Pulmonary toxicity and translocation of nanodiamonds in mice

Abstract

Because of the possible health threat of nanodiamonds (NDs) to organisms, the pulmonary toxicity and translocation of NDs in different sizes in mice were investigated after intratracheal instillation administration. Biochemical assays, ultrastructural and histopathological evaluations of the lungs of the control and the ND exposed mice were carried out at 1, 7, 14 or 28 days post-exposure. Exposure to 1.0 mg/kg NDs with an average diameter of 4 nm produced a temporary lung index increase at 1 day post-exposure. NDs did not have evident adverse effects in the lungs within the studied period according to histopathological and ultrastructural investigations. Furthermore, no lipid peroxidation of the lung was observed. On the whole, intratracheally instilled NDs are of low pulmonary toxicity. In addition, the amount of NDs in alveolar decreased with time elapsed and the macrophages burdened with NDs were clearly observed in the bronchia from 1 day to 28 days post-exposure. Thus we affirm the critical role of alveolar macrophages in the main excretion pathway of NDs from the lungs, i.e. they engulf the NDs, migrate upward to the trachea by escalator/mucociliary system and finally enter the pharynx.

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Pulmonary toxicity and translocation of nanodiamonds in mice
Yuan Yuana, Xiang Wangb, Guang Jiab, Jia-Hui Liua, Tiancheng Wangc, Yiqun Gud, Sheng-Tao Yanga,
Sen Zhenb, Haifang Wanga,e,⁎, Yuanfang Liua,e
aBeijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
bDepartment of Occupational and Environmental Health Sciences, School of Public Health, Peking University, Beijing 100083, China
cDepartment of Clinical Laboratory, Third Hospital of Peking University, Beijing 100083, China
dMaternity Hospital of Haidian District, Beijing 100080, China
eInstitute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444, China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 30 April 2009
Received in revised form 12 November 2009
Accepted 30 November 2009
Available online 5 December 2009
Keywords:
Nanodiamond
Pulmonary toxicity
Intratracheal instillation
Translocation
Because of the possible health threat of nanodiamonds (NDs) to organisms, the pulmonary toxicity and
translocation of NDs in different sizes in mice were investigated after intratracheal instillation
administration. Biochemical assays, ultrastructural and histopathological evaluations of the lungs of the
control and the ND exposed mice were carried out at 1, 7, 14 or 28 days post-exposure. Exposure to 1.0 mg/kg
NDs with an average diameter of 4 nm produced a temporary lung index increase at 1 day post-exposure. NDs
did not have evident adverse effects in the lungs within the studied period according to histopathological and
ultrastructural investigations. Furthermore, no lipid peroxidation of the lung was observed. On the whole,
intratracheally instilled NDs are of low pulmonary toxicity. In addition, the amount of NDs in alveolar
decreased with time elapsed and the macrophages burdened with NDs were clearly observed in the bronchia
from 1 day to 28 days post-exposure. Thus we affirm the critical role of alveolar macrophages in the main
excretion pathway of NDs from the lungs, i.e. they engulf the NDs, migrate upward to the trachea by escalator/
mucociliary system and finally enter the pharynx.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Nowadays, nanomaterials have been used in various fields due to
their attractive properties. However, as the production and use of
them are expanding rapidly, the intensive investigation on the
potential impact of these nanomaterials on the environment and
human health is imperative [1]. Among the various nanomaterials,
nanodiamond (ND), an allotropeof carbondiscoveredseveraldecades
ago, regains much attention because of its extreme chemical
inertness, optical transparency, exceptional hardness and good
biocompatibility. Currently it is widely used in diverse areas, such as
electrochemical coatings, polymer compositions, antifriction coatings,
polishing, lubricants, biosensors, imaging probes, implants and drug
carriers [2–9]. Hundreds of tons of NDs are produced worldwide
annually.
ND is a type of ashy material with very low density thus it diffuses
in air easily during its producing and processing procedures. The
airborne ND undoubtedly increases the risk to human health, because
respiratorysystemisoneof theinevitableexposureroutesthatpeople
may encounter nanoparticles [10]. And epidemiological and experi-
mental studies have shown a positive correlation between increases
in atmospheric particulate concentrations and short-term increases in
morbidity and mortality [11]. Therefore, a thorough understanding of
the basic biological consequences of the inhaled NDs is vital.
