 0.51
σ is a capture cross section of the neutron; T1/2 is the halflife of the isotope; nγ is the quantum yield (number of photons) per decay; Eγ is energy of γradiation of the isotope; M is the molar mass of the isotope. * The microelements entering into the composition of the preparations of NPs used. ** The reference microelement used to estimate the content of blood in the brain. *** The background element impeding the activation analysis of the target microelements.
where me is the known mass of the element in the ref erence sample, Aγ, x and Aγ, e is the γactivity measured in the sample studied and the reference sample, respectively. The content of the chemical elements analyzed in the organs was expressed in nanograms (ng) per whole organ (tissue) and per mass unit (g) of the organ (tis sue). In evaluating the total content of the elements in the blood, correction for its incomplete sampling was made: the total blood mass of the animal was consid ered to be equal to 6% of the body mass. For every index, the mean value (M) with a standard error (m) was calculated. The statistical processing of the results was carried out in the SPSS 17.0 program. In assessing the sensitivity of the detection of Ag and Au NPs using the NAA, the detection limit (ISO 11929, 2010) was used, which represented the count rate so that the desired signal exceeded back ground fluctuations with a probability of 50%. With minor simplifications, the formula to calculate the detection limit (in units of the count rate of the decays recorded) is the following:
Lc = 2.33 × Rb T , where Lc is the detection limit, cps; Rb is the sum of the count rates of the ambient background and Compton background, cps; T is measurement time of the sample (3600 s). RESULTS AND DISCUSSION Throughout the whole period, when the NPs were introduced into the animals, they were characterized by uniform growth and normal appearance and behav ior. In all experimental groups of rats, the weight gain unsignificantly differed from that of the control ani
mals. Morbidity and mortality of the animals were not observed. The main problem in γspectrometric analysis of the activated biological samples was the high level of the continuous Compton background in all the ranges in which measurements were made. This background was caused by the presence in the sample of 24Na and 65Zn radionuclides emerging upon activation of bio logical samples containing natural sodium and zinc, which enter into the composition of all animal tissues. To overcome the effect caused by the sodium isotope, its relatively short halflife (0.623 days) was used, which was significantly smaller than the halflives of the marker isotopes used, 198Ag and 110mAg. The 65Zn isotope characterized by a long lifetime (245 days) determines the Compton background and basic mea surement error, then as 24Na having decayed. Together with the analysis of the activities of the Ag and Au target nucleotides, γspectrometry made it possible to assess the content of a number of isotopes formed from the biogenic microelements present in the samples naturally, including 59Fe, the additional information obtained being used to assess the possibil ity of the penetration of NPs from the bloodstream to the brain through the blood–brain barrier. Table 2 gives the results of the accumulation of Ag and Au NPs in rat tissues and organs. From the data present, it can be seen that the highest accumulation of Ag NPs was found in the liver and then in descend ing order in the blood, spleen, gonads, kidneys, brain, and heart. Introducing Au NPs into the rat stomach, the greatest mass of them is registered in the kidneys and a significantly smaller amount is found in the liver, blood, and spleen. The accumulation of Au NPs in the brain and gonads was small (in absolute magnitude) as compared to that in the organs mentioned above. BIOLOGY BULLETIN
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Table 2. The results of neutron activation analysis of Ag and Au in organs and tissues of experimental and control groups of rats expressed in the mass of the element of the whole organ Content, ng (M ± m)* Organs, tissue
experience (administration of NPs) Ag
Liver Kidneys Spleen Heart Gonads Blood Brain
Au
768 ± 246 39.5 ± 12.3*** 110 ± 14 11 ± 2.4 40.2 ± 6.2 198 ± 54*** 66.4 ± 5.6
75.1 ± 8.3 456 ± 34 43.2 ± 4.4 4.29 ± 1.25 8.12 ± 1.94 45.7 ± 10.6**** 2.52 ± 0.64
control (administration of water) Ag**
Au**
