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Particle and Fibre Toxicology

Research
Open Access

Bioavailability, distribution and clearance of tracheally-instilled and gavaged uncoated or silica-coated zinc oxide nanoparticles

  • Nagarjun V Konduru1,
  • Kimberly M Murdaugh1,
  • Georgios A Sotiriou1,
  • Thomas C Donaghey1,
  • Philip Demokritou1,
  • Joseph D Brain1 and
  • Ramon M Molina1Email author
Particle and Fibre Toxicology201411:44

https://doi.org/10.1186/s12989-014-0044-6

©  Konduru et al.; licensee BioMed Central Ltd. 2014

Received: 15 April 2014

Accepted: 20 August 2014

Published: 3 September 2014

Abstract

Background

Nanoparticle pharmacokinetics and biological effects are influenced by several factors. We assessed the effects of amorphous SiO2 coating on the pharmacokinetics of zinc oxide nanoparticles (ZnO NPs) following intratracheal (IT) instillation and gavage in rats.

Methods

Uncoated and SiO2-coated ZnO NPs were neutron-activated and IT-instilled at 1 mg/kg or gavaged at 5 mg/kg. Rats were followed over 28 days post-IT, and over 7 days post-gavage. Tissue samples were analyzed for 65Zn radioactivity. Pulmonary responses to instilled NPs were also evaluated at 24 hours.

Results

SiO2-coated ZnO elicited significantly higher inflammatory responses than uncoated NPs. Pulmonary clearance of both 65ZnO NPs was biphasic with a rapid initial t1/2 (0.2 - 0.3 hours), and a slower terminal t1/2 of 1.2 days (SiO2-coated ZnO) and 1.7 days (ZnO). Both NPs were almost completely cleared by day 7 (>98%). With IT-instilled 65ZnO NPs, significantly more 65Zn was found in skeletal muscle, liver, skin, kidneys, cecum and blood on day 2 in uncoated than SiO2-coated NPs. By 28 days, extrapulmonary levels of 65Zn from both NPs significantly decreased. However, 65Zn levels in skeletal muscle, skin and blood remained higher from uncoated NPs. Interestingly, 65Zn levels in bone marrow and thoracic lymph nodes were higher from coated 65ZnO NPs. More 65Zn was excreted in the urine from rats instilled with SiO2-coated 65ZnO NPs. After 7 days post-gavage, only 7.4% (uncoated) and 6.7% (coated) of 65Zn dose were measured in all tissues combined. As with instilled NPs, after gavage significantly more 65Zn was measured in skeletal muscle from uncoated NPs and less in thoracic lymph nodes. More 65Zn was excreted in the urine and feces with coated than uncoated 65ZnO NPs. However, over 95% of the total dose of both NPs was eliminated in the feces by day 7.

Conclusions

Although SiO2-coated ZnO NPs were more inflammogenic, the overall lung clearance rate was not affected. However, SiO2 coating altered the tissue distribution of 65Zn in some extrapulmonary tissues. For both IT instillation and gavage administration, SiO2 coating enhanced transport of 65Zn to thoracic lymph nodes and decreased transport to the skeletal muscle.

Keywords

Zinc oxideNanoparticlesPharmacokineticsBioavailabilitySilica coatingNanotoxicology

Background

Zinc oxide nanoparticles (ZnO NPs) are widely used in consumer products, including ceramics, cosmetics, plastics, sealants, toners and foods [1]. They are a common component in a range of technologies, including sensors, light emitting diodes, and solar cells due to their semiconducting and optical properties [2]. ZnO NPs filter both UV-A and UV-B radiation but remain transparent in the visible spectrum [3]. For this reason, ZnO NPs are commonly added to sunscreens [4] and other cosmetic products. Furthermore, advanced technologies have made the large-scale production of ZnO NPs possible [5]. Health concerns have been raised due to the growing evidence of the potential toxicity of ZnO NPs. Reduced pulmonary function in humans was observed 24 hours after inhalation of ultrafine (<100 nm) ZnO [6]. It has also been shown to cause DNA damage in HepG2 cells and neurotoxicity due to the formation of reactive oxygen species (ROS) [7],[8]. Recently, others and we have demonstrated that ZnO NPs can cause DNA damage in TK6 and H9T3 cells [9],[10]. ZnO NPs dissolve in aqueous solutions, releasing Zn2+ ions that may in turn cause cytotoxicity and DNA damage to cells [9],[11]–[13].

Studies have shown that changing the surface characteristics of certain NPs may alter the biologic responses of cells [14],[15]. Developing strategies to reduce the toxicity of ZnO NPs without changing their core properties (safer-by-design approach) is an active area of research. Xia et al. [16] showed that doping ZnO NPs with iron could reduce the rate of ZnO dissolution and the toxic effects in zebra fish embryos and rat and mouse lungs [16]. We also showed that encapsulation of ZnO NPs with amorphous SiO2 reduced the dissolution of Zn2+ ions in biological media, and reduced cell cytotoxicity and DNA damage in vitro [17]. Surface characteristics of NPs, such as their chemical and molecular structure, influence their pharmacokinetic behavior [18]–[20]. Surface chemistry influences the adsorption of phospholipids, proteins and other components of lung surfactants in the formation of a particle corona, which may regulate the overall nanoparticle pharmacokinetics and biological responses [19]. Coronas have been shown to influence the dynamics of cellular uptake, localization, biodistribution, and biological effects of NPs [21],[22].

Coating of NPs with amorphous silica is a promising technique to enhance colloidal stability and biocompatibility for theranostics [23],[24]. A recent study by Chen et al. showed that coating gold nanorods with silica can amplify the photoacoustic response without altering optical absorption [25]. Furthermore, coating magnetic NPs with amorphous silica enhances particle stability and reduces its cytotoxicity in a human bronchial epithelium cell line model [26]. Amorphous SiO2 is generally considered relatively biologically inert [27], and is commonly used in cosmetic and personal care products, and as a negative control in some nanoparticle toxicity screening assays [28]. However, Napierska et al. demonstrated the size-dependent cytotoxic effects of amorphous silica in vitro[29]. They concluded that the surface area of amorphous silica is an important determinant of cytotoxicity. An in vivo study using a rat model demonstrated that the pulmonary toxicity and inflammatory responses to amorphous silica are transient [30]. Moreover, SiO2-coated nanoceria induced minimal lung injury and inflammation [31]. It has also been demonstrated that SiO2 coating improves nanoparticle biocompatibility in vitro for a variety of nanomaterials, including Ag [32], Y2O3[33], and ZnO [17]. We have recently developed methods for the gas-phase synthesis of metal and metal oxide NPs by a modified flame spray pyrolysis (FSP) reactor. Coating metal oxide NPs with amorphous SiO2 involves the encapsulation of the core NPs in flight with a nanothin amorphous SiO2 layer [34]. An important advantage of flame-made NPs is their high purity. Flame synthesis is a high-temperature process that leaves no organic contamination on the particle surface. Furthermore, the presence of SiO2 does not influence the optoelectronic properties of the core ZnO nanorods. Thus, they retain their desired high transparency in the visible spectrum and UV absorption rendering them suitable for UV blocking applications [17]. The SiO2 coating has been demonstrated to reduce ZnO nanorod toxicity by mitigating their dissolution and generation of ions in solutions, and by preventing the immediate contact between the core particle and mammalian cells. For ZnO NPs, such a hermetic SiO2 coating reduces ZnO dissolution while preserving the optical properties and band-gap energy of the ZnO core [17].

Studies examining nanoparticle structure-pharmacokinetic relationships have established that plasma protein binding profiles correlate with circulation half-lives [27]. However, studies evaluating the relationship between surface modifications, lung clearance kinetics, and pulmonary effects are lacking. Thus, we sought to study the effects of amorphous SiO2 coating on ZnO pulmonary effects and on pharmacokinetics of 65Zn when radioactive 65ZnO and SiO2-coated 65ZnO nanorods are administered by intratracheal instillation (IT) and gavage. We explored how the SiO2 coating affected acute toxicity and inflammatory responses in the lungs, as well as 65Zn clearance and tissue distribution after IT instillation over a period of 28 days. The translocation of the 65Zn from the stomach to other organs was also quantified for up to 7 days after gavage. Finally, we examined how the SiO2 coating affected the urinary and fecal excretion of 65Zn during the entire observation period.