Several studies have been performed at the cellular level to get the
basic information of the toxicity of NDs [12–19]. It has been found that
NDs were non-toxic to various cells [12,14,16] and more biocompatible
than other carbon nanomaterials, such as carbon black, multi-walled
carbon nanotubes or single-walled carbon nanotubes (SWNTs) [17].
As for the toxicity of NDs in vivo, the knowledge is very limited at
present. Bakowicz and Mitura reported that there were no immune
responses in rats at 10 days after intraperitoneal injection of NDs [20].
No inflammatory symptoms in mice were observed after three
months of subcutaneous exposure to NDs, but several blood
biochemical indices of rabbit were affected after intravenous
administration of NDs [21]. Very recently, we reported the distribu-
tion of NDs in mice and no adverse effects were observed during the
whole experiment period [22]. As far as we know, the toxicity and
translocation of NDs in the lungs have not been touched yet.
The aims of this work are to investigate the pulmonary toxicity of
NDs and explore the translocation of NDs in the lungs. NDs with an
average diameter of 4 nm (NDs-4) and 50 nm (NDs-50) were
employed to test the pulmonary responses in ICR mice via intra-
tracheally instilled (i.t.) exposure.
Diamond & Related Materials 19 (2010) 291–299
⁎ Corresponding author. Beijing National Laboratory for Molecular Sciences,
Department of Chemical Biology, College of Chemistry and Molecular Engineering,
Peking University, Beijing 100871, China. Tel./fax: +86 21 66135276x103.
E-mail address: hwang@shu.edu.cn (H. Wang).
0925-9635/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.diamond.2009.11.022
Contents lists available at ScienceDirect
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journal homepage: www.elsevier.com/locate/diamond

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2. Methods and materials
2.1. Materials
NDs-4 (Jin Gang Yuan New Material Development Co., Shenzhen,
China) were synthesized by detonation. NDs-50 (Advapowder Co.,
Guangzhou, China) were produced by the destruction of larger
artificial diamond crystals, which were synthesized by a high-
temperature–high-pressure (HTHP) method. ND samples were used
without further purification. All chemicals were of analytical grade or
better.
2.2. ND characterization
The morphological characterization of NDs was performed with
high-resolution transmission electron microscopy (HRTEM, JEM-
2010, JEOL, Japan, 120 keV). Other chemical and physical properties
of the NDs were characterized by a variety of methods, including
elemental analysis (Elementar Vario MICRO CUBE, Germany), X-ray
fluorescence spectrometry (XRF, S4-Explorer, Bruker), as well as
Brunauer–Emmett–Teller surface area measurement (BET, ASAP2010,
Micromeritics, USA).
2.3. Animals
All animal experiments were carried out in compliance with the
regulations of the local ethics committee.
Healthy adult male ICR mice (approximately 8 weeks old, 25 g)
were purchased from Animal Center of Peking University, Beijing,
China. The mice were housed in polycarbonate cages with wooden
chip bedding and received food and water ad libitum. They were
treated and used humanely according to the Animal Care and Use
Program guidelines of Peking University. After acclimation, the mice
were randomized into groups (6 mice per group).
2.4. Intratracheal instillation
After being anesthetized with pentobarbital sodium solution
(80 mg/kg, intraperitoneal injection), each mouse was secured on
an inclined ligneous platform. Before instillation, all the ND–PBS
suspensions were sonicated for 10–20 s for better dispersion. The
trachea was exposed by a 0.5 cm incision on the ventral neck skin, and
a small hole was made in the trachea close to the larynx. Then a
blunted needle was inserted inside the trachea and the prefilled 50 µl
of 50 or 500 µg/ml NDs in PBS and PBS was rapidly propelled into the
lungs. The neck incision was then sutured and swabbed with iodine.
Twenty minutes later, the mice recovered and were active. The
incision healed within one day, and the mice were observed daily
until their scheduled termination. At 1, 7, 14 and 28 days post-
exposure, the blood samples and tissues (lungs, livers and spleens)
were collected from mice for the following biochemical assay,
histopathological and ultrastructural investigations.
2.5. Biochemical assay
Serum was obtained from blood sample after centrifugation at
4000 rpm for 10 min. All biochemical assays were performed using a
Hitachi 7170A clinical automatic chemistry analyzer. Lactate dehy-
drogenase (LDH) and alkaline phosphatase (ALP) were measured
using the commercial kits (Bühlmann Laboratories, Switzerland).
