Toxicology Letters 230 (2014) 10–18
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Effects of nanoparticle size and gestational age on maternal biodistribution and toxicity of gold nanoparticles in pregnant mice Hui Yang a,b,1, Libo Du c,1, Xin Tian a,d, Zhenlin Fan a , Cuiji Sun a , Yang Liu c , Jeffrey A. Keelan e, * , Guangjun Nie a, * a
CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China Immunology Department, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China d School for Radiological & Interdisciplinary Sciences, Soochow University, Suzhou 215123, China e School of Women’s and Infant’s Health, University of Western Australia, Perth, Western Australia, Australia b c
H I G H L I G H T S
Biodistribution of PEGylated GNPs in pregnant mice is size-dependent. GNPs exhibited distinct biodistribution profiles regardless of gestational ages. The main factor controlling GNPs clearance routes and rates was nanomaterial size. No adverse effect was found on pregnant mice by i.v. injection except 30 nm GNPs. This study laid a foundation for GNPs pregnancy application as drug vehicles.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 June 2014 Received in revised form 24 July 2014 Accepted 30 July 2014 Available online 4 August 2014
Gold nanoparticles (GNPs) have considerable applications in biomedicine, such as in bio-sensing, bio-imaging, drug delivery and photothermal therapeutics. However, currently there are limited information regarding the impact of pregnancy on their biodistribution, elimination and toxicity. In this study, we investigated the biodistribution and potential toxic effects of different-sized GNPs (1.5, 4.5, 13, 30 and 70 nm in diameter) in non-pregnant and pregnant mice at different gestational ages (E5.5, 7.5, 9.5, 11.5 and 13.5). 5 h after intravenous injection, GNPs exhibited size-dependent biodistribution profiles; however, regardless of size, no significant biodistribution changes were observed between non-pregnant and pregnant mice. Kinetic studies showed that 4.5 nm GNPs were primarily excreted through urine within 5 h, whereas 30 nm GNPs had a more prolonged blood circulation time. No apparent toxic effects (e.g., increased mortality, altered behavior, reduced animal weight, abnormal organ morphology or reduced pregnancy duration) were observed with different-sized GNPs in pregnant mice. However, treatment with 30 nm GNPs induced mild emphysema-like changes in lungs of pregnant mice. These results indicated that the maternal biodistribution patterns of GNPs in pregnant mice depended on particle size, but not gestational age; organ-specific adverse effects may arise with treatment with some GNPs according to their size. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gold nanoparticles Nanotoxicity Pregnancy Biodistribution Size effects
1. Introduction
* Corresponding authors. E-mail addresses: [email protected] (J.A. Keelan), [email protected] (G. Nie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.07.030 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Gold nanoparticles (GNPs) hold great promise in biomedicine as carriers of pharmaceuticals or as novel diagnostic and therapeutic agents (Caruthers et al., 2007; Dreaden et al., 2012). At present, the likelihood of exposure to GNPs is increasing with the rapid development of nanotechnology (Cherukuri et al., 2010; Cobley et al., 2010; Keelan, 2011). Some of the beneficial characteristics of GNPs include their straight forward synthesis, high stability, low
H. Yang et al. / Toxicology Letters 230 (2014) 10–18
toxicity in vivo and ability to selectively incorporate recognition molecules such as peptides or proteins (Pissuwan et al., 2006). These properties make them well suited for biomedical and pharmaceutical applications, and ideal nanomaterials with which to evaluate the biological effects and safety of spherical nanoparticles in different settings. Such studies require the assessment of the biodistribution, pharmacokinetics and local or systemic toxicity of GNPs after systematic administration, and the influence of specific pathological and physiological states on these factors (Saunders, 2009; Holcberg et al., 2003; Menezes et al., 2011). Considerable research has been carried out on biodistribution, cellular uptake and toxicity of gold nanoparticles in recent years (Alkilany and Murphy, 2010; Khlebtsov and Dykman, 2011). Nanoparticle size had proved to be one of the most important factors in influencing biodistribution, tissue uptake and applications in the biomedical fields (Arvizo et al., 2012; Yu et al., 2012; Wang et al., 2010). Particle size-dependent biodistribution and toxicity of GNPs has been widely studied in vivo (Hillyer and Albrecht, 2001; Sonavane et al., 2008; De Jong et al., 2008; Semmler-Behnke et al., 2008; Hainfeld et al., 2006). The in vivo biodistribution of the GNP influences their accumulation by secondary organs, which may eventually cause diverse health effects. In most studies, systemically administrated GNPs are primarily taken up by liver and spleen, with small amounts distributed in the lung, kidney, heart, and brain. Hyllier and Albertch showed that orally administered colloidal GNPs (58, 28,10 and 4 nm in diameter) can be detected in various tissues in mice and that the amount of absorption and distribution in the body inversely correlated with particle size (Hillyer and Albrecht, 2001). In De Jong’s study, after intravenous injection into rats, spherical GNPs ranging from 10 to 250 nm in diameter were shown to be taken up primarily by the liver and spleen, with the 10 nm nanoparticles more broadly distributed in various organs (De Jong et al., 2008). GNPs of 12.5 nm in diameter do not exert toxic effects of in the liver, lungs, kidneys, spleen or brain (Lasagna-Reeves et al., 2010; Schmid, 2008). Toxicity assessments of GNPs of different sizes (3,10, 50, and 100 nm) in zebrafish embryos showed only minimal sub-lethal toxic effects with no size effect (Bar-Ilan et al., 2009). In contrast, size-dependent in vitro toxicity was found to occur with 1.4 nm GNP, but not 0.8 or 15 nm GNP (Pan et al., 2007). In spite of a number of studies investigating the in vivo size effects of GNPs, little is known about the effects of size on biodistribution, pharmacokinetics and toxicity of GNPs in pregnant females and their fetuses after systematic administration. Pregnancy represents
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a unique physiological state with altered hemodynamics and pharmacokinetics in which the biodistribution and toxicity of nanoparticles is unpredictable (Abduljalil et al., 2012). Maternal physiological changes begin early in gestation and are most pronounced in the third trimester (Federiksen, 2001). Plasma volume increases by about 40–50% during pregnancy and the total plasma concentrations of albumin-bound drugs decrease due to hemodilution (Frederiksen, 2001; Dawes and Chowienczyk, 2001; Loebstein and Koren, 2002; Loebstein et al.,1997). Hepatic metabolic ability changes during pregnancy in response to the increase in estrogens and progesterone (Dawes and Chowienczyk, 2001; Loebstein and Koren, 2002; Loebstein et al., 1997). In pregnancy, renal blood flow and glomerular filtration rate increase, leading to enhanced elimination of some albumin-bound drugs (Jeyabalan and Conrad, 2007; Morgan, 1997). However, the renal clearance of nanoparticles in pregnancy has not been investigated, and it is still not clear whether the physiological changes exhibited by the main organs such as liver and kidney in pregnancy affect the biodistribution and in vivo metabolism dynamics of nanoparticles or how these adaptations change as pregnancy progresses. To address these questions, we applied a series of PEGylated GNPs from 1.5 to 70 nm in diameter to pregnant mice (or non-pregnant female controls) by intravenous injection at different gestational ages, and assessed their biodistribution and in vivo maternal dynamics throughout pregnancy; we also assessed the acute and sub-acute toxicities of GNPs in pregnant mice compared to non-pregnant females using a variety of methods. Our aim was to provide fundamental information on maternal GNPs biodistribution and toxicity in pregnancy. Data on fetal and placental biodistribution and toxicity will be presented in a subsequent study. 2. Materials and methods 2.1. Synthesis of different-sized GNPs capped with PEG-5000 15 nm GNPs: triphenylphosphine (TPP) stabilized gold nanoparticles (5 nm Au-TPP) were synthesized as previously described (Hutchison et al., 2004). 20 mg of the thiolyated PEG-5000 was added to 5 mL solution including 20 mg of 5 nm Au-TPP in dichloromethane and stirred rapidly for 24 h to obtain the PEGylated particles. 4.5 nm GNPs: Au5-PEG was prepared according to reference Manna et al. (2003). The resulting crude GNPs were redissolved and purified by column chromatography using Sephadex LH-20
Fig. 1. Characterization of PEGylated GNPs with different sizes. PEGylated GNPs with diameters of 1.5, 4.5, 13, 30 and 70 nm were examined under TEM. Scale bars are 10 nm for images of 1.5 and 4.5 nm GNPs and 50 nm for images of 13, 30, and 70 nm GNPs. The lower panel shows the zeta-potential of GNPs.
