Bulletin of Experimental Biology and Medicine, Vol. 157, No. 3, July, 2014
383
NANOTECHNOLOGY Penetration of Pegylated Gold Nanoparticles through Rat Placental Barrier N. A. Tsyganova, R. M. Khairullin, G. S. Terentyuk*, B. N. Khlebtsov**, V. A. Bogatyrev*,**, L. A. Dykman**, S. N. Erykov, and N. G. Khlebtsov*,** Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 3, pp. 366-369, March, 2014 Original article submitted November 20, 2012 Penetration of pegylated (enveloped in polyethylene glycol) gold nanoparticles 5 and 30 nm in diameter through the placental barrier was studied in pregnant rats injected intravenously with these particles in a dose of about 0.8 mg Au/kg on day 10 of gestation. The particles were visualized in tissues by silver nitrate autometallography; the total content of gold in the fetuses was evaluated by atomic adsorption spectroscopy. Gold nanoparticles were detected in the fetal liver macrophages and in the spleens; high total content of gold in the fetuses was demonstrated for particles of both sizes. The data suggest that gold nanoparticles penetrate through rat placental barrier in vivo. No morphological changes were detected in the liver, kidneys, spleen, and brain of fetuses. Key Words: gold nanoparticles; biodistribution; placental barrier The blood-tissue barriers in living organisms are structural and functional mechanisms with regulatory and protective functions, including protection from various nanoparticles. Nanomaterials of different size and shape of the particles, surface modification, and hence, different biodistribution, kinetics of the particles accumulation and discharge, potential recirculation from tissues to blood and vice versa have been actively studied in biomedicine over the recent 10-15 years [9]. A prospective nanomaterial is gold nanoparticles (GNP) used for diagnosis, addressed drug delivery, visualization of cell structures, photothermal and photodynamic therapy [2,6]. In photodynamic therapy, GNP are used in the form of nanocomposites conInstitute of Medicine, Ecology, and Physical Education, Ul’yanovsk State University; *N. G. Chernyshevsky Saratov State University; **Institute of Biochemistry and Physiology of Plants and Microorganism, the Russian Academy of Sciences, Saratov, Russia. Address for correspondence: [email protected]. R. M. Khairullin
sisting of a gold particle coated with a mesoporous layer of silicon dioxide doped with a photodynamic stain [10]. Despite wide use of GNP, the possibility of their penetration through the blood-tissue barriers is little studied [9]. According to some data, 2-20-nm GNP in size penetrate through the blood-brain barrier [4,8,12,14], blood–retinal [13], and blood–testis [5] barriers. Dose dependence of 13-nm GNP penetration into the brain has been detected [11]. In vitro experiments on human placenta have shown that 10-30-nm GNP do not pass through the placental barrier [12]. GNP 2 and 40 nm in size have not been detected in the fetuses of pregnant C57Bl/6 mice [14], but penetrate through the blood-brain barrier. However, the penetration of the placental barrier of rats has not been studied in vivo. Hence, despite a vast scope of data on the GNP biodistribution and toxicity [1,9], their effects on the fetuses of pregnant women are in fact unstudied. We studied the permeability of the placental barrier for GNP of 5 and 30 nm in laboratory rats.
0007-4888/14/15730383 © 2014 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 157, No. 3, July, 2014 NANOTECHNOLOGY
MATERIALS AND METHODS The study was carried out on pregnant outbred laboratory rats. Suspension of GNP (57 μg/ml) with a mean diameter of 5 and 30 nm was injected (1.5 ml) into the caudal vein twice at 24-h interval during stage II of hemochorial placenta development (day 10 of gestation). The particles were synthesized by citrate reduction of HAuCl4 by the method of France [2] and functionalized with thiolated polyethylene glycol molecules (PEG-SH; Nektar) for improving GNP bioavailability and stability in the bloodflow [6]. The mean size of the particles was evaluated under a Libra-120 electron microscope (Carl Zeiss). According to electron microscopy of 300-400 images of the particles, their mean sizes were 5.1±0.6 and 32.0±3.6 nm, respectively (mean±standard deviation). The females were divided at random into control and experimental groups, 5 per group. Pregnant controls were injected with physiological saline according to the same protocol as GNP. The animals were sacrificed under chloroform narcosis 24 h after the second injection of GNP or physiological saline. All experiments and handling of animals were carried out in accordance with the Directive No. 63 from September 22, 2010 of the Presidium and Parliament of Europe “On Protection of Animals Used for Research”, “Sanitary Regulations of Organization, Equipment, and Use of Experimental Biological Clinics” from April 06, 1993, and Order No. 267 “On Laboratory Practice Regulations” of the Ministry of Health of the Russian Federation from June 19, 2003. Penetration of GNP through the placenta and all the subsequent relevant effects on biological tissues were evaluated by the morphological changes in fetal liver, kidneys, spleen, and brain. Experimental material was fixed in 10% neutral formalin, embedded in paraffin, and analyzed by the standard histological methods.
Accumulation of GNP in cell and tissue structures was visualized by silver nitrate autometallography [7] in paraffin histological sections. The integral evaluation of nanoparticles accumulation in the fetuses was carried out by atomic adsorption spectroscopy on a Dual Atomizer Zeeman AA Spectrometer iCE 3500 (Thermo Scientific, Inc.). The same method was used for GNP measurements in the organs of pregnant females. The data were processed using Statistica 6.0 software (StatSoft Inc.).