2.6. Histopathological and ultrastructural analysis
Tissue sections dissected from the lungs, liver and spleen were
fixed with formalin, embedded in paraffin, and thin-sectioned. The
thin-sections were mounted on glass microscope slides using
standard histopathological techniques, stained with hematoxylin–
eosin and then examined by light microscopy.
Representative portions of the lungs were prefixed with 2%
glutaraldehyde at 4 °C overnight. After being washed, they were
postfixed with 1% osmium tetroxide at 4 °C for 3 h and washed with
0.1 M cacodylate buffer. The lung samples were dehydrated, embed-
ded in resin, sectioned to 70 nm thick, stained with uranyl acetate and
lead citrate, and then examined with HRTEM.
2.7. HRTEM investigation of NDs in digested organs
About 1 g lung or liver tissue was digested in a 4 ml mixture of 65%
HClO4and 30% H2O2(v:v=1:1) and heated. Five milliliters of nitric
acid was added after the tissue was dissolved. The solution was boiled
for 20 min and then 30% H2O2was added dropwise to make the
solution transparent. After neutralization by sodium bicarbonate, the
digested solution was dialyzed against deionized water to remove
salts. Then, the dialyzed solution was concentrated for HRTEM
observation.
2.8. Determination of reduced glutathione (GSH) and lipid peroxidation
Lung homogenates were prepared from 0.08 to 0.12 g lung
samples. The tissues were washed three times by cold physiological
saline (0.9% NaCl), and homogenized in 4 °C water for 1 min. Then the
homogenates were centrifuged at 2000 rpm for 10 min to obtain the
supernatants. The protein concentration of lung extracts was
determined according to the method of Bradford, using bovine
serum albumin (BSA) as a standard [23].
The level of GSH in lung tissue supernatant was examined using
spectrophotometric diagnostic kits (Nanjing Jiancheng Biotechnology
Institute, China) based on the method of Jollow et al. [24]. The level of
their thiol groups was measured at 412 nm based on the reduction of
5,5′-dithiobis-2-nitrobenzoic acid (DTNB) to 2-nitro-5-thiobenzoate
anion (NTP).
The level of lipid peroxidation in terms of malondialdehyde (MDA)
was determined using the method of thiobarbituric acid reactive
species(TBARS) [25].Results of MDAwereexpressed asnanomoleper
milligram protein (nmol/mg pr.), using 1,1,3,3-tetraethoxypropane
(TEP) as a standard.
2.9. Statistical analysis
All data are presented as the mean of four individual observations
with the standard deviation. Significance has been calculated using
Student's t-test. Difference was considered significant if pb0.05.
3. Results
3.1. Characterization of NDs
The size of NDs-4 distributes in the range of 2–8 nm with an
average diameter of 4 nm (Fig. 1A), while the size of NDs-50 ranges
between 30 and 80 nm with an average diameter of 50 nm (Fig. 1B).
The interlayer spacing of both kinds of NDs is 0.202 nm (Fig. S1 in
Supplementary material (SM)) that is consistent with that of the
diamond (111) planes, indicating the characteristic diamond struc-
ture of NDs.
Table 1 shows the composition of the two kinds of NDs. The carbon
contents of NDs-4 and NDs-50 are 89.65% and 93.85% (w/w),
respectively. The most abundant impurity in NDs-4 is chlorine,
which was introduced during the manufacture process. Iron and
tungsten are the main impurities in NDs-50.
The BET surface area of NDs-4 is 264.1 m2/g, whereas that of NDs-
50 is 70.4 m2/g.
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3.2. Clinical symptoms and body weights
There was no difference of body weight gain between all exposed
groups and the PBS group during the post-exposure period (pN0.05)
(data not shown). Besides, the mice of all groups did not show any
clinical symptoms and signs during the post-exposure period.
3.3. Organ indices
The organ indices were recorded to provide a general impression
of the toxicity (Table 2). The lung indices are the ratio of the wet
weight of the lung to the whole body weight (g/g). The decrease of the
organ indices means organ shrinking or function weakening, while
the increase usually means that the organ is congested, swelled,
proliferated or function is strengthened. Only the lung indices of mice
exposed to 1.0 mg/kg NDs-4 at 1 day post-exposure increase
compared with that of the PBS groups (pb0.05). Seven days later,
these indices decrease and go back to the PBS control level. Compared
with PBS controls, no significant increases were observed in the lung
indices of other exposed groups at each measured time point.