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Fig. 2. Au accumulation in pregnant mice organs after a single dose of GNPs at E9.5. Different-sized GNPs (1.5, 4.5, 13, 30 and 70 nm) were injected i.v. into non-pregnant or E9.5 pregnant mice at 0.9 mg Au/kg body weight. 5 h after injection, the major organs were collected and digested in aqua fortis to detect the Au concentration in each tissue sample by ICP-MS assay. Each data represents the mean s.d. (n = 4 per group). **p < 0.01 by ANOVA with Tukey’s test post-hoc.
(eluant 1:1, dichloromethane:methanol) to remove by-products and excess reactant. 13, 30 and 70 nm GNPs were prepared by the citrate reduction method according to reference Frens (1973). A 100 mL aqueous solution of HAuCl4 (1 mM) was brought to a reflux with vigorous stirring, and then different amounts of 38.8 mM trisodium citrate solution (13 nm: 10 mL; 30 nm: 0.7 mL; 70 nm: 0.3 mL) was added quickly. After the solution color changed to deep red, the solution was refluxed for an additional 15 min, then allowed to cool to room temperature and subsequently filtered through 0.45 mm membrane (Millipore). 10 mg of PEG-5000 was added to 10 mL of above aqueous GNP solution, and stirred for 24 h to complete the ligand exchange (citrate to PEG). All the PEGylated GNPs were stored at 20 C before use. 2.2. Characterization of different sizes of GNPs The morphology and size of the GNPs were evaluated using transmission electron microscopy (TEM, JEM-200CX, Jeol Ltd., Japan). The nanoparticles’ surface charge (zeta potential, mV) and size distribution were determined using a ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd., Malvern, UK). All PEGylated GNP suspensions were sonicated for 5 min before use. 2.3. Animals and GNP treatment CD-1 mice (20–25 g) were obtained from Beijing Vital River Laboratories, Beijing, China. The animal experiments were approved by the Animal Ethics Committee of the Medical School, Beijing University. After acclimatization for 1 week, the mice were
mated and the presence of vaginal plug was confirmed 12 h later, designated as embryonic day 0.5 (E0.5). The mice had free access to food and water and were maintained on a 12 h dark/light cycle in a room with controlled temperature (22 2 C). Pregnant mice (gestational ages: E5.5, E7.5, E9.5, E11.5 and E13.5) and non-pregnant female mice (n = 4 per group) were given injections via tail vein of 100–200 mL GNPs (0.9 mg Au/g body weight) diluted
Table 1 Au concentrations in organs after administration of 4.5 nm (A) and 30 nm (B) GNPs to female mice at different gestational ages. A
N.P. E5.5 E7.5 E9.5 E11.5 E13.5
Heart
Lung
Liver
Kidney
129 7 140 43 120 24 130 10 131 18 177 53*
223 65 241 103 147 60 200 78 351 184 191 34
409 148 314 64 318 39 256 106 498 119 512 241
1781 741 1683 578 2295 295 2193 956 3352 1367* 2285 619
B
N.P. E5.5 E7.5 E9.5 E11.5 E13.5
Heart
Lung
Liver
Kidney
870 52 801 370 1056 318 1201 116* 1026 99 1051 55
1928 108 1820 228 2098 374 2204 526 2085 856 1893 226
1650 320 1667 403 2728 843* 2076 846 1894 295 1449 413
822 103 958 236 1022 69 867 95 872 196 895 75
No statistical difference was found among the same organs of different gestational ages and non-pregnant (N.P.) female mice after GNP administration (n = 4 per group).
H. Yang et al. / Toxicology Letters 230 (2014) 10–18
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Fig. 3. Relative accumulation of Au in major maternal organs. Total Au content within the major organs (heart, liver, spleen, lung and kidney) 5 h after GNPs administration was expressed as the percentage of total administered Au dose in non-pregnant or pregnant mice. Each data represents the mean s.d. (n = 4 for non-pregnant mice, n = 20 for pregnant mice of E5.5 to E13.5 gestational ages). No statistical difference existed between pregnant and non-pregnant female mice for each GNP formulation. **p < 0.01 by ANOVA with Tukey’s test post-hoc.
Fig. 4. Dynamic changes in Au concentrationin blood and excretions. The dynamic changes of Au concentration in blood (A), urine (B) and feces (C) were measured after administration of 30 nm or 4.5 nm GNPs to non-pregnant and E9.5 pregnant mice. Each data represents the mean s.d. (n = 4). Statistical difference between pregnant and non-pregnant female mice existed at some time points. **p < 0.01 by t-test.
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in distilled water (184 mg/mL); controls received saline injections. After administration of GNPs, animals were examined daily for survival and evident behavioral or motor impairments. Mice were also weighed daily. 2.4. Determination of Au content in tissues by ICP-MS The tissue concentrations of GNPs were assessed by quantitative inductively coupled plasma mass spectrometry (ICP-MS). 5 h after GNP injection, mice were anesthetized, euthanized and the tissues were excised and weighed, then digested in aqua fortis (nitric acid: hydrochloric acid 3:1). After adjusting the solution volume to 3 mL using 2% nitric acid and 1% hydrochloride acid (1:1), Au content assays were performed using an ELAN DRC e ICP-MS instrument (PerkinElmer, Massachusetts, USA).
Table 2 The pups born date and pups number after administration of GNP to female mice. N.P.