Results

Synthesis and characterization of ZnO and SiO2-coated ZnO NPs

Uncoated and SiO2-coated ZnO NPs were made by flame spray pyrolysis using the Versatile Engineered Nanomaterial Generation System at Harvard University [35],[17]. The detailed physicochemical and morphological characterization of these NPs was reported earlier [36],[17]. The ZnO primary NPs had a rod-like shape with an aspect ratio of 2:1 to 8:1 (Figure 1) [37],[17]. Flame-made nanoparticles typically exhibit a lognormal size distribution with a geometric standard deviation of σ g = 1.45 [38]. To create the SiO2-coated ZnO nanorods, a nanothin (~4.6 ± 2.5 nm) amorphous SiO2 layer encapsulated the ZnO core [17] (Figure 1B). The amorphous nature of the silica coating was verified by X-ray diffraction (XRD) and electron microscopy analyses [17]. The average crystal size of uncoated and SiO2-coated NPs were 29 and 28 nm, respectively [39]. Their specific surface areas (SSA) were 41 m2/g (uncoated) and 55 m2/g (SiO2-coated) [40]. The lower density of SiO2 compared to ZnO contributes to the higher SSA of the SiO2-coated ZnO than uncoated NPs. The extent of the SiO2 coating was assessed by X-ray photoelectron spectroscopy and photocatalytic experiments. These data showed that less than 5% of ZnO NPs were uncoated, as some of the freshly-formed core ZnO NPs may escape the coating process [41],[17]. Furthermore, the ZnO dissolution of the SiO2-coated nanorods was significantly lower than the uncoated NPs in culture medium over 24 h [17]. The Zn2+ ion concentration reached equilibrium after 6 hours for the coated NPs (~20%), while the uncoated ones dissolved at a constant rate up to 24 hours [17]. For both IT and gavage routes, the NPs were dispersed in deionized water by sonication at 242 J/ml. The hydrodynamic diameters were 165 ± 3 nm (SiO2-coated) and 221 ± 3 nm (uncoated). The zeta potential values in these suspensions were 23 ± 0.4 mV (uncoated) and −16.2 ± 1.2 (SiO2-coated). The zeta potential differences between these two types of NPs were observed at a pH range of 2.5-8.0 [17], which includes the pH conditions in the airways/alveoli and small and large intestines. The post-irradiation hydrodynamic diameter and zeta potential in water suspension were similar to those of pristine NPs used in the lung toxicity/inflammation experiments.
Figure 1

Physicochemical characterization of test materials. Transmission electron micrograph of uncoated ZnO (A) and SiO2-coated ZnO (B) NPs. The thin silica coating of approximately 5 nm is shown in B, inset.

Pulmonary responses to intratracheally instilled ZnO and SiO2-coated ZnO

We compared the pulmonary responses to uncoated versus SiO2-coated ZnO NPs at 24 hours after IT instillation in rats. Groups of 4–6 rats received 0, 0.2 or 1 mg/kg of either type of NP. We found that IT-instilled coated and uncoated ZnO NPs induced a dose-dependent injury and inflammation evident by increased neutrophils, elevated levels of myeloperoxidase (MPO), albumin and lactate dehydrogenase (LDH) in the bronchoalveolar lavage (BAL) fluid at 24 hours post-instillation (Figure 2). At the lower dose of 0.2 mg/kg, only the SiO2-coated ZnO instilled rats (n = 4) showed elevated neutrophils, LDH, MPO, and albumin levels. But at 1 mg/kg, both types of NPs induced injury and inflammation to the same extent, except that MPO was higher in rats instilled with SiO2-coated ZnO NPs.
Figure 2

Cellular and biochemical parameters of lung injury and inflammation in bronchoalveolar lavage (BAL). Tracheally instilled ZnO and SiO2-coated ZnO induced a dose-dependent lung injury and inflammation at 24 hours. (A) Significant increases in BAL neutrophils were observed at 1 mg/kg of both NPs (n = 6/group). At the lower dose of 0.2 mg/kg (n = 4-6/group), only the SiO2-coated ZnO (n = 4) induced significant neutrophil influx in the lungs. (B) Similarly, significant increases in LDH, myeloperoxidase and albumin were observed at 1 mg/kg of both NPs, and at 0.2 mg of SiO2-coated ZnO. (*P < 0.05, vs. control, #P < 0.05, SiO2-coated ZnO versus ZnO).

Pharmacokinetics of intratracheally-instilled uncoated or SiO2-coated 65ZnO NPs

Clearance of instilled uncoated or SiO2-coated 65ZnO NPs from the lungs is shown in Figure 3. Overall, both 65ZnO NPs and SiO2-coated 65ZnO NPs exhibited a biphasic clearance with a rapid initial phase (t1/2: 65ZnO = 0.3 hours; SiO2-coated 65ZnO = 0.2 hours) and a slower terminal phase (t1/2: 65ZnO = 42 hours; SiO2-coated 65ZnO = 29 hours). No significant difference was observed on the initial clearance between the two types of NPs. At 2 days, 18.1 ± 2.1% and 16.1 ± 2.0% remained in the lungs for the SiO2-coated and uncoated 65ZnO NPs, respectively. At 7 and 28 days post-IT instillation, we observed statistically significant but small (in magnitude) differences. At 28 days, only 0.14 ± 0.01% of SiO2-coated 65ZnO and 0.28 ± 0.05% of the uncoated 65ZnO NPs remained in the lungs.
Figure 3

Lung clearance of 65 Zn post-IT instillation of 65 ZnO and SiO 2 -coated 65 ZnO NPs. The percentages of instilled 65Zn measured in the whole lungs are shown over a period of 28 days. The clearance of 65Zn was rapid with only 16-18% of dose remaining at 2 days. By day 7, only 1.1% (SiO2-coated 65ZnO NPs) and 1.9% (65ZnO NPs) were measured in the lungs. And by the end of experiment, 65Zn was nearly gone (less than 0.3% of dose). Although statistically higher levels of 65ZnO NPs than of SiO2-coated 65ZnO NPs remained in the lungs at 7 and 28 days, the graphs show nearly identical clearance kinetics. (n = 8 rats at 5 minutes, 2 days, and 7 days, n = 5 at 28 days).

However, analyses of the selected extrapulmonary tissues showed significant differences (Figure 4). Even at the earliest time point of 5 minutes post-IT instillation, significantly more 65Zn was detected in the blood (0.47% vs. 0.25%) and heart (0.03% vs. 0.01%) of rats instilled with the uncoated 65ZnO NPs. These tissue differences became more pronounced at later time points. At 2 days post-IT instillation, more 65Zn from uncoated 65ZnO NPs translocated to the blood, skeletal muscle, kidneys, heart, liver and cecum than from SiO2-coated 65ZnO NPs (Table 1). At 7 and 28 days, the overall differences in the 65Zn contents in these tissues remained the same. As shown in Tables 2 and 3, significantly higher fractions of the 65Zn from uncoated 65ZnO NPs than from SiO2-coated 65ZnO NPs were found in the blood, skeletal muscle, heart, liver and skin. Interestingly, higher percentages of 65Zn dose from the SiO2-coated 65ZnO NPs were found in the thoracic lymph nodes and bone marrow (Tables 2 and 4). Radioactive 65Zn levels decreased from 2 to 28 days in all tissues except bone, where it increased for both types of NPs. Additionally, we found that the total recovered 65Zn in examined tissues, feces and urine was significantly higher in uncoated than SiO2-coated 65ZnO NPs (Tables 1, 23 and Figure 5). Since the thoracic lymph nodes had higher 65Zn in the latter group at all time points (Tables 1, 2 and 3), we speculate that the unaccounted radioactivity may have been in other lymph nodes as well as organs not analyzed such as adipose tissue, pancreas, adrenals, teeth, nails, tendons, and nasal tissues.
Figure 4

Extrapulmonary distribution of 65 Zn post-IT instillation of 65 ZnO and SiO 2 -coated 65 ZnO NPs. Data are % of instilled dose recovered in all secondary tissues examined. It included blood, thoracic lymph nodes, bone, bone marrow, skin, brain, skeletal muscle, testes, kidneys, heart, liver, and the gastrointestinal tract. There was a rapid absorption and accumulation of 65Zn in secondary tissues. At day 2, 59-72% of the dose was detected in extrapulmonary organs. Then, 65Zn levels decreased over time to 25-37% by day 28. Significantly more 65Zn was detected in secondary organs at all time points in rats instilled with uncoated 65ZnO NPs.

Table 1

Tissue distribution of 65 Zn at 2 days after intratracheal instillation of 65 ZnO or SiO 2 -coated 65 ZnO NPs in rats

 

ZnO

SiO2-coated ZnO

 

Mean ± SE

Mean ± SE

Lungs

16.08 ± 2.00

18.09 ± 2.17

Blood

2.54 ± 0.07*

2.22 ± 0.08

Lymph nodes

0.63 ± 0.23

0.49 ± 0.04

Bone marrow

3.37 ± 0.32

2.89 ± 0.15

Bone

9.59 ± 0.46

9.25 ± 0.29

Skin

11.30 ± 1.00

12.52 ± 0.77

Brain

0.17 ± 0.01

0.19 ± 0.01 #

Skeletal muscle

14.22 ± 0.77*

6.39 ± 2.44

Testes

0.84 ± 0.05

0.78 ± 0.04

Kidneys

2.05 ± 0.07*

1.74 ± 0.03

Spleen

0.50 ± 0.02

0.44 ± 0.02

Heart

0.47 ± 0.04*

0.36 ± 0.01

Liver

12.23 ± 0.24*

9.88 ± 0.38

Stomach

1.01 ± 0.14

0.78 ± 0.02

Small intestine

7.37 ± 0.32

6.89 ± 0.20

Large intestine

2.08 ± 0.21

1.91 ± 0.12

Cecum

3.42 ± 0.22*

2.35 ± 0.25

Total recovered

87.78 ± 2.35*

76.78 ± 2.84

Data are mean ± SE% instilled dose, n = 8/group.

Total recovered = sum of 65Zn in analyzed organs, feces and urine.

*P < 0.05, ZnO > SiO2-coated ZnO.