RESULTS The total content of GNP 30 nm in diameter in experimental fetuses (about 0.25±0.08 μg/g) significantly (~6-fold) surpassed the control (p
383
NANOTECHNOLOGY Penetration of Pegylated Gold Nanoparticles through Rat Placental Barrier N. A. Tsyganova, R. M. Khairullin, G. S. Terentyuk*, B. N. Khlebtsov**, V. A. Bogatyrev*,**, L. A. Dykman**, S. N. Erykov, and N. G. Khlebtsov*,** Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 3, pp. 366-369, March, 2014 Original article submitted November 20, 2012 Penetration of pegylated (enveloped in polyethylene glycol) gold nanoparticles 5 and 30 nm in diameter through the placental barrier was studied in pregnant rats injected intravenously with these particles in a dose of about 0.8 mg Au/kg on day 10 of gestation. The particles were visualized in tissues by silver nitrate autometallography; the total content of gold in the fetuses was evaluated by atomic adsorption spectroscopy. Gold nanoparticles were detected in the fetal liver macrophages and in the spleens; high total content of gold in the fetuses was demonstrated for particles of both sizes. The data suggest that gold nanoparticles penetrate through rat placental barrier in vivo. No morphological changes were detected in the liver, kidneys, spleen, and brain of fetuses. Key Words: gold nanoparticles; biodistribution; placental barrier The blood-tissue barriers in living organisms are structural and functional mechanisms with regulatory and protective functions, including protection from various nanoparticles. Nanomaterials of different size and shape of the particles, surface modification, and hence, different biodistribution, kinetics of the particles accumulation and discharge, potential recirculation from tissues to blood and vice versa have been actively studied in biomedicine over the recent 10-15 years [9]. A prospective nanomaterial is gold nanoparticles (GNP) used for diagnosis, addressed drug delivery, visualization of cell structures, photothermal and photodynamic therapy [2,6]. In photodynamic therapy, GNP are used in the form of nanocomposites conInstitute of Medicine, Ecology, and Physical Education, Ul’yanovsk State University; *N. G. Chernyshevsky Saratov State University; **Institute of Biochemistry and Physiology of Plants and Microorganism, the Russian Academy of Sciences, Saratov, Russia. Address for correspondence: [email protected]. R. M. Khairullin
sisting of a gold particle coated with a mesoporous layer of silicon dioxide doped with a photodynamic stain [10]. Despite wide use of GNP, the possibility of their penetration through the blood-tissue barriers is little studied [9]. According to some data, 2-20-nm GNP in size penetrate through the blood-brain barrier [4,8,12,14], blood–retinal [13], and blood–testis [5] barriers. Dose dependence of 13-nm GNP penetration into the brain has been detected [11]. In vitro experiments on human placenta have shown that 10-30-nm GNP do not pass through the placental barrier [12]. GNP 2 and 40 nm in size have not been detected in the fetuses of pregnant C57Bl/6 mice [14], but penetrate through the blood-brain barrier. However, the penetration of the placental barrier of rats has not been studied in vivo. Hence, despite a vast scope of data on the GNP biodistribution and toxicity [1,9], their effects on the fetuses of pregnant women are in fact unstudied. We studied the permeability of the placental barrier for GNP of 5 and 30 nm in laboratory rats.
0007-4888/14/15730383 © 2014 Springer Science+Business Media New York
384
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 3, July, 2014 NANOTECHNOLOGY
MATERIALS AND METHODS The study was carried out on pregnant outbred laboratory rats. Suspension of GNP (57 μg/ml) with a mean diameter of 5 and 30 nm was injected (1.5 ml) into the caudal vein twice at 24-h interval during stage II of hemochorial placenta development (day 10 of gestation). The particles were synthesized by citrate reduction of HAuCl4 by the method of France [2] and functionalized with thiolated polyethylene glycol molecules (PEG-SH; Nektar) for improving GNP bioavailability and stability in the bloodflow [6]. The mean size of the particles was evaluated under a Libra-120 electron microscope (Carl Zeiss). According to electron microscopy of 300-400 images of the particles, their mean sizes were 5.1±0.6 and 32.0±3.6 nm, respectively (mean±standard deviation). The females were divided at random into control and experimental groups, 5 per group. Pregnant controls were injected with physiological saline according to the same protocol as GNP. The animals were sacrificed under chloroform narcosis 24 h after the second injection of GNP or physiological saline. All experiments and handling of animals were carried out in accordance with the Directive No. 63 from September 22, 2010 of the Presidium and Parliament of Europe “On Protection of Animals Used for Research”, “Sanitary Regulations of Organization, Equipment, and Use of Experimental Biological Clinics” from April 06, 1993, and Order No. 267 “On Laboratory Practice Regulations” of the Ministry of Health of the Russian Federation from June 19, 2003. Penetration of GNP through the placenta and all the subsequent relevant effects on biological tissues were evaluated by the morphological changes in fetal liver, kidneys, spleen, and brain. Experimental material was fixed in 10% neutral formalin, embedded in paraffin, and analyzed by the standard histological methods.
Accumulation of GNP in cell and tissue structures was visualized by silver nitrate autometallography [7] in paraffin histological sections. The integral evaluation of nanoparticles accumulation in the fetuses was carried out by atomic adsorption spectroscopy on a Dual Atomizer Zeeman AA Spectrometer iCE 3500 (Thermo Scientific, Inc.). The same method was used for GNP measurements in the organs of pregnant females. The data were processed using Statistica 6.0 software (StatSoft Inc.).
RESULTS The total content of GNP 30 nm in diameter in experimental fetuses (about 0.25±0.08 μg/g) significantly (~6-fold) surpassed the control (p