1.5 nm 4.5 nm 13 nm 30 nm 70 nm
E9.5
Born date
Pups number
Born date
Pups number
E19 E19 E19 E19 E19
11.6 1.1 11.0 1.2 11.0 1.0 11.0 0.7 10.6 1.1
E19 E19 E19 E19 E19
11.0 1.2 10.8 1.3 11.2 0.8 11.0 1.0 11.0 0.7
The E9.5 pregnant mice (n = 5) were injected with 30 nm GNPs at 0.9 mg/kg body weight or an equal volume of saline. The treated pregnant mice were fed normally and checked daily till the pups were born.
3. Results and discussions 3.1. Characterization of GNPs
2.5. Determination of changes in circulation and excretion of GNPs GNPs (0.9 mg Au/g body weight) were administered to E9.5 pregnant mice. At different time points after injection (from 2 to 72 h), feces and urine were collected by putting individual mice on a piece of clean membrane to excrete freely; blood was collected from the angular vein. Digestion and analysis by ICP-MS was carried out as described above.
Transmission electron microscopy (TEM) showed that all PEGylated nanoparticle formulations were monodispersed and exhibited the expected particle sizes (Fig. 1). The zeta potentials (z potentials) of 1.5, 4.5, 13, 30 and 70 nm PEGylated GNPs were determined to be 0, 4.5, 6.0, 3.3 and 0 mV, respectively, indicating that introduction of a PEG layer to the GNPs masked the negative charge on the particle surface and resulted in almost neutral particles.
2.6. Toxicological studies 3.2. Tissue distribution of GNPs 2.6.1. General examination After administration of GNPs, animals were weighed daily and visually examined for gross anomalies in behavior or movement. To examine organ morphological and pathological changes, the lungs, liver, spleen, heart, and kidneys were removed 72 h after GNP injection, weighed and processed for histological analysis as outlined below. 2.6.2. Histopathological examination Tissues were fixed in 10% formalin for 48 h, embedded in paraffin blocks, and then sectioned at 5 mm on Leica CM1850 microtome. Bright-field images of hematoxylin–eosin (H&E) stained sections were acquired on a Leica DM1–3000B microscope equipped with a Nikon DXM1200 color CCD camera (Nikon Instruments Inc., Melville, NY). 2.7. Statistical analysis All results are presented as mean standard deviation (s.d.). Statistical significance between different groups was evaluated by LSD t-test or Tukey’s method after analysis of variance (ANOVA).
Nanoparticle size affects the accessibility of target organs, the mode of cellular uptake, and efficiency of particle processing via endocytic pathways, thus is one of the most important factors for determining nanoparticle deposition and fate. In this study, we examined the gold content in various pregnant mice tissues after GNP administration and found that each sized GNP had a distinct biodistribution profile (Fig 2). The deposition of GNPs in the heart was generally low, but showed a rising tendency with increasing size (Fig 2A). The Au content in lungs was significantly increased when GNP size exceeded 13 nm. Interestingly, the lung Au content was higher with exposure to 30 nm GNPs compared to 13 or 70 nm GNPs (Fig 2B). Lung is well known to be sensitive to xenobiotics; the accumulation of GNPs in lung might initiate inflammation or some abnormality (Di Gioacchino et al., 2011). Except for 4.5 nm GNP, all sized GNPs accumulated largely in the liver (Fig 2C). In contrast, 4.5 nm GNP showed higher accumulation in kidney, whereas other sized GNPs showed relatively low amounts (Fig 2D). These findings were observed in both non-pregnant mice and pregnant mice; there was little or no evidence that pregnancy altered the biodistribution patterns of GNPs.
Fig. 5. Weight increases in non-pregnant and E9.5 pregnant mice after GNPs administration. Different-sized GNPs were injected into E9.5 pregnant mice or non-pregnant female mice. The body weights were checked every 24 h. Data are shown as mean s.d. (n = 4).
H. Yang et al. / Toxicology Letters 230 (2014) 10–18
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Table 3 Weight coefficient of heart, liver, spleen, lung, and kidney after exposure to GNPs. E9.5
control 1.5 nm 4.5 nm 13 nm 30 nm 70 nm
N.P.
Heart (mg/g)
Liver (mg/g)
Spleen (mg/g)
Lung (mg/g)
Kidney (mg/g)
Heart (mg/g)
Liver (mg/g)
Spleen (mg/g)
Lung (mg/g)
Kidney (mg/g)
3.6 0.3 4.1 0.2* 3.8 0.2 3.4 0.3 3.6 0.1 3.9 0.2
57.2 2.3 63.3 2.6* 56.6 3.5 53.5 2.0 50.7 6.2* 62.4 2.6
8.5 0.3 7.3 1.1 9.0 0.4 7.9 2.2 9.5 0.7 8.8 0.4
5.3 0.6 5.7 0.1 5.2 0.8 5.3 0.6 5.7 0.4 6.3 0.2*
9.2 0.3 10.6 0.4** 9.7 0.5 9.3 0.7 9.6 0.8 9.6 0.3
4.5 0.3 4.2 4.9 0.2* 5.0 0.2* 4.5 0.2 4.9 0.2*
45.2 1.9 42.3 5.5 43.3 2.3 42.4 2.7 49.8 4.9 50.9 1.4*
5.5 0.2 5.4 1.2 5.7 0.6 5.2 1.2 6.2 0.9 4.2 0.2*
7.1 0.7 6.1 0.4* 8.0 0.7* 7.6 0.7 7.0 0.2 7.1 0.3
12.3 0.5 11.0 0.3 13.1 1.8 12.7 0.5 11.3 1.5 12.4 0.6
Data represent the mean s.d. (n = 4 per group). * p < 0.05 compared to saline control by ANOVA followed by Tukey’s test. ** p < 0.01 compared to saline control by ANOVA followed by Tukey’s test.