# P <0.05, SiO2-coated ZnO > ZnO.

Table 2

Tissue distribution of 65 Zn at 7 days after intratracheal instillation of 65 ZnO or SiO 2 -coated 65 ZnO NPs in rats

 

65ZnO

SiO2-coated65ZnO

 

Mean ± SE

Mean ± SE

Lungs

1.90 ± 0.18

1.05 ± 0.04

Blood

2.13 ± 0.05*

1.79 ± 0.07

Lymph nodes

0.18 ± 0.02

0.31 ± 0.05 #

Bone marrow

3.70 ± 0.28

3.38 ± 0.16

Bone

12.12 ± 0.53

12.21 ± 0.84

Skin

10.02 ± 0.49

10.55 ± 0.80

Brain

0.25 ± 0.01

0.27 ± 0.01

Skeletal muscle

19.81 ± 0.84*

8.34 ± 3.45

Testes

1.27 ± 0.06

1.23 ± 0.03

Kidneys

0.75 ± 0.03

0.69 ± 0.02

Spleen

0.21 ± 0.01

0.20 ± 0.01

Heart

0.27 ± 0.01*

0.23 ± 0.01

Liver

5.80 ± 0.13*

5.19 ± 0.25

Stomach

0.66 ± 0.02

0.66 ± 0.03

Small intestine

2.83 ± 0.10

2.53 ± 0.12

Large intestine

0.84 ± 0.07

0.83 ± 0.05

Cecum

1.15 ± 0.06

1.04 ± 0.09

Total recovered

81.33 ± 6.51*

69.91 ± 3.33

Data are mean ± SE% instilled dose, n = 8/group.

Total recovered = sum of 65Zn in analyzed organs, feces and urine.

*P < 0.05, ZnO > SiO2-coated ZnO.

# P <0.05, SiO2-coated ZnO > ZnO.

Table 3

Tissue distribution of 65 Zn at 28 days after intratracheal instillation of 65 ZnO or SiO 2 -coated 65 ZnO NPs in rats

 

65ZnO

SiO2-coated65ZnO

 

Mean ± SE

Mean ± SE

Lungs

0.28 ± 0.05*

0.14 ± 0.01

Blood

0.79 ± 0.05*

0.61 ± 0.02

Lymph nodes

0.03 ± 0.005

0.12 ± 0.01 #

Bone marrow

2.30 ± 0.12

3.33 ± 0.15 #

Bone

12.40 ± 0.36

13.59 ± 0.52

Skin

4.72 ± 0.60*

3.26 ± 0.13

Brain

0.18 ± 0.02

0.15 ± 0.01

Skeletal muscle

13.27 ± 3.02*

0.66 ± 0.04

Testes

0.49 ± 0.03

0.46 ± 0.03

Kidneys

0.19 ± 0.01

0.17 ± 0.005

Spleen

0.05 ± 0.005

0.04 ± 0.003

Heart

0.07 ± 0.004*

0.06 ± 0.002

Liver

1.32 ± 0.09

1.14 ± 0.02

Stomach

0.20 ± 0.01

0.20 ± 0.01

Small intestine

0.63 ± 0.05

0.55 ± 0.01

Large intestine

0.19 ± 0.02

0.25 ± 0.01 #

Cecum

0.23 ± 0.02

0.24 ± 0.01

Total recovered

88.20 ± 4.36*

72.81 ± 0.53

Data are mean ± SE% instilled dose, n = 5/group.

Total recovered = sum of 65Zn in analyzed organs, feces and urine.

*P < 0.05, ZnO > SiO2-coated ZnO.

# P <0.05, SiO2-coated ZnO > ZnO.

Table 4

Distribution of 65 Zn 7 days after gavage administration of 65 ZnO or SiO 2 -coated 65 ZnO NPs in rats

 

65ZnO

SiO2-coated65ZnO

 

Mean ± SE

Mean ± SE

Lungs

0.04 ± 0.01

0.06 ± 0.01

Blood

0.23 ± 0.03

0.22 ± 0.04

Lymph nodes

0.02 ± 0.003

0.06 ± 0.01 #

Bone marrow

0.49 ± 0.06

0.47 ± 0.09

Bone

1.62 ± 0.26

2.20 ± 0.45

Skin

1.13 ± 0.14

1.77 ± 0.29

Brain

0.03 ± 0.002

0.04 ± 0.005

Skeletal muscle

2.45 ± 0.36*

0.20 ± 0.03

Testes

0.14 ± 0.01

0.19 ± 0.03

Kidneys

0.08 ± 0.01

0.09 ± 0.01

Spleen

0.02 ± 0.004

0.02 ± 0.004

Heart

0.03 ± 0.003

0.02 ± 0.004

Liver

0.58 ± 0.07

0.71 ± 0.09

Stomach

0.07 ± 0.01

0.09 ± 0.01

Small intestine

0.25 ± 0.03

0.36 ± 0.05

Large intestine

0.09 ± 0.02

0.11 ± 0.01

Cecum

0.13 ± 0.02

0.12 ± 0.02

Total recovered

100.59 ± 2.56*

83.40 ± 2.42

Data are mean ± SE% gavaged dose, n = 5/group.

Total recovered = sum of 65Zn in analyzed organs, feces and urine.

*P < 0.05, ZnO > SiO2-coated ZnO.

# P <0.05, SiO2-coated ZnO > ZnO.

Urinary excretion of 65Zn was much lower than fecal excretion in both groups. The urinary excretion of 65Zn in rats instilled with SiO2-coated 65ZnO NPs was significantly higher than in those instilled with uncoated 65ZnO NPs (Figure 5B). Although the fecal excretion rates appeared similar, slightly but significantly more 65Zn (50.04 ± 0.96% vs. 46.68 ± 0.76%) was eliminated via the feces over 28 days in rats instilled with uncoated 65ZnO NPs (Figure 5A).
Figure 5

Fecal and urinary excretion of 65 Zn post-IT instillation of 65 ZnO and SiO 2 -coated 65 ZnO NPs. Data are estimated cumulative urinary or fecal excretion of 65Zn over 28 days. The predominant excretion pathway was via the feces. Approximately half of the instilled 65Zn was excreted in the feces in both groups over 28 days (A). Only about 1% of the 65Zn dose was excreted in the urine (B).

Pharmacokinetics of gavaged uncoated or SiO2-coated 65ZnO NPs

Absorption of 65Zn from the gut was studied at 5 minutes and 7 days post-gavage of uncoated or SiO2-coated 65ZnO NPs. Nearly 100% of the dose was recovered at 5 minutes in the stomach for both types of NPs (Figure 6A). The 65Zn levels in tissues other than the gastrointestinal tract were much lower (0.3% for uncoated, 0.05% for coated 65ZnO NPs). However, significantly higher percentages of total dose were still detected in the blood, bone marrow, skin, testes, kidneys, spleen and liver in rats instilled with uncoated 65ZnO NPs (data not shown). After 7 days, low levels of 65Zn from both types of NPs (<1% original dose) were measured in all organs except the bone, skeletal muscle and skin (Figure 6B, Table 4). Higher levels of 65Zn were observed in the skeletal muscle from uncoated than from coated 65ZnO NPs at this time point (Table 4). However, similar to the IT-instillation data, the thoracic lymph nodes retained more 65Zn from the SiO2-coated than the uncoated 65ZnO NPs. Urinary excretion of 65Zn was also much lower than fecal excretion post-gavage. The urinary excretion of 65Zn in rats gavaged with SiO2-coated 65ZnO NPs was significantly higher than in rats gavaged with uncoated 65ZnO NPs (Figure 7B). The fecal excretion in the gavaged rats was higher than in IT-instilled rats. Despite a significant difference in fecal excretion during the first day post-gavage, nearly 95% of the dose for both types of NPs was excreted in the feces by day 7 (Figure 7A).
Figure 6

Tissue distribution of 65 Zn post-gavage of 65 ZnO and SiO 2 -coated 65 ZnO NPs. Data are % dose of administered 65Zn in different organs. (A) At 5 minutes post-gavage, the 65Zn levels in tissues other than the gastrointestinal tract were much lower (0.3% for uncoated, 0.05% for coated 65ZnO NPs). (B) At day 7, significantly more 65Zn was absorbed and retained in non-GIT tissues (6.9% for uncoated, 6.0% for coated 65ZnO NPs). Significantly more 65Zn was measured in skeletal muscle in rat gavaged with uncoated versus coated 65ZnO NPs. (Note: RBC: red blood cell; sk muscl: skeletal muscle; sm int: small intestine: large int: large intestine).

Figure 7

Fecal and urinary excretion of 65 Zn post-gavage of 65 ZnO and SiO 2 -coated 65 ZnO NPs. Data are estimated cumulative urinary or fecal excretion of 65Zn over 7 days. Similar to the IT-instilled groups, the predominant excretion pathway was via the feces. Ninety five % of the instilled 65Zn was excreted in both groups by day 7 (A). Only 0.1% of the 65Zn dose was excreted in the urine (B).