3.4. Effects of NDs on serum biochemistry of mice
No considerable enhancement in ALP values of the ND exposed
mice relative to that of PBS controls is observed during the post-
exposure period (pN0.05, Fig. 2A).
One day after the mice were treated with NDs, both kinds of NDs
induce a slight increase in serum LDH values compared with the PBS
groups (pN0.05), but the LDH values of all treated groups (including
PBS group)descendgraduallyas timeelapses (Fig. 2B). The temporary
increase in LDH might come from the surgery damage of intratracheal
instillation. As the mice recovered from the surgery, the LDH level
decreased accordingly. There is no significant difference between the
values of the ND and PBS treated mice during the time period from
1 day to 14 days (pN0.05). However, at 28 days post instillation, the
LDH values of all the ND treated groups, except the 1 mg/kg ND-50
Fig. 1. Representative TEM images of NDs-4 (A) and NDs-50 (B).
Table 1
The composition of NDs-4 and NDs-50 examined by elemental analysis (EA) and X-ray
fluorescence spectrometry (XRF). The impurities with content less than 0.1% were not
shown.
SampleContent (% by weight)
Examined by EA Examined by XRF
CNHS Cl FeW
NDs-4
NDs-50
89.65
93.85
2.07
–
1.90
–
0.695
–
4.43
0.253
0.413
3.83
–
1.25
Table 2
Lung indices of mice after exposure to NDs and PBS at different time points (mean±SD,
n=4).
Dose
group
(mg/kg)
Time points
1 day 7 days 14 days28 days
PBS
NDs-50 0.1
0.0059±0.0002
0.0061±0.0005
0.0060±0.0004
0.0063±0.0005
0.0073±0.0004⁎0.0062±0.0005 0.0059±0.0003 0.0060±0.0002
0.0060±0.0006 0.0058±0.0005 0.0057±0.0007
0.0061±0.0004 0.0057±0.0002 0.0056±0.0004
0.0058±0.0003 0.0057±0.0004 0.0056±0.0004
0.0064±0.0009 0.0063±0.0005 0.0056±0.0002
1.0
0.1
1.0
NDs-4
⁎Indicates pb0.05 versus the PBS group.
Fig. 2. Bioactivities of ALP (A) and LDH (B) in the serum of mice exposed to different
doses of NDs at each post-exposure time point. PBS was used as control. Data are
presented as the mean±SD (n=4). **indicates pb0.05 versus the PBS group.
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treated group, are significantly greater than that of the corresponding
PBS controls (pb0.05).
3.5. Histopathological and ultrastructural analysis
The histopathological observations were carried out to determine
the organic damage induced by NDs-4 and NDs-50 at the high dose of
1 mg/kg and find the possible elimination of NDs from the lungs.
Representative light micrographs of lung sections of mice instilled
with 1.0 mg/kg NDs-4, NDs-50 as well as PBS at 1 day post-exposure
are presented in Fig. 3. No significant inflammatory response in the
lungs of the ND treated groups is observed compared with the PBS
controls. Pathologic changes such as alveolar wall thickening and
neutrophils and alveolar macrophage accumulation are negligible.
The lungs from the control groups which were instilled with PBS
are very clean and we do not find any particles with the microscope
(Fig. 3A and B). On the contrary, there are many spots which have
totallydifferentcolorscomparedwiththestainedtissueinthealveolar
and bronchia under the microscope (magnification=400×, pictures
not shown). Taking higher magnification (1000×, Fig. 3D and F), we
candiscriminatethespotsfromthetissuethroughtheirspecialshining
color,uniformsmallsize,anddistinctlocationinthecytoplasmaround
the nuclei. The structural determination of the particles was carried
out with TEM (Fig. 7). TEM images of these particles show the faceted
structure anduniquelatticefringes ofNDs.Soweconcludethesespots
should be the administrated NDs.
At 1 day post-exposure, most of the NDs are engulfed by
macrophages in the respiratory bronchiole, alveolar macrophages
(AMs) and alveolar epithelia while no particles are observed in the
controls (Figs. 3, S2 and S3 in SM). Macrophages are usually round or
oval shaped. When their phagocytic ability is activated, the macro