A more detailed analysis of the data showed that after tail vein injection of GNPs to pregnant mice, there were no statistical differences between the same organs at different gestational ages or non-pregnancy state (Table 1), which indicated pregnancy onset and gestational age have no significant influence on GNP distribution. For different sized GNPs, there was a trend for the percent of total dose recovered in the main organs to increase with size, with the 70 nm GNP having higher overall levels than the smaller GNPs (Fig 3). These changes showed no statistical difference between pregnant and non-pregnant animals, consistent with the interpretation that the accumulation and clearance of GNPs are similar in the pregnant and non-pregnant states. These findings suggest that the major determinant of overall accumulation and selective organ uptake is the size of the particle, with pregnancy having little if any effect. It has been speculated that, during gestation, pregnancy-specific changes in maternal hemodynamics and excretion due to the presence of the fetoplacental unit and adaptations of the reticuloendothelial system (RES) might alter the maternal biodistribution of nanoparticles in pregnancy (Nicklin and Billington, 1979; Gerentyuk et al., 2009). Our studies suggest that GNP accumulation in all of the major organs is not influenced by the onset or stage of pregnancy, irrespective of particle size (Fig. 2). 3.3. Metabolism of GNPs after administration in gestation To further explore the clearance of GNPs within pregnant and non-pregnant mice, 4.5 and 30 nm GNPs were administrated by tail vein injection to non-pregnant and E9.5 pregnant mice and gold concentrations in blood, urine and feces were determined by ICP-MS up to 72 h after injection. GNPs of 4.5 and 30 nm had markedly different blood concentrations and clearance rates (Fig 4A). The initial (t = 0 h) Au concentration in blood of all tested mice was 9000 ng/mL. Serum concentrations of 30 nm GNPs remained elevated for 5 h after injection, gradually declining to approximately 10% of the initial concentration over the following 20 h (Fig. 4A, right); in contrast, serum concentrations of 4.5 nm GNPs dropped by 99% by the time of the first measurement (2 h) to less than 100 ng/mL (Fig. 4A, left). The data in Fig 4B suggests that the rapid clearance of the 4.5 nm GNPs was due to efficient renal clearance. Gold levels increased from zero to 2000 to 4000 ng/mL in urine 2 h after 4.5 nm GNPs injection, and declined gradually to zero level within 24 h (Fig 4B, left). In contrast, following administration of the 30 nm GNPs, urine gold levels remained low for 72 h after injection (Fig 4B, right). Interestingly, at 2 and 5 h after 4.5 nm GNPs injection, urine gold concentration was significantly higher in non-pregnant mice than in pregnant mice (p < 0.01, t-test). This might reflect enhanced dilution in pregnancy associated with the well-documented increase in urine output with pregnancy, or a general reduction in GNP clearance with pregnancy. The Au concentration in feces was very low after
administration of both the 4.5 and 30 nm GNPs (Fig. 4C), consistent with the urine data which suggests that clearance is primarily via the renal route. 3.4. Toxicological studies We tested if GNP exposure produced acute or sub-acute toxicity in pregnant mice compared with non-pregnant mice. Our previously published work has shown that, 13 nm GNP does not disturb fetal development and offspring growth, and induce no change on gestational period and offspring birth rate (Yang et al., 2012). In the current study, irrespective of GNP size, no evidence of organ atrophy or inflammation was observed, and no gross behavioral changes were observed. GNP treatment did not influence overall body weight in non-pregnant mice or weight gain in the E9.5 pregnant mice compared with controls without GNPs exposure (Fig. 5). The pregnant mice receiving different-sized GNPs delivered spontaneously at term, with pup number comparable with control (Table 2). No obvious GNPs size-dependent reductions in organ weight were observed in GNP-treated non-pregnant and E9.5 pregnant mice (Table 3). Furthermore, tissue color and morphology remain unchanged after GNPs treatment; representative images of tissues from non-pregnant and E9.5 pregnant mice after 30 nm GNP treatment are shown in Fig 6. To explore the potential toxicity of GNPs on tissue and cellular levels, histological examinations of various organs of E9.5 pregnant mice were performed 72 h after GNPs treatment. No acute or sub-acute tissue injury was observed in the sections of kidney, liver and spleen after pregnant mice exposed to GNPs (all sizes). However, histological differences were observed in lung sections from mice treated with 13 and 30 nm GNPs (Fig. 7). While the 1.5 and 4.5 nm GNPs did not induce any pulmonary injury, in the 13 and 30 nm GNP-treated sections, enlarged airway cavities and
Fig. 6. Morphologies of the major organs in mice treated with GNPs. 30 nm GNP or saline control was injected into E9.5 pregnant mice or non-pregnant female mice. 72 h after the treatment, mice were sacrificed and the main organs were examined.
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H. Yang et al. / Toxicology Letters 230 (2014) 10–18
Fig. 7. Histological analysis of the major organs in pregnant mice after GNPs treatment. Different sized GNPs from 1.5 to 70 nm (or saline control) were injected into E9.5 pregnant mice or non-pregnant female mice. 72 h after the treatment, mice were sacrificed, organs were fixed and embedded in paraffin blocks and sectioned at 5 mm. The bright-field images of H&E staining sections were acquired with a Leica DM1–3000B microscope equipped with a Nikon DXM1200 color CCD camera (Nikon Instruments Inc., Melville, NY). The scale bar is 100 mm.
Fig. 8. Histological analysis of lung tissues after different doses of 30 nm GNPs treatment on pregnant mice. Thirty nm GNPs were injected to E9.5 pregnant mice on the final concentration from 0.02 mg/kg to 4.5 mg/kg. 72 h after injection, mice were sacrificed and the blocks of lungs were fixed, embedded in paraffin blocks and sectioned at 5 mm. The bright-field images of H&E staining sections were conducted as above. The scale bar is 100 mm.
H. Yang et al. / Toxicology Letters 230 (2014) 10–18 Table 4 Marking scale for lung injury. Grade
Score
Feature
Normal
1 2 3
Dense and uniform Dense with intervals Dense with more intervals
Mild injury
4 5 6
Small or middle cavity, no fusion Small or middle cavity, little fusion Small or middle cavity, more fusion
Moderate injury
7 8 9
Middle or large cavity, fusion Middle or large cavity, more fusion Middle or large cavity, full fusion
Severe injury
10 11 12
Large cavity, full fusion Large cavity, inflammatory cell infiltation Large cavity, severe inflammatory cell infiltation
mild epithelial hyperplasia could be seen, consistent with emphysema-like changes. Similar effects have been reported by Terentyuk et al. on rat after tail vein injection of 15 and 50 nm PEG-coated colloidal gold particles (Terentyuk et al., 2009). In 70 nm GNP-treated lung sections, epithelial hyperplasia was evident but less severe compared with sections from the 30 nm GNP-treated tissues (Fig. 7). In light of the increased accumulation of 30 nm (and to a lesser extent 13 and 70 nm) GNPs in the lungs (Fig. 2B), it appears that some adverse effects are associated with lung accumulation of 30 nm GNPs. We attempted to assess the extent of infiltration of inflammatory cells in lungs by CD45 immunohistochemistry, but failed to detect any immunopositive cells. To further explore this issue, we performed a dose response study at E9.5 with 30 nm GNPs to clarify the concentrationdependence of these effects on maternal lung tissues. GNPs were administered at different doses from 0.02 to 4.5 mg/kg body weight. It was found that with increased GNP dose, injuries in pregnant mice lungs became more extensive, as evidenced by the appearance and enlargement of cavities, thinning of the alveolar cell layer, and the increased proportion of lesions in the tissue (Fig. 8). Low doses GNPs initiated little or no damage effect on lung cavity structure; while lung damage became fairly obvious at GNP concentrations in excess of 0.9 mg/kg body weight. Table 5 Evaluation of lung injury in pregnant mice treated with 30 nm GNPs. Control (saline) Num. Score Section 1 scores 2 3 4 5 6 7 8 9 10 Mean score SD P Class
1.4 1.4 1.6 1.2 1.4 2.2 1.6 1.6 1.8 1.8 2.0 1.1 No injury
GNP (0.9 mg/kg) Num. Score Num. Score
Num. Score
11 12 13 14 15 16 17 18 19 20
11 12 13 14 15 16 17 18 19 20
1.8 1.4 1.4 1.6 1.2 1.4 2.6 3.8 4.8 4.8
1 2 3 4 5 6 7 8 9 10
4.0 4.2 4.8 5.6 4.4 5.0 5.8 5.4 6.2 6.4
5.4 4.4 4.2 3.2 4.0 4.2 4.6 5.0 4.8 4.8
4.8 0.8
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Effects of nanoparticle size and gestational age on maternal biodistribution and toxicity of gold nanoparticles in pregnant mice Hui Yang a,b,1, Libo Du c,1, Xin Tian a,d, Zhenlin Fan a , Cuiji Sun a , Yang Liu c , Jeffrey A. Keelan e, * , Guangjun Nie a, * a
CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China Immunology Department, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China d School for Radiological & Interdisciplinary Sciences, Soochow University, Suzhou 215123, China e School of Women’s and Infant’s Health, University of Western Australia, Perth, Western Australia, Australia b c
H I G H L I G H T S
Biodistribution of PEGylated GNPs in pregnant mice is size-dependent. GNPs exhibited distinct biodistribution profiles regardless of gestational ages. The main factor controlling GNPs clearance routes and rates was nanomaterial size. No adverse effect was found on pregnant mice by i.v. injection except 30 nm GNPs. This study laid a foundation for GNPs pregnancy application as drug vehicles.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 June 2014 Received in revised form 24 July 2014 Accepted 30 July 2014 Available online 4 August 2014
Gold nanoparticles (GNPs) have considerable applications in biomedicine, such as in bio-sensing, bio-imaging, drug delivery and photothermal therapeutics. However, currently there are limited information regarding the impact of pregnancy on their biodistribution, elimination and toxicity. In this study, we investigated the biodistribution and potential toxic effects of different-sized GNPs (1.5, 4.5, 13, 30 and 70 nm in diameter) in non-pregnant and pregnant mice at different gestational ages (E5.5, 7.5, 9.5, 11.5 and 13.5). 5 h after intravenous injection, GNPs exhibited size-dependent biodistribution profiles; however, regardless of size, no significant biodistribution changes were observed between non-pregnant and pregnant mice. Kinetic studies showed that 4.5 nm GNPs were primarily excreted through urine within 5 h, whereas 30 nm GNPs had a more prolonged blood circulation time. No apparent toxic effects (e.g., increased mortality, altered behavior, reduced animal weight, abnormal organ morphology or reduced pregnancy duration) were observed with different-sized GNPs in pregnant mice. However, treatment with 30 nm GNPs induced mild emphysema-like changes in lungs of pregnant mice. These results indicated that the maternal biodistribution patterns of GNPs in pregnant mice depended on particle size, but not gestational age; organ-specific adverse effects may arise with treatment with some GNPs according to their size. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gold nanoparticles Nanotoxicity Pregnancy Biodistribution Size effects
1. Introduction
* Corresponding authors. E-mail addresses: [email protected] (J.A. Keelan), [email protected] (G. Nie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.07.030 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Gold nanoparticles (GNPs) hold great promise in biomedicine as carriers of pharmaceuticals or as novel diagnostic and therapeutic agents (Caruthers et al., 2007; Dreaden et al., 2012). At present, the likelihood of exposure to GNPs is increasing with the rapid development of nanotechnology (Cherukuri et al., 2010; Cobley et al., 2010; Keelan, 2011). Some of the beneficial characteristics of GNPs include their straight forward synthesis, high stability, low
H. Yang et al. / Toxicology Letters 230 (2014) 10–18
toxicity in vivo and ability to selectively incorporate recognition molecules such as peptides or proteins (Pissuwan et al., 2006). These properties make them well suited for biomedical and pharmaceutical applications, and ideal nanomaterials with which to evaluate the biological effects and safety of spherical nanoparticles in different settings. Such studies require the assessment of the biodistribution, pharmacokinetics and local or systemic toxicity of GNPs after systematic administration, and the influence of specific pathological and physiological states on these factors (Saunders, 2009; Holcberg et al., 2003; Menezes et al., 2011). Considerable research has been carried out on biodistribution, cellular uptake and toxicity of gold nanoparticles in recent years (Alkilany and Murphy, 2010; Khlebtsov and Dykman, 2011). Nanoparticle size had proved to be one of the most important factors in influencing biodistribution, tissue uptake and applications in the biomedical fields (Arvizo et al., 2012; Yu et al., 2012; Wang et al., 2010). Particle size-dependent biodistribution and toxicity of GNPs has been widely studied in vivo (Hillyer and Albrecht, 2001; Sonavane et al., 2008; De Jong et al., 2008; Semmler-Behnke et al., 2008; Hainfeld et al., 2006). The in vivo biodistribution of the GNP influences their accumulation by secondary organs, which may eventually cause diverse health effects. In most studies, systemically administrated GNPs are primarily taken up by liver and spleen, with small amounts distributed in the lung, kidney, heart, and brain. Hyllier and Albertch showed that orally administered colloidal GNPs (58, 28,10 and 4 nm in diameter) can be detected in various tissues in mice and that the amount of absorption and distribution in the body inversely correlated with particle size (Hillyer and Albrecht, 2001). In De Jong’s study, after intravenous injection into rats, spherical GNPs ranging from 10 to 250 nm in diameter were shown to be taken up primarily by the liver and spleen, with the 10 nm nanoparticles more broadly distributed in various organs (De Jong et al., 2008). GNPs of 12.5 nm in diameter do not exert toxic effects of in the liver, lungs, kidneys, spleen or brain (Lasagna-Reeves et al., 2010; Schmid, 2008). Toxicity assessments of GNPs of different sizes (3,10, 50, and 100 nm) in zebrafish embryos showed only minimal sub-lethal toxic effects with no size effect (Bar-Ilan et al., 2009). In contrast, size-dependent in vitro toxicity was found to occur with 1.4 nm GNP, but not 0.8 or 15 nm GNP (Pan et al., 2007). In spite of a number of studies investigating the in vivo size effects of GNPs, little is known about the effects of size on biodistribution, pharmacokinetics and toxicity of GNPs in pregnant females and their fetuses after systematic administration. Pregnancy represents
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a unique physiological state with altered hemodynamics and pharmacokinetics in which the biodistribution and toxicity of nanoparticles is unpredictable (Abduljalil et al., 2012). Maternal physiological changes begin early in gestation and are most pronounced in the third trimester (Federiksen, 2001). Plasma volume increases by about 40–50% during pregnancy and the total plasma concentrations of albumin-bound drugs decrease due to hemodilution (Frederiksen, 2001; Dawes and Chowienczyk, 2001; Loebstein and Koren, 2002; Loebstein et al.,1997). Hepatic metabolic ability changes during pregnancy in response to the increase in estrogens and progesterone (Dawes and Chowienczyk, 2001; Loebstein and Koren, 2002; Loebstein et al., 1997). In pregnancy, renal blood flow and glomerular filtration rate increase, leading to enhanced elimination of some albumin-bound drugs (Jeyabalan and Conrad, 2007; Morgan, 1997). However, the renal clearance of nanoparticles in pregnancy has not been investigated, and it is still not clear whether the physiological changes exhibited by the main organs such as liver and kidney in pregnancy affect the biodistribution and in vivo metabolism dynamics of nanoparticles or how these adaptations change as pregnancy progresses. To address these questions, we applied a series of PEGylated GNPs from 1.5 to 70 nm in diameter to pregnant mice (or non-pregnant female controls) by intravenous injection at different gestational ages, and assessed their biodistribution and in vivo maternal dynamics throughout pregnancy; we also assessed the acute and sub-acute toxicities of GNPs in pregnant mice compared to non-pregnant females using a variety of methods. Our aim was to provide fundamental information on maternal GNPs biodistribution and toxicity in pregnancy. Data on fetal and placental biodistribution and toxicity will be presented in a subsequent study. 2. Materials and methods 2.1. Synthesis of different-sized GNPs capped with PEG-5000 15 nm GNPs: triphenylphosphine (TPP) stabilized gold nanoparticles (5 nm Au-TPP) were synthesized as previously described (Hutchison et al., 2004). 20 mg of the thiolyated PEG-5000 was added to 5 mL solution including 20 mg of 5 nm Au-TPP in dichloromethane and stirred rapidly for 24 h to obtain the PEGylated particles. 4.5 nm GNPs: Au5-PEG was prepared according to reference Manna et al. (2003). The resulting crude GNPs were redissolved and purified by column chromatography using Sephadex LH-20
Fig. 1. Characterization of PEGylated GNPs with different sizes. PEGylated GNPs with diameters of 1.5, 4.5, 13, 30 and 70 nm were examined under TEM. Scale bars are 10 nm for images of 1.5 and 4.5 nm GNPs and 50 nm for images of 13, 30, and 70 nm GNPs. The lower panel shows the zeta-potential of GNPs.