Discussion

Nanoparticles can be released into the workplace environment during production and handling of nanomaterials [42]. For example, studies have shown that ZnO NPs were released during an abrasion test of commercially available two-pack polyurethane coatings with ZnO NPs [43]. This suggests the likelihood of emission of NPs during activities related to handling of nano-enabled products. In this study we describe the acute pulmonary responses to ZnO NPs and the pharmacokinetics of Zn from ZnO or SiO2-coated ZnO NPs in male Wistar Han rats. To track Zn for biokinetic studies in rats, we neutron activated the NPs to change the stable element 64Zn into radioactive 65Zn, suitable for detection over long-term studies. The agglomerate size and zeta potential in water suspension were similar to those of pristine ZnO NPs. Using these radioactive NPs, we evaluated the influence of an amorphous silica coating on the clearance, bioavailability and excretion of 65Zn following intratracheal instillation and gavage of 65ZnO and Si-coated 65ZnO NPs. We have shown previously that the hermetic encapsulation of ZnO NPs with a thin layer of amorphous SiO2 reduces the dissolution of Zn2+ ions in biological media, DNA damage in vitro [17] and cellular toxicity [36]. Since the SiO2 coating does not affect the core ZnO NP optoelectronic properties, these coatings may be employed in sunscreens and UV filters. This could be a strategy to reduce ZnO toxicity while maintaining the intended performance of ZnO NPs.

Intratracheal instillation differs from inhalation exposure in terms of particle distribution, dose rate, clearance, NP agglomerate surface properties, and pattern of injury [44],[45]. A study by Baisch et al. reported a higher inflammatory response following intratracheal instillation compared to whole body inhalation for single and repeated exposures of titanium dioxide NPs when deposited doses were held constant [46]. Although IT instillation does not directly model inhalation exposure, it is a reliable method for administering a precise dose to the lungs for biokinetic studies. We hypothesized that silica coating may alter zinc-induced lung injury and inflammation by reducing the available zinc ions based on our previous data [17]. We have also shown that pulmonary toxicity in rats exposed to nanoceria via inhalation was reduced when exposed to the same nanoceria with amorphous SiO2 coating. Surprisingly, the in vivo lung responses in the present study showed the opposite. That amorphous silica can cause injury and inflammation when inhaled at high doses has been shown in several previous studies [47]–[51]. However, it has also been shown that the lung injury and inflammatory responses to amorphous silica are transient [27]. In this study, SiO2-coated ZnO NPs induce more lung injury/inflammation than uncoated ZnO, even at a low dose at which uncoated ZnO had no effects. Considering that the effective density of ZnO NPs is reduced by silica coating (ZnO: 5.6 g/cm3 vs. SiO2-coated ZnO: estimated 4.1 g/cm3), it is possible that the coated particle number concentration is higher for an equivalent mass of NP. It is also likely that the silica coating elicits more inflammation than the ZnO NPs. Silica may act in concert with dissolved Zn ions, causing more lung injury. Furthermore, surface coating with amorphous silica also changed the zeta-potential of ZnO NPs from positive (23.0 ± 0.4 mV, uncoated ZnO NPs) to negative (−16.2 ± 1.2 mV, SiO2-coated ZnO NPs), decreasing the likelihood of agglomeration and sedimentation of SiO2-coated NP suspension in aqueous systems. The reduced NP agglomeration of the SiO2-coated ZnO NPs may increase the available NP surface area that may facilitate biointeractions with lung cells and thus induces a higher toxic/inflammatory response. It has also been reported that surface charge may influence the lung translocation rates of NPs [52]. For example, the adsorption of endogenous proteins like albumin to the surface of charged NPs increases their hydrodynamic diameter and alters their translocation rate [53]. It was also showed that NPs with zwitterionic cysteine and polar PEG ligands on the surface cause their rapid translocation to the mediastinal lymph nodes. Additionally, a higher surface charge density has been shown to cause an increased adsorption of proteins on NPs [54] while zwitterionic or neutral organic coatings have been shown to prevent adsorption of serum proteins [18]. A recent study also showed that nanoparticle protein corona can alter their uptake by macrophages [55].

Our results demonstrate that ZnO and SiO2-coated ZnO NPs are both cleared rapidly and completely from the lungs by 28 days after IT instillation. In the lungs, NPs may be cleared via different pathways. They may be cleared by dissolution before or after alveolar macrophage uptake, by phagocytic cells in the lymph nodes, or by translocation across the alveolar epithelium into the blood circulation [56]. Since ZnO NPs have been shown to dissolve in culture medium and in endosomes [57], it is not surprising that lung clearance of 65ZnO NPs was rapid compared to that of poorly soluble NPs of cerium oxide [58] and titanium dioxide [59]. The clearance of radioactive 65Zn from the lungs includes translocation of the NPs themselves as well as dissolution of 65ZnO which is an important clearance mechanism [60]. As shown previously, the silica coating reduced the dissolution of ZnO NPs in culture medium [17], suggesting that dissolution and clearance in vivo may also be reduced. However, the silica coating appeared to very modestly but significantly enhance the amount of cleared 65Zn at day 7 and 28. The significance of this observation needs further investigation.

Despite similar clearance from the lungs over 28 days, translocation of 65Zn from uncoated ZnO NPs is significantly higher than from coated ZnO NPs in some of the examined extrapulmonary tissues, especially skeletal muscle. In these extrapulmonary tissues, the measured 65Zn is more likely to be dissolved Zn, rather than intact 65ZnO. The amount of 65Zn was greatest in the skeletal muscle, liver, skin, and bone from both particle types. The selective retention of 65Zn into those tissues might be explained, in part, by the fact that 85% of the total body zinc is present in skeletal muscle and bone [61]. There was clearance of 65Zn from most of the extrapulmonary tissues we examined over time (day 2 to day 28), except in bone where 65Zn levels increased. The skin and skeletal muscle exhibited faster clearance with coated than with uncoated NPs. 65Zn from both particle types was largely excreted in the feces, presumably via pancreato-biliary secretion, and to a lesser extent via mucociliary clearance of instilled NPs [62]. A study investigating the pharmacokinetic behavior of inhaled iridium NPs showed that they accumulated in soft connective tissue (2%) and bone, including bone marrow (5%) [63].

Although this study indicates that the SiO2 coating modestly reduces the translocation of 65Zn to the blood, skin, kidneys, heart, liver and skeletal muscle, it is unclear whether the SiO2-coated ZnO NPs dissolve at a different rate in vivo, and whether 65Zn is in particulate or ionic form when it reaches the circulation and bone. ZnO NPs have been shown to rapidly dissolve under acidic conditions (pH 4.5) and are more likely to remain intact around neutral pHs [64]. It is likely that the ZnO NPs entering phagolysosomal compartments of alveolar macrophages or neutrophils may encounter conditions favorable for dissolution. Our previous study suggested that the SiO2 coating is stable in vitro and exhibits low dissolution in biological media (<8% over 24 hours) [17]. Thus, it is possible that the SiO2-coated NPs remain in particulate form for a longer period of time. There are data showing that translocation of gold, silver, TiO2, polystyrene and carbon particles in the size range of 5–100 nm crossing the air-blood barrier and reaching blood circulation and extrapulmonary organs can occur [65]–[71].

The SiO2 coating significantly increased the levels of 65Zn in the bone and bone marrow (Table 3). We note that zinc is essential to the development and maintenance of bone. Zinc is known to play a major role in bone growth in mammals [72], and is required for protein synthesis in osteoblasts [73]. It can also inhibit the development of osteoclasts from bone marrow cells, thereby reducing bone resorption and bone growth [74],[75]. Radioactive 65Zn from uncoated and coated 65ZnO NPs also translocated to the skin, skeletal muscle, liver, heart, small intestine, testes, and brain (but to a lesser extent than the bone and bone marrow). It is important to note that of the 16 extrapulmonary tissues examined at 28 days after IT instillation, 4 had a higher 65Zn content from uncoated ZnO than coated ZnO (blood, skin, skeletal muscle and heart) (Table 3). This suggests that amorphous silica coating of NPs may reduce Zn retention and its potential toxicity when accumulated at high levels in those organs. Whether coating modifications like the use of thicker or different coatings can further reduce Zn bioavailability warrants further investigation. There was significantly more 65Zn from SiO2-coated ZnO excreted in the urine, which was more likely the ionic form of Zn.

The oral exposure to ZnO NPs is relevant from an environmental health perspective. ZnO is widely used as a nutritional supplement and as a food additive [76]. Because it is an essential trace element, zinc is routinely added to animal food products and fertilizer [75]. Due to its antimicrobial properties, there is increasing interest in adding ZnO to polymers in food packaging and preservative films to prevent bacterial growth [77]. It is possible that ZnO in sunscreens, ointments, and other cosmetics can be accidentally ingested, especially by children. The biokinetic behavior of NPs in the gastrointestinal tract may be influenced by particle surface charge. Positively charged particles are attracted to negatively charged mucus, while negatively charged particles directly contact epithelial cell surfaces [78]. A study by Paek et al. investigating the effect of surface charge on the biokinetics of Zn over 4 hours after oral administration of ZnO NPs showed that negatively charged NPs were absorbed more than positively charged ZnO NPs [79]. However, no effect on tissue distribution was observed. This is in contrast to our findings at 7 days post-gavage when coating of ZnO NPs with amorphous SiO2 (with negative zeta potential) increased the retention in thoracic lymph nodes compared to uncoated ZnO NPs (with positive zeta potential). Our study also showed that low levels of 65Zn were retained in the blood, skeletal muscle, bone and skin from both coated and uncoated 65ZnO NPs (Table 4). Most of the gavaged dose (over 90%) was excreted in the feces by day 3 indicating a rapid clearance of ZnO NPs, consistent with previous reports. Another study reported the pharmacokinetics of ZnO NPs (125, 250 and 500 mg/kg) after a single and repeated dose oral administration (90-day) [80]. They found that plasma Zn concentration significantly increased in a dose-dependent manner, but significantly decreased within 24 hours post-oral administration, suggesting that the systemic clearance of ZnO NPs is rapid even at these high doses. In another study, Baek et al. examined the pharmacokinetics of 20 nm and 70 nm citrate-modified ZnO NPs at doses of 50, 300 and 2000 mg/kg [81]. Similar to our results, they showed that ZnO NPs were not readily absorbed into the bloodstream after single-dose oral administration. The tissue distributions of Zn from both 20 nm and 70 nm ZnO NPs were similar and mainly to the liver, lung and kidneys. The study also reported predominant excretion of Zn in the feces, with smaller 20 nm particles being cleared more rapidly than the 70 nm NPs.

In summary, the results presented here show that uncoated 65Zn NPs resulted in higher levels of 65Zn in multiple organs following intratracheal instillation or gavage, particularly in skeletal muscle. This suggests that coating with amorphous silica can reduce tissue Zn concentration and its potential toxicity. Interestingly, the bioavailability of Zn from SiO2-coated 65ZnO was higher in thoracic lymph nodes and bone. Additionally, the excretion of 65Zn was higher from SiO2-coated 65ZnO NPs from both routes suggesting enhanced hepatobiliary excretion. Our data indicate that silica coating alters the pharmacokinetic behavior of ZnO NPs, but the effect was not as dramatic as anticipated. With increasing trends in physicochemical modifications of NPs for special applications, it is necessary to understand their influence on the fate, metabolism and toxicity of these nanoparticles.

Conclusions

We examined the influence of a 4.5 nm SiO2 coating on ZnO NPs on the 65Zn pharmacokinetics following IT instillation and gavage of neutron activated NPs. The SiO2 coating does not affect the clearance of 65Zn from the lungs. However, the extrapulmonary translocation and distribution of 65Zn from coated versus uncoated 65ZnO NPs were significantly altered in some tissues. The SiO2 coating resulted in lower translocation of instilled 65Zn to the skeletal muscle, skin and heart. The SiO2 coating also reduced 65Zn translocation to skeletal muscle post-gavage. For both routes of administration, the SiO2 coating enhanced the transport of 65Zn to the thoracic lymph nodes.

Methods

Synthesis of ZnO and SiO2-coated ZnO NPs

The synthesis of these NPs was reported in detail elsewhere [17]. In brief, uncoated and SiO2-coated ZnO particles were synthesized by flame spray pyrolysis (FSP) of zinc naphthenate (Sigma-Aldrich, St. Louis, MO, USA) dissolved in ethanol (Sigma-Aldrich) at a precursor molarity of 0.5 M. The precursor solution was fed through a stainless steel capillary at 5 ml/min, dispersed by 5 L/min O2 (purity > 99%, pressure drop at nozzle tip: pdrop = 2 bar) (Air Gas, Berwyn, PA, USA) and combusted. A premixed methane-oxygen (1.5 L/min, 3.2 L/min) supporting flame was used to ignite the spray. Oxygen (Air Gas, purity > 99%) sheath gas was used at 40 L/min. Core particles were coated in-flight by the swirl-injection of hexamethyldisiloxane (HMDSO) (Sigma Aldrich) through a torus ring with 16 jets at an injection height of 200 mm above the FSP burner. A total gas flow of 16 L/min, consisting of N2 carrying HMDSO vapor and pure N2, was injected through the torus ring jets. HMDSO vapor was obtained by bubbling N2 gas through liquid HMDSO (500 ml), maintained at a controlled temperature using a temperature-controlled water bath.

Characterization of ZnO and SiO2-coated ZnO NPs

The morphology of these NPs was examined by electron microscopy. Uncoated and SiO2-coated ZnO NPs were dispersed in ethanol at a concentration of 1 mg/ml in 50 ml polyethylene conical tubes and sonicated at 246 J/ml (Branson Sonifier S-450A, Swedesboro, NJ, USA). The samples were deposited onto lacey carbon TEM grids. All grids were imaged with a JEOL 2100. The primary particle size was determined by X-ray diffraction (XRD). XRD patterns for uncoated ZnO and SiO2-coated ZnO NPs were obtained using a Scintag XDS2000 powder diffractometer (Cu Kα, λ = 0.154 nm, 40 kV, 40 mA, stepsize = 0.02°). One hundred mg of each sample was placed onto the diffractometer stage and analyzed from a range of 2θ = 20-70°. Major diffraction peaks were identified using the Inorganic Crystal Structure Database (ICSD) for wurtzite (ZnO) crystals. The crystal size was determined by applying the Debye-Scherrer Shape Equation to the Gaussian fit of the major diffraction peak. The specific surface area was obtained using the Brunauer-Emmet-Teller (BET) method. The samples were degassed in N2 for at least 1 hour at 150°C before obtaining five-point N2 adsorption at 77 K (Micrometrics Tristar 3000, Norcross, GA, USA).

Neutron activation of NPs

The NPs with and without the SiO2 coating were neutron-activated at the Massachusetts Institute of Technology (MIT) Nuclear Reactor Laboratory (Cambridge, MA). Samples were irradiated with a thermal neutron flux of 5 × 1013 n/cm2s for 120 hours. The resulting 65Zn radioisotope has a half-life of 244.3 days and a primary gamma energy peak of 1115 keV. The relative specific activities for 65Zn were 37.7 ± 5.0 kBq/mg for SiO2-coated 65ZnO and 41.7 ± 7.2 kBq/mg for 65ZnO NPs.

Preparation and characterization of ZnO and SiO2-coated ZnO nanoparticle suspensions

Uncoated and SiO2-coated ZnO NPs were dispersed using a protocol previously described [82],[36]. The NPs were dispersed in deionized water at a concentration of 0.66 mg/ml (IT) or 10 mg/ml (gavage). Sonication was performed in deionized water to minimize the formation of reactive oxygen species. Samples were thoroughly mixed immediately prior to instillation. Dispersions of NPs were analyzed for hydrodynamic diameter (dH), polydispersity index (PdI), and zeta potential (ζ) by DLS using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK).

Animals

The protocols used in this study were approved by the Harvard Medical Area Animal Care and Use Committee. Nine-week-old male Wistar Han rats were purchased from Charles River Laboratories (Wilmington, MA). Rats were housed in pairs in polypropylene cages and allowed to acclimate for 1 week before the studies were initiated. Rats were maintained on a 12-hour light/dark cycle. Food and water were provided ad libitum.

Pulmonary responses – Bronchoalveolar lavage and analyses

This experiment was performed to determine pulmonary responses to instilled NPs. A group of rats (mean wt. 264 ± 15 g) was intratracheally instilled with either an uncoated ZnO or SiO2 -coated ZnO NP suspension at a 0, 0.2 or 1.0 mg/kg dose. The particle suspensions were delivered to the lungs through the trachea in a volume of 1.5 ml/kg. Twenty-four hours later, rats were euthanized via exsanguination with a cut in the abdominal aorta while under anesthesia. The trachea was exposed and cannulated. The lungs were then lavaged 12 times, with 3 ml of 0.9% sterile PBS, without calcium and magnesium ions. The cells of all washes were separated from the supernatant by centrifugation (350 × g at 4°C for 10 min). Total cell count and hemoglobin measurements were made from the cell pellets. After staining the cells, a differential cell count was performed. The supernatant of the two first washes was clarified via centrifugation (14,500 × g at 4°C for 30 min), and used for standard spectrophotometric assays for lactate dehydrogenase (LDH), myeloperoxidase (MPO) and albumin.

Pharmacokinetics of 65Zn

The mean weight of rats at the start of the experiment was 285 ± 3 g. Two groups of rats (29 rats/NP) were intratracheally instilled with 65ZnO NPs or with SiO2-coated 65ZnO NPs at a 1 mg/kg dose (1.5 ml/kg, 0.66 mg/ml). Rats were placed in metabolic cages containing food and water, as previously described. Twenty four-hour samples of feces and urine were collected at selected time points (0–24 hours, 2–3 days, 6–7 days, 9–10 days, 13–14 days, 20–21 days, and 27–28 days post-IT instillation). Fecal/urine collection was accomplished by placing each rat in individual metabolic cage containing food and water during each 24-hour period. All samples were analyzed for total 65Zn activity, and expressed as % of instilled 65Zn dose. Fecal and urine clearance curves were generated and were used to estimate the daily cumulative excretion. Groups of 8 rats were humanely sacrificed at 5 minutes, 2 days, 7 days, and 5 rats/group at 28 days. Therefore, the number of collected fecal/urine samples decreased over time.

Another cohort of 20 rats was dosed with 65ZnO (n = 10) or SiO2-coated 65ZnO (n = 10) by gavage at a 5 mg/kg dose (0.5 ml/kg, 10 mg/ml). One group of 5 rats was humanely sacrificed at 5 minutes and immediately dissected. Another group of 5 rats was individually placed in metabolic cages, as previously described, and 24-hour samples of urine and feces were collected at 0–1 day, 2–3 days, and 6–7 days post-gavage. The remaining rats were sacrificed at 7 days.