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Fig. 2. Au accumulation in pregnant mice organs after a single dose of GNPs at E9.5. Different-sized GNPs (1.5, 4.5, 13, 30 and 70 nm) were injected i.v. into non-pregnant or E9.5 pregnant mice at 0.9 mg Au/kg body weight. 5 h after injection, the major organs were collected and digested in aqua fortis to detect the Au concentration in each tissue sample by ICP-MS assay. Each data represents the mean s.d. (n = 4 per group). **p < 0.01 by ANOVA with Tukey’s test post-hoc.
(eluant 1:1, dichloromethane:methanol) to remove by-products and excess reactant. 13, 30 and 70 nm GNPs were prepared by the citrate reduction method according to reference Frens (1973). A 100 mL aqueous solution of HAuCl4 (1 mM) was brought to a reflux with vigorous stirring, and then different amounts of 38.8 mM trisodium citrate solution (13 nm: 10 mL; 30 nm: 0.7 mL; 70 nm: 0.3 mL) was added quickly. After the solution color changed to deep red, the solution was refluxed for an additional 15 min, then allowed to cool to room temperature and subsequently filtered through 0.45 mm membrane (Millipore). 10 mg of PEG-5000 was added to 10 mL of above aqueous GNP solution, and stirred for 24 h to complete the ligand exchange (citrate to PEG). All the PEGylated GNPs were stored at 20 C before use. 2.2. Characterization of different sizes of GNPs The morphology and size of the GNPs were evaluated using transmission electron microscopy (TEM, JEM-200CX, Jeol Ltd., Japan). The nanoparticles’ surface charge (zeta potential, mV) and size distribution were determined using a ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd., Malvern, UK). All PEGylated GNP suspensions were sonicated for 5 min before use. 2.3. Animals and GNP treatment CD-1 mice (20–25 g) were obtained from Beijing Vital River Laboratories, Beijing, China. The animal experiments were approved by the Animal Ethics Committee of the Medical School, Beijing University. After acclimatization for 1 week, the mice were
mated and the presence of vaginal plug was confirmed 12 h later, designated as embryonic day 0.5 (E0.5). The mice had free access to food and water and were maintained on a 12 h dark/light cycle in a room with controlled temperature (22 2 C). Pregnant mice (gestational ages: E5.5, E7.5, E9.5, E11.5 and E13.5) and non-pregnant female mice (n = 4 per group) were given injections via tail vein of 100–200 mL GNPs (0.9 mg Au/g body weight) diluted
Table 1 Au concentrations in organs after administration of 4.5 nm (A) and 30 nm (B) GNPs to female mice at different gestational ages. A
N.P. E5.5 E7.5 E9.5 E11.5 E13.5
Heart
Lung
Liver
Kidney
129 7 140 43 120 24 130 10 131 18 177 53*
223 65 241 103 147 60 200 78 351 184 191 34
409 148 314 64 318 39 256 106 498 119 512 241
1781 741 1683 578 2295 295 2193 956 3352 1367* 2285 619
B
N.P. E5.5 E7.5 E9.5 E11.5 E13.5
Heart
Lung
Liver
Kidney
870 52 801 370 1056 318 1201 116* 1026 99 1051 55
1928 108 1820 228 2098 374 2204 526 2085 856 1893 226
1650 320 1667 403 2728 843* 2076 846 1894 295 1449 413
822 103 958 236 1022 69 867 95 872 196 895 75
No statistical difference was found among the same organs of different gestational ages and non-pregnant (N.P.) female mice after GNP administration (n = 4 per group).
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Fig. 3. Relative accumulation of Au in major maternal organs. Total Au content within the major organs (heart, liver, spleen, lung and kidney) 5 h after GNPs administration was expressed as the percentage of total administered Au dose in non-pregnant or pregnant mice. Each data represents the mean s.d. (n = 4 for non-pregnant mice, n = 20 for pregnant mice of E5.5 to E13.5 gestational ages). No statistical difference existed between pregnant and non-pregnant female mice for each GNP formulation. **p < 0.01 by ANOVA with Tukey’s test post-hoc.
Fig. 4. Dynamic changes in Au concentrationin blood and excretions. The dynamic changes of Au concentration in blood (A), urine (B) and feces (C) were measured after administration of 30 nm or 4.5 nm GNPs to non-pregnant and E9.5 pregnant mice. Each data represents the mean s.d. (n = 4). Statistical difference between pregnant and non-pregnant female mice existed at some time points. **p < 0.01 by t-test.
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in distilled water (184 mg/mL); controls received saline injections. After administration of GNPs, animals were examined daily for survival and evident behavioral or motor impairments. Mice were also weighed daily. 2.4. Determination of Au content in tissues by ICP-MS The tissue concentrations of GNPs were assessed by quantitative inductively coupled plasma mass spectrometry (ICP-MS). 5 h after GNP injection, mice were anesthetized, euthanized and the tissues were excised and weighed, then digested in aqua fortis (nitric acid: hydrochloric acid 3:1). After adjusting the solution volume to 3 mL using 2% nitric acid and 1% hydrochloride acid (1:1), Au content assays were performed using an ELAN DRC e ICP-MS instrument (PerkinElmer, Massachusetts, USA).
Table 2 The pups born date and pups number after administration of GNP to female mice. N.P.