At each endpoint, rats were euthanized and dissected, and the whole brain, spleen, kidneys, heart, liver, lungs, GI tract, testes, thoracic lymph nodes, blood (10 ml, separated into plasma and RBC), bone marrow (from femoral bones), bone (both femurs), skin (2 × 3 inches), and skeletal muscle (from 4 sites) were collected. The 65Zn radioactivity present in each sample was measured with a WIZARD Gamma Counter (PerkinElmer, Inc., Waltham, MA). The number of disintegrations per minute was determined from the counts per minute and the counting efficiency. The efficiency of the gamma counter was derived from counting multiple aliquots of NP samples and relating them to the specific activities measured at Massachusetts Institute of Technology Nuclear Reactor. We estimated that the counter had an efficiency of ~52%. The 65Zn radioactivity was expressed as kBq/g tissue and the percentage of administered dose in each organ. All radioactivity data were adjusted for physical decay over the entire observation period. The radioactivity in organs and tissues not measured in their entirety was estimated as a percentage of total body weight as: skeletal muscle, 40%; bone marrow, 3.2%; peripheral blood, 7%; skin, 19%; and bone, 6% [83],[84]. Based on the 65Zn specific activity (kBq/mg NP) and tissue 65Zn concentration, the amount of Zn derived from each NP was calculated for each tissue examined (ng Zn/g tissue).

Statistical analyses

Differences in the 65Zn tissue distribution and in cellular and biochemical parameters measured in bronchoalveolar lavage between groups were analyzed using multivariate analysis of variance (MANOVA) with REGWQ (Ryan-Einot-Gabriel-Welch based on range) and Tukey post hoc tests using SAS Statistical Analysis software (SAS Institute, Cary, NC). The lung clearance half-life was estimated by a two-phase estimation by a biexponential model using the R Program v. 3.1.0 [85].

Declarations

Acknowledgements

This research was supported by NSF (1235806) and NIEHS grant (ES 0000002). GAS was supported by the Swiss National Science foundation for the Advanced Researcher fellowship (grant no. 145392). KMM received a Graduate Research Fellowship from the National Science Foundation (DGE-1144152). We thank Dr. Evelyn Hu (Harvard School of Engineering and Applied Sciences) for helpful discussions and Melissa Curran for editorial assistance.

Authors’ Affiliations

(1)
Center for Nanotechnology and Nanotoxicology, Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, School of Public Health, Harvard University, Boston, USA