1.5 nm 4.5 nm 13 nm 30 nm 70 nm
E9.5
Born date
Pups number
Born date
Pups number
E19 E19 E19 E19 E19
11.6 1.1 11.0 1.2 11.0 1.0 11.0 0.7 10.6 1.1
E19 E19 E19 E19 E19
11.0 1.2 10.8 1.3 11.2 0.8 11.0 1.0 11.0 0.7
The E9.5 pregnant mice (n = 5) were injected with 30 nm GNPs at 0.9 mg/kg body weight or an equal volume of saline. The treated pregnant mice were fed normally and checked daily till the pups were born.
3. Results and discussions 3.1. Characterization of GNPs
2.5. Determination of changes in circulation and excretion of GNPs GNPs (0.9 mg Au/g body weight) were administered to E9.5 pregnant mice. At different time points after injection (from 2 to 72 h), feces and urine were collected by putting individual mice on a piece of clean membrane to excrete freely; blood was collected from the angular vein. Digestion and analysis by ICP-MS was carried out as described above.
Transmission electron microscopy (TEM) showed that all PEGylated nanoparticle formulations were monodispersed and exhibited the expected particle sizes (Fig. 1). The zeta potentials (z potentials) of 1.5, 4.5, 13, 30 and 70 nm PEGylated GNPs were determined to be 0, 4.5, 6.0, 3.3 and 0 mV, respectively, indicating that introduction of a PEG layer to the GNPs masked the negative charge on the particle surface and resulted in almost neutral particles.
2.6. Toxicological studies 3.2. Tissue distribution of GNPs 2.6.1. General examination After administration of GNPs, animals were weighed daily and visually examined for gross anomalies in behavior or movement. To examine organ morphological and pathological changes, the lungs, liver, spleen, heart, and kidneys were removed 72 h after GNP injection, weighed and processed for histological analysis as outlined below. 2.6.2. Histopathological examination Tissues were fixed in 10% formalin for 48 h, embedded in paraffin blocks, and then sectioned at 5 mm on Leica CM1850 microtome. Bright-field images of hematoxylin–eosin (H&E) stained sections were acquired on a Leica DM1–3000B microscope equipped with a Nikon DXM1200 color CCD camera (Nikon Instruments Inc., Melville, NY). 2.7. Statistical analysis All results are presented as mean standard deviation (s.d.). Statistical significance between different groups was evaluated by LSD t-test or Tukey’s method after analysis of variance (ANOVA).
Nanoparticle size affects the accessibility of target organs, the mode of cellular uptake, and efficiency of particle processing via endocytic pathways, thus is one of the most important factors for determining nanoparticle deposition and fate. In this study, we examined the gold content in various pregnant mice tissues after GNP administration and found that each sized GNP had a distinct biodistribution profile (Fig 2). The deposition of GNPs in the heart was generally low, but showed a rising tendency with increasing size (Fig 2A). The Au content in lungs was significantly increased when GNP size exceeded 13 nm. Interestingly, the lung Au content was higher with exposure to 30 nm GNPs compared to 13 or 70 nm GNPs (Fig 2B). Lung is well known to be sensitive to xenobiotics; the accumulation of GNPs in lung might initiate inflammation or some abnormality (Di Gioacchino et al., 2011). Except for 4.5 nm GNP, all sized GNPs accumulated largely in the liver (Fig 2C). In contrast, 4.5 nm GNP showed higher accumulation in kidney, whereas other sized GNPs showed relatively low amounts (Fig 2D). These findings were observed in both non-pregnant mice and pregnant mice; there was little or no evidence that pregnancy altered the biodistribution patterns of GNPs.
Fig. 5. Weight increases in non-pregnant and E9.5 pregnant mice after GNPs administration. Different-sized GNPs were injected into E9.5 pregnant mice or non-pregnant female mice. The body weights were checked every 24 h. Data are shown as mean s.d. (n = 4).
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Table 3 Weight coefficient of heart, liver, spleen, lung, and kidney after exposure to GNPs. E9.5
control 1.5 nm 4.5 nm 13 nm 30 nm 70 nm
N.P.
Heart (mg/g)
Liver (mg/g)
Spleen (mg/g)
Lung (mg/g)
Kidney (mg/g)
Heart (mg/g)
Liver (mg/g)
Spleen (mg/g)
Lung (mg/g)
Kidney (mg/g)
3.6 0.3 4.1 0.2* 3.8 0.2 3.4 0.3 3.6 0.1 3.9 0.2
57.2 2.3 63.3 2.6* 56.6 3.5 53.5 2.0 50.7 6.2* 62.4 2.6
8.5 0.3 7.3 1.1 9.0 0.4 7.9 2.2 9.5 0.7 8.8 0.4
5.3 0.6 5.7 0.1 5.2 0.8 5.3 0.6 5.7 0.4 6.3 0.2*
9.2 0.3 10.6 0.4** 9.7 0.5 9.3 0.7 9.6 0.8 9.6 0.3
4.5 0.3 4.2 4.9 0.2* 5.0 0.2* 4.5 0.2 4.9 0.2*
45.2 1.9 42.3 5.5 43.3 2.3 42.4 2.7 49.8 4.9 50.9 1.4*
5.5 0.2 5.4 1.2 5.7 0.6 5.2 1.2 6.2 0.9 4.2 0.2*
7.1 0.7 6.1 0.4* 8.0 0.7* 7.6 0.7 7.0 0.2 7.1 0.3
12.3 0.5 11.0 0.3 13.1 1.8 12.7 0.5 11.3 1.5 12.4 0.6
Data represent the mean s.d. (n = 4 per group). * p < 0.05 compared to saline control by ANOVA followed by Tukey’s test. ** p < 0.01 compared to saline control by ANOVA followed by Tukey’s test.
A more detailed analysis of the data showed that after tail vein injection of GNPs to pregnant mice, there were no statistical differences between the same organs at different gestational ages or non-pregnancy state (Table 1), which indicated pregnancy onset and gestational age have no significant influence on GNP distribution. For different sized GNPs, there was a trend for the percent of total dose recovered in the main organs to increase with size, with the 70 nm GNP having higher overall levels than the smaller GNPs (Fig 3). These changes showed no statistical difference between pregnant and non-pregnant animals, consistent with the interpretation that the accumulation and clearance of GNPs are similar in the pregnant and non-pregnant states. These findings suggest that the major determinant of overall accumulation and selective organ uptake is the size of the particle, with pregnancy having little if any effect. It has been speculated that, during gestation, pregnancy-specific changes in maternal hemodynamics and excretion due to the presence of the fetoplacental unit and adaptations of the reticuloendothelial system (RES) might alter the maternal biodistribution of nanoparticles in pregnancy (Nicklin and Billington, 1979; Gerentyuk et al., 2009). Our studies suggest that GNP accumulation in all of the major organs is not influenced by the onset or stage of pregnancy, irrespective of particle size (Fig. 2). 3.3. Metabolism of GNPs after administration in gestation To further explore the clearance of GNPs within pregnant and non-pregnant mice, 4.5 and 30 nm GNPs were administrated by tail vein injection to non-pregnant and E9.5 pregnant mice and gold concentrations in blood, urine and feces were determined by ICP-MS up to 72 h after injection. GNPs of 4.5 and 30 nm had markedly different blood concentrations and clearance rates (Fig 4A). The initial (t = 0 h) Au concentration in blood of all tested mice was 9000 ng/mL. Serum concentrations of 30 nm GNPs remained elevated for 5 h after injection, gradually declining to approximately 10% of the initial concentration over the following 20 h (Fig. 4A, right); in contrast, serum concentrations of 4.5 nm GNPs dropped by 99% by the time of the first measurement (2 h) to less than 100 ng/mL (Fig. 4A, left). The data in Fig 4B suggests that the rapid clearance of the 4.5 nm GNPs was due to efficient renal clearance. Gold levels increased from zero to 2000 to 4000 ng/mL in urine 2 h after 4.5 nm GNPs injection, and declined gradually to zero level within 24 h (Fig 4B, left). In contrast, following administration of the 30 nm GNPs, urine gold levels remained low for 72 h after injection (Fig 4B, right). Interestingly, at 2 and 5 h after 4.5 nm GNPs injection, urine gold concentration was significantly higher in non-pregnant mice than in pregnant mice (p < 0.01, t-test). This might reflect enhanced dilution in pregnancy associated with the well-documented increase in urine output with pregnancy, or a general reduction in GNP clearance with pregnancy. The Au concentration in feces was very low after
administration of both the 4.5 and 30 nm GNPs (Fig. 4C), consistent with the urine data which suggests that clearance is primarily via the renal route. 3.4. Toxicological studies We tested if GNP exposure produced acute or sub-acute toxicity in pregnant mice compared with non-pregnant mice. Our previously published work has shown that, 13 nm GNP does not disturb fetal development and offspring growth, and induce no change on gestational period and offspring birth rate (Yang et al., 2012). In the current study, irrespective of GNP size, no evidence of organ atrophy or inflammation was observed, and no gross behavioral changes were observed. GNP treatment did not influence overall body weight in non-pregnant mice or weight gain in the E9.5 pregnant mice compared with controls without GNPs exposure (Fig. 5). The pregnant mice receiving different-sized GNPs delivered spontaneously at term, with pup number comparable with control (Table 2). No obvious GNPs size-dependent reductions in organ weight were observed in GNP-treated non-pregnant and E9.5 pregnant mice (Table 3). Furthermore, tissue color and morphology remain unchanged after GNPs treatment; representative images of tissues from non-pregnant and E9.5 pregnant mice after 30 nm GNP treatment are shown in Fig 6. To explore the potential toxicity of GNPs on tissue and cellular levels, histological examinations of various organs of E9.5 pregnant mice were performed 72 h after GNPs treatment. No acute or sub-acute tissue injury was observed in the sections of kidney, liver and spleen after pregnant mice exposed to GNPs (all sizes). However, histological differences were observed in lung sections from mice treated with 13 and 30 nm GNPs (Fig. 7). While the 1.5 and 4.5 nm GNPs did not induce any pulmonary injury, in the 13 and 30 nm GNP-treated sections, enlarged airway cavities and
Fig. 6. Morphologies of the major organs in mice treated with GNPs. 30 nm GNP or saline control was injected into E9.5 pregnant mice or non-pregnant female mice. 72 h after the treatment, mice were sacrificed and the main organs were examined.
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Fig. 7. Histological analysis of the major organs in pregnant mice after GNPs treatment. Different sized GNPs from 1.5 to 70 nm (or saline control) were injected into E9.5 pregnant mice or non-pregnant female mice. 72 h after the treatment, mice were sacrificed, organs were fixed and embedded in paraffin blocks and sectioned at 5 mm. The bright-field images of H&E staining sections were acquired with a Leica DM1–3000B microscope equipped with a Nikon DXM1200 color CCD camera (Nikon Instruments Inc., Melville, NY). The scale bar is 100 mm.
Fig. 8. Histological analysis of lung tissues after different doses of 30 nm GNPs treatment on pregnant mice. Thirty nm GNPs were injected to E9.5 pregnant mice on the final concentration from 0.02 mg/kg to 4.5 mg/kg. 72 h after injection, mice were sacrificed and the blocks of lungs were fixed, embedded in paraffin blocks and sectioned at 5 mm. The bright-field images of H&E staining sections were conducted as above. The scale bar is 100 mm.
H. Yang et al. / Toxicology Letters 230 (2014) 10–18 Table 4 Marking scale for lung injury. Grade
Score
Feature
Normal
1 2 3
Dense and uniform Dense with intervals Dense with more intervals
Mild injury
4 5 6
Small or middle cavity, no fusion Small or middle cavity, little fusion Small or middle cavity, more fusion
Moderate injury
7 8 9
Middle or large cavity, fusion Middle or large cavity, more fusion Middle or large cavity, full fusion
Severe injury
10 11 12
Large cavity, full fusion Large cavity, inflammatory cell infiltation Large cavity, severe inflammatory cell infiltation
mild epithelial hyperplasia could be seen, consistent with emphysema-like changes. Similar effects have been reported by Terentyuk et al. on rat after tail vein injection of 15 and 50 nm PEG-coated colloidal gold particles (Terentyuk et al., 2009). In 70 nm GNP-treated lung sections, epithelial hyperplasia was evident but less severe compared with sections from the 30 nm GNP-treated tissues (Fig. 7). In light of the increased accumulation of 30 nm (and to a lesser extent 13 and 70 nm) GNPs in the lungs (Fig. 2B), it appears that some adverse effects are associated with lung accumulation of 30 nm GNPs. We attempted to assess the extent of infiltration of inflammatory cells in lungs by CD45 immunohistochemistry, but failed to detect any immunopositive cells. To further explore this issue, we performed a dose response study at E9.5 with 30 nm GNPs to clarify the concentrationdependence of these effects on maternal lung tissues. GNPs were administered at different doses from 0.02 to 4.5 mg/kg body weight. It was found that with increased GNP dose, injuries in pregnant mice lungs became more extensive, as evidenced by the appearance and enlargement of cavities, thinning of the alveolar cell layer, and the increased proportion of lesions in the tissue (Fig. 8). Low doses GNPs initiated little or no damage effect on lung cavity structure; while lung damage became fairly obvious at GNP concentrations in excess of 0.9 mg/kg body weight. Table 5 Evaluation of lung injury in pregnant mice treated with 30 nm GNPs. Control (saline) Num. Score Section 1 scores 2 3 4 5 6 7 8 9 10 Mean score SD P Class
1.4 1.4 1.6 1.2 1.4 2.2 1.6 1.6 1.8 1.8 2.0 1.1 No injury
GNP (0.9 mg/kg) Num. Score Num. Score
Num. Score
11 12 13 14 15 16 17 18 19 20
11 12 13 14 15 16 17 18 19 20
1.8 1.4 1.4 1.6 1.2 1.4 2.6 3.8 4.8 4.8
1 2 3 4 5 6 7 8 9 10
4.0 4.2 4.8 5.6 4.4 5.0 5.8 5.4 6.2 6.4
5.4 4.4 4.2 3.2 4.0 4.2 4.6 5.0 4.8 4.8
4.8 0.8