References

  1. Sun B, Sirringhaus H: Solution-processed zinc oxide field-effect transistors based on self-assembly of colloidal nanorods. Nano Lett 2005, 5: 2408–2413. 10.1021/nl051586wView ArticlePubMedGoogle Scholar
  2. Su YK, Peng SM, Ji LW, Wu CZ, Cheng WB, Liu CH: Ultraviolet ZnO nanorod photosensors. Langmuir 2010, 26: 603–606. 10.1021/la902171jView ArticlePubMedGoogle Scholar
  3. Djurisic AB, Leung YH: Optical properties of ZnO nanostructures. Small 2006, 2: 944–961. 10.1002/smll.200600134View ArticlePubMedGoogle Scholar
  4. Nohynek GJ, Dufour EK, Roberts MS: Nanotechnology, cosmetics and the skin: is there a health risk? Skin Pharmacol Physiol 2008, 21: 136–149. 10.1159/000131078View ArticlePubMedGoogle Scholar
  5. Fan Z, Lu JG: Zinc oxide nanostructures: synthesis and properties. J Nanosci Nanotechnol 2005, 5: 1561–1573. 10.1166/jnn.2005.182View ArticlePubMedGoogle Scholar
  6. Beckett WS, Chalupa DF, Pauly-Brown A, Speers DM, Stewart JC, Frampton MW, Utell MJ, Huang LS, Cox C, Zareba W, Oberdorster G: Comparing inhaled ultrafine versus fine zinc oxide particles in healthy adults: a human inhalation study. Am J Respir Crit Care Med 2005, 171: 1129–1135. 10.1164/rccm.200406-837OCPubMed CentralView ArticlePubMedGoogle Scholar
  7. Sharma V, Anderson D, Dhawan A: Zinc oxide nanoparticles induce oxidative stress and genotoxicity in human liver cells (HepG2). J Biomed Nanotechnol 2011, 7: 98–99. 10.1166/jbn.2011.1220View ArticlePubMedGoogle Scholar
  8. Valdiglesias V, Costa C, Kilic G, Costa S, Pasaro E, Laffon B, Teixeira JP: Neuronal cytotoxicity and genotoxicity induced by zinc oxide nanoparticles. Environ Int 2013, 55: 92–100. 10.1016/j.envint.2013.02.013View ArticlePubMedGoogle Scholar
  9. Alarifi S, Ali D, Alkahtani S, Verma A, Ahamed M, Ahmed M, Alhadlaq HA: Induction of oxidative stress, DNA damage, and apoptosis in a malignant human skin melanoma cell line after exposure to zinc oxide nanoparticles. Int J Nanomed 2013, 8: 983–993. 10.2217/nnm.13.80View ArticleGoogle Scholar
  10. Watson C, Ge J, Cohen J, Pyrgiotakis G, Engelward BP, Demokritou P: High-throughput screening platform for engineered nanoparticle-mediated genotoxicity using CometChip technology. ACS Nano 2014, 8: 2118–2133. 10.1021/nn404871pPubMed CentralView ArticlePubMedGoogle Scholar
  11. De Berardis B, Civitelli G, Condello M, Lista P, Pozzi R, Arancia G, Meschini S: Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells. Toxicol Appl Pharmacol 2010, 246: 116–127. 10.1016/j.taap.2010.04.012View ArticlePubMedGoogle Scholar
  12. Vandebriel RJ, De Jong WH: A review of mammalian toxicity of ZnO nanoparticles. Nanotechnol Sci Appl 2012, 5: 61–71. 10.2147/NSA.S23932PubMed CentralView ArticlePubMedGoogle Scholar
  13. Warheit DB, Sayes CM, Reed KL: Nanoscale and fine zinc oxide particles: can in vitro assays accurately forecast lung hazards following inhalation exposures? Environ Sci Technol 2009, 43: 7939–7945. 10.1021/es901453pView ArticlePubMedGoogle Scholar
  14. Moghimi SM, Davis SS: Innovations in avoiding particle clearance from blood by Kupffer cells: cause for reflection. Crit Rev Ther Drug Carrier Syst 1994, 11: 31–59.PubMedGoogle Scholar
  15. Sund J, Alenius H, Vippola M, Savolainen K, Puustinen A: Proteomic characterization of engineered nanomaterial-protein interactions in relation to surface reactivity. ACS Nano 2011, 5: 4300–4309. 10.1021/nn101492kView ArticlePubMedGoogle Scholar
  16. Xia T, Zhao Y, Sager T, George S, Pokhrel S, Li N, Schoenfeld D, Meng H, Lin S, Wang X, Wang M, Ji Z, Zink JI, Madler L, Castranova V, Nel AE: Decreased dissolution of ZnO by iron doping yields nanoparticles with reduced toxicity in the rodent lung and zebrafish embryos. ACS Nano 2011, 5: 1223–1235. 10.1021/nn1028482PubMed CentralView ArticlePubMedGoogle Scholar
  17. Sotiriou GA, Watson C, Murdaugh KM, Darrah TH, Pyrgiotakis G, Elder A, Brain JD, Demokritou P: Engineering safer-by-design, transparent, silica-coated ZnO nanorods with reduced DNA damage potential. Environ Sci Nano 2014, 1: 144–153. 10.1039/c3en00062aPubMed CentralView ArticlePubMedGoogle Scholar
  18. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Itty Ipe B, Bawendi MG, Frangioni JV: Renal clearance of quantum dots. Nat Biotechnol 2007, 25: 1165–1170. 10.1038/nbt1340PubMed CentralView ArticlePubMedGoogle Scholar
  19. Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA: Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A 2008, 105: 14265–14270. 10.1073/pnas.0805135105PubMed CentralView ArticlePubMedGoogle Scholar
  20. Moghimi SM, Hunter AC, Andresen TL: Factors controlling nanoparticle pharmacokinetics: an integrated analysis and perspective. Annu Rev Pharmacol Toxicol 2012, 52: 481–503. 10.1146/annurev-pharmtox-010611-134623View ArticlePubMedGoogle Scholar
  21. Lynch I, Salvati A, Dawson KA: Protein-nanoparticle interactions: what does the cell see? Nat Nanotechnol 2009, 4: 546–547. 10.1038/nnano.2009.248View ArticlePubMedGoogle Scholar
  22. Monopoli MP, Aberg C, Salvati A, Dawson KA: Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol 2012, 7: 779–786. 10.1038/nnano.2012.207View ArticlePubMedGoogle Scholar
  23. Alwi R, Telenkov S, Mandelis A, Leshuk T, Gu F, Oladepo S, Michaelian K: Silica-coated super paramagnetic iron oxide nanoparticles (SPION) as biocompatible contrast agent in biomedical photoacoustics. Biomed Opt Express 2012, 3: 2500–2509. 10.1364/BOE.3.002500PubMed CentralView ArticlePubMedGoogle Scholar
  24. Jana NR, Yu HH, Ali EM, Zheng Y, Ying JY: Controlled photostability of luminescent nanocrystalline ZnO solution for selective detection of aldehydes. Chem Commun (Camb) 2007, 1406–1408.Google Scholar
  25. Chen Y-S, Wolfgang F, Seungsoo K, Pieter K, Kimberly H, Stanislav E: Silica-coated gold nanorods as photoacoustic signal nano-amplifiers. Nano Lett 2011, 11: 348–354. 10.1021/nl1042006PubMed CentralView ArticlePubMedGoogle Scholar
  26. Baber O, Jang M, Barber D, Powers K: Amorphous silica coatings on magnetic nanoparticles enhance stability and reduce toxicity to in vitro BEAS-2B cells. Inhal Toxicol 2011, 23: 532–543. 10.3109/08958378.2011.592869View ArticlePubMedGoogle Scholar
  27. Moghimi SM, Hunter AC, Murray JC: Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol Rev 2001, 53: 283–318.PubMedGoogle Scholar
  28. Brunner TJ, Wick P, Manser P, Spohn P, Grass RN, Limbach LK, Bruinink A, Stark WJ: In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environ Sci Technol 2006, 40: 4374–4381. 10.1021/es052069iView ArticlePubMedGoogle Scholar
  29. Napierska D, Thomassen LC, Rabolli V, Lison D, Gonzalez L, Kirsch-Volders M, Martens JA, Hoet PH: Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small 2009, 5: 846–853. 10.1002/smll.200800461View ArticlePubMedGoogle Scholar
  30. Warheit DB, McHugh TA, Hartsky MA: Differential pulmonary responses in rats inhaling crystalline, colloidal or amorphous silica dusts. Scand J Work Environ Health 1995,21(Suppl 2):19–21.PubMedGoogle Scholar
  31. Demokritou P, Gass S, Pyrgiotakis G, Cohen JM, Goldsmith W, McKinney W, Frazer D, Ma J, Schwegler-Berry D, Brain J, Castranova V: An in vivo and in vitro toxicological characterisation of realistic nanoscale CeO(2) inhalation exposures. Nanotoxicology 2013, 7: 1338–1350. 10.3109/17435390.2012.739665PubMed CentralView ArticlePubMedGoogle Scholar
  32. Sotiriou GA, Sannomiya T, Teleki A, Krumeich F, Voros J, Pratsinis SE: Non-toxic dry-coated nanosilver for plasmonic biosensors. Adv Funct Mater 2010, 20: 4250–4257. 10.1002/adfm.201000985PubMed CentralView ArticlePubMedGoogle Scholar
  33. Sotiriou GA, Franco D, Poulikakos D, Ferrari A: Optically stable biocompatible flame-made SiO2-coated Y2O3:Tb3+ nanophosphors for cell imaging. ACS Nano 2012, 6: 3888–3897. 10.1021/nn205035pPubMed CentralView ArticlePubMedGoogle Scholar
  34. Teleki A, Heine MC, Krumeich F, Akhtar MK, Pratsinis SE: In situ coating of flame-made TiO2 particles with nanothin SiO2 films. Langmuir 2008, 24: 12553–12558. 10.1021/la801630zView ArticlePubMedGoogle Scholar
  35. Demokritou P, Buchel R, Molina RM, Deloid GM, Brain JD, Pratsinis SE: Development and characterization of a Versatile Engineered Nanomaterial Generation System (VENGES) suitable for toxicological studies. Inhal Toxicol 2010,22(Suppl 2):107–116. 10.3109/08958378.2010.499385PubMed CentralView ArticlePubMedGoogle Scholar
  36. Gass S, Cohen JM, Pyrgiotakis G, Sotiriou GA, Pratsinis SE, Demokritou P: A safer formulation concept for flame-generated engineered nanomaterials. ACS Sustain Chem Eng 2013, 1: 843–857.PubMed CentralPubMedGoogle Scholar
  37. Hembram K, Sivaprakasam D, Rao TN, Wegner K: Large-scale manufacture of ZnO nanorods by flame spray pyrolysis. J Nanopart Res 2013, 15: 1461–1464. 10.1007/s11051-013-1461-4View ArticleGoogle Scholar
  38. Beaucage G, Kammler HK, Pratsinis SE: Particle size distributions from small-angle scattering using global scattering functions. J Appl Crystallogr 2004, 37: 523–535. 10.1107/S0021889804008969View ArticleGoogle Scholar
  39. Height MJ, Madler L, Pratsinis SE: Nanorods of ZnO made by flame spray pyrolysis. Chem Mater 2006, 18: 572–578. 10.1021/cm052163yView ArticleGoogle Scholar
  40. Sotiriou GA, Schneider M, Pratsinis SE: Green, silica-coated monoclinic Y 2 O 3 :Tb 3+ nanophosphors: flame synthesis and characterization. J Phys Chem C 2012, 116: 4493–4499. 10.1021/jp211722zView ArticleGoogle Scholar
  41. Buesser B, Pratsinis SE: Design of gas-phase synthesis of core-shell particles by computational fluid - aerosol dynamics. AIChE J 2011, 57: 3132–3142. 10.1002/aic.12512PubMed CentralView ArticlePubMedGoogle Scholar
  42. Brouwer D: Exposure to manufactured nanoparticles in different workplaces. Toxicology 2010, 269: 120–127. 10.1016/j.tox.2009.11.017View ArticlePubMedGoogle Scholar
  43. Vorbau M, Hillemann L, Stintz M: Method for the characterization of the abrasion induced nanoparticle release into air from surface coatings. Aerosol Sci 2009, 40: 209–217. 10.1016/j.jaerosci.2008.10.006View ArticleGoogle Scholar
  44. Brain JD, Knudson DE, Sorokin SP, Davis MA: Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ Res 1976, 11: 13–33. 10.1016/0013-9351(76)90107-9View ArticlePubMedGoogle Scholar
  45. Osier M, Oberdorster G: Intratracheal inhalation vs intratracheal instillation: differences in particle effects. Fundam Appl Toxicol 1997, 40: 220–227. 10.1006/faat.1997.2390View ArticlePubMedGoogle Scholar
  46. Baisch BL, Corson NM, Wade-Mercer P, Gelein R, Kennell AJ, Oberdorster G, Elder A: Equivalent titanium dioxide nanoparticle deposition by intratracheal instillation and whole body inhalation: the effect of dose rate on acute respiratory tract inflammation. Part Fibre Toxicol 2014, 11: 5. 10.1186/1743-8977-11-5PubMed CentralView ArticlePubMedGoogle Scholar
  47. Du Z, Zhao D, Jing L, Cui G, Jin M, Li Y, Liu X, Liu Y, Du H, Guo C, Zhou X, Sun Z: Cardiovascular toxicity of different sizes amorphous silica nanoparticles in rats after intratracheal instillation. Cardiovasc Toxicol 2013, 13: 194–207. 10.1007/s12012-013-9198-yView ArticlePubMedGoogle Scholar
  48. Johnston CJ, Driscoll KE, Finkelstein JN, Baggs R, O’Reilly MA, Carter J, Gelein R, Oberdorster G: Pulmonary chemokine and mutagenic responses in rats after subchronic inhalation of amorphous and crystalline silica. Toxicol Sci 2000, 56: 405–413. 10.1093/toxsci/56.2.405View ArticlePubMedGoogle Scholar
  49. McCarthy J, Inkielewicz-Stepniak I, Corbalan JJ, Radomski MW: Mechanisms of toxicity of amorphous silica nanoparticles on human lung submucosal cells in vitro: protective effects of fisetin. Chem Res Toxicol 2012, 25: 2227–2235. 10.1021/tx3002884View ArticlePubMedGoogle Scholar
  50. Merget R, Bauer T, Kupper HU, Philippou S, Bauer HD, Breitstadt R, Bruening T: Health hazards due to the inhalation of amorphous silica. Arch Toxicol 2002, 75: 625–634. 10.1007/s002040100266View ArticlePubMedGoogle Scholar
  51. Warheit DB, Webb TR, Reed KL: Pulmonary toxicity screening studies in male rats with TiO2 particulates substantially encapsulated with pyrogenically deposited, amorphous silica. Part Fibre Toxicol 2006, 3: 3. 10.1186/1743-8977-3-3PubMed CentralView ArticlePubMedGoogle Scholar
  52. Braakhuis HM, Park MV, Gosens I, De Jong WH, Cassee FR: Physicochemical characteristics of nanomaterials that affect pulmonary inflammation. Part Fibre Toxicol 2014, 11: 18. 10.1186/1743-8977-11-18PubMed CentralView ArticlePubMedGoogle Scholar
  53. Choi HS, Ashitate Y, Lee JH, Kim SH, Matsui A, Insin N, Bawendi MG, Semmler-Behnke M, Frangioni JV, Tsuda A: Rapid translocation of nanoparticles from the lung airspaces to the body. Nat Biotechnol 2010, 28: 1300–1303. 10.1038/nbt.1696PubMed CentralView ArticlePubMedGoogle Scholar
  54. Gessner A, Lieske A, Paulke B, Muller R: Influence of surface charge density on protein adsorption on polymeric nanoparticles: analysis by two-dimensional electrophoresis. Eur J Pharm Biopharm 2002, 54: 165–170. 10.1016/S0939-6411(02)00081-4View ArticlePubMedGoogle Scholar
  55. Lartigue L, Wilhelm C, Servais J, Factor C, Dencausse A, Bacri JC, Luciani N, Gazeau F: Nanomagnetic sensing of blood plasma protein interactions with iron oxide nanoparticles: impact on macrophage uptake. ACS Nano 2012, 6: 2665–2678. 10.1021/nn300060uView ArticlePubMedGoogle Scholar
  56. Buzea C, Pacheco II, Robbie K: Nanomaterials and nanoparticles: sources and toxicity. Biointerphases 2007, 2: MR17–71. 10.1116/1.2815690View ArticlePubMedGoogle Scholar
  57. Xia T, Kovochich M, Liong M, Madler L, Gilbert B, Shi H, Yeh JI, Zink JI, Nel AE: Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2008, 2: 2121–2134. 10.1021/nn800511kPubMed CentralView ArticlePubMedGoogle Scholar
  58. He X, Zhang H, Ma Y, Bai W, Zhang Z, Lu K, Ding Y, Zhao Y, Chai Z: Lung deposition and extrapulmonary translocation of nano-ceria after intratracheal instillation. Nanotechnology 2010, 21: 285103. 10.1088/0957-4484/21/28/285103View ArticlePubMedGoogle Scholar
  59. Cullen RT, Tran CL, Buchanan D, Davis JM, Searl A, Jones AD, Donaldson K: Inhalation of poorly soluble particles. I. Differences in inflammatory response and clearance during exposure. Inhal Toxicol 2000, 12: 1089–1111. 10.1080/08958370050166787View ArticlePubMedGoogle Scholar
  60. Adamcakova-Dodd A, Stebounova LV, Kim JS, Vorrink SU, Ault AP, O’Shaughnessy PT, Grassian VH, Thorne PS: Toxicity assessment of zinc oxide nanoparticles using sub-acute and sub-chronic murine inhalation models. Part Fibre Toxicol 2014, 11: 15. 10.1186/1743-8977-11-15PubMed CentralView ArticlePubMedGoogle Scholar
  61. Mineral Tolerance of Animals. The National Academic Press, Washington. D.C; 2005.Google Scholar
  62. Environmental Health Criteria 221 Zinc. Worl Health Organization, Geneva; 2001.Google Scholar
  63. Kreyling WG, Semmler-Behnke M, Seitz J, Scymczak W, Wenk A, Mayer P, Takenaka S, Oberdörster G: Size and material dependency of translocation of inhaled iridium or carbon nanoparticles from the lungs of rats to blood. Inhal Toxicol 2009, 21: 55–60. 10.1080/08958370902942517View ArticlePubMedGoogle Scholar
  64. Cho WS, Duffin R, Howie SE, Scotton CJ, Wallace WA, Macnee W, Bradley M, Megson IL, Donaldson K: Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes. Part Fibre Toxicol 2011, 8: 27. 10.1186/1743-8977-8-27PubMed CentralView ArticlePubMedGoogle Scholar
  65. Berry JP, Arnoux B, Stanislas G, Galle P, Chretien J: A microanalytic study of particles transport across the alveoli: role of blood platelets. Biomedicine 1977, 27: 354–357.PubMedGoogle Scholar
  66. Cohen JM, Derk R, Wang L, Godleski J, Kobzik L, Brain J, Demokritou P: Tracking translocation of industrially relevant engineered nanomaterials (ENMs) across alveolar epithelial monolayers in vitro. Nanotoxicology 2014, 8: 216–225. 10.3109/17435390.2013.879612PubMed CentralView ArticlePubMedGoogle Scholar
  67. Geiser M, Kreyling WG: Deposition and biokinetics of inhaled nanoparticles. Part Fibre Toxicol 2010, 7: 2. 10.1186/1743-8977-7-2PubMed CentralView ArticlePubMedGoogle Scholar
  68. Geiser M, Rothen-Rutishauser B, Kapp N, Schurch S, Kreyling W, Schulz H, Semmler M, Im Hof V, Heyder J, Gehr P: Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ Health Perspect 2005, 113: 1555–1560. 10.1289/ehp.8006PubMed CentralView ArticlePubMedGoogle Scholar
  69. Oberdorster G, Sharp Z, Atudorei V, Elder A, Gelein R, Lunts A, Kreyling W, Cox C: Extrapulmonary translocation of ultrafine carbon particles following whole-body inhalation exposure of rats. J Toxicol Environ Health A 2002, 65: 1531–1543. 10.1080/00984100290071658View ArticlePubMedGoogle Scholar
  70. Semmler M, Seitz J, Erbe F, Mayer P, Heyder J, Oberdorster G, Kreyling WG: Long-term clearance kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient translocation into secondary organs. Inhal Toxicol 2004, 16: 453–459. 10.1080/08958370490439650View ArticlePubMedGoogle Scholar
  71. Takenaka S, Karg E, Roth C, Schulz H, Ziesenis A, Heinzmann U, Schramel P, Heyder J: Pulmonary and systemic distribution of inhaled ultrafine silver particles in rats. Environ Health Perspect 2001,109(Suppl 4):547–551. 10.1289/ehp.01109s4547PubMed CentralView ArticlePubMedGoogle Scholar
  72. Burch RE, Hahn HK, Sullivan JF: Newer aspects of the roles of zinc, manganese, and copper in human nutrition. Clin Chem 1975, 21: 501–520.PubMedGoogle Scholar
  73. Seo J-H, Cho Y-E, Kim T, Shin H-I, Kwun I-S: Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic. Nutr Res Pract 2010, 4: 356–361. 10.4162/nrp.2010.4.5.356PubMed CentralView ArticlePubMedGoogle Scholar
  74. Hashizume M, Yamaguchi M: Stimulatory effect of beta-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells. Mol Cell Biochem 1993, 122: 59–64. 10.1007/BF00925737View ArticlePubMedGoogle Scholar
  75. Yamaguchi M, Igarashi A, Uchiyama S: Bioavailability of zinc yeast in rats: stimulatory effect of bone calcification in vivo. J Health Sci 2004,50(1):75–81. 10.1248/jhs.50.75View ArticleGoogle Scholar
  76. Hilty FM, Arnold M, Hilbe M, Teleki A, Knijnenburg JTN, Ehrensperger F, Hurrell RF, Pratsinis SE, Langhans W, Zimmermann MB: Iron from nanocompounds containing iron and zinc is highly bioavailable in rats without tissue accumulation. Nat Nanotechnol 2010, 5: 374–380. 10.1038/nnano.2010.79View ArticlePubMedGoogle Scholar
  77. Espita PJP, Soares NFF, Coimbra JSR, de Andrade NJ, Medeiros EAA: Zinc oxide nanoparticles: synthesis, antimicrobial activity and food packaging applications. Food Bioprocess Technol 2012, 5: 1447–1464. 10.1007/s11947-012-0797-6View ArticleGoogle Scholar
  78. Lai SK, Wang YY, Hanes J: Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv Drug Deliv Rev 2009, 61: 158–171. 10.1016/j.addr.2008.11.002PubMed CentralView ArticlePubMedGoogle Scholar
  79. Paek HJ, Lee YJ, Chung HE, Yoo NH, Lee JA, Kim MK, Lee JK, Jeong J, Choi SJ: Modulation of the pharmacokinetics of zinc oxide nanoparticles and their fates in vivo. Nanoscale 2013, 5: 11416–11427. 10.1039/c3nr02140hView ArticlePubMedGoogle Scholar
  80. Chung HE, Yu J, Baek M, Lee JA, Kim MS, Kim SH, Maeng EH, Lee JK, Jeong J, Choi SJ: Toxicokinetics of zinc oxide nanoparticles in rats. J Phys: Conf Ser 2013, 429: 012037.Google Scholar
  81. Baek M, Chung HE, Yu J, Lee JA, Kim TH, Oh JM, Lee WJ, Paek SM, Lee JK, Jeong J, Choy JH, Choi SJ: Pharmacokinetics, tissue distribution, and excretion of zinc oxide nanoparticles. Int J Nanomedicine 2012, 7: 3081–3097.PubMed CentralPubMedGoogle Scholar
  82. Cohen J, Deloid G, Pyrgiotakis G, Demokritou P: Interactions of engineered nanomaterials in physiological media and implications for in vitro dosimetry. Nanotoxicology 2013, 7: 417–431. 10.3109/17435390.2012.666576PubMed CentralView ArticlePubMedGoogle Scholar
  83. Brown RP, Delp MD, Lindstedt SL, Rhomberg LR, Beliles RP: Physiological parameter values for physiologically based pharmacokinetic models. Toxicol Ind Health 1997, 13: 407–484. 10.1177/074823379701300401View ArticlePubMedGoogle Scholar
  84. Schoeffner DJ, Warren DA, Muralidara S, Bruckner JV, Simmons JE: Organ weights and fat volume in rats as a function of strain and age. J Toxicol Environ Health A 1999, 56: 449–462. 10.1080/009841099157917View ArticlePubMedGoogle Scholar
  85. Jaki T, Wolfsegger MJ: Estimation of pharmacokinetic parameters with the R package PK. Pharm Stat 2011, 10: 288–294. 10.1002/pst.449View ArticleGoogle Scholar

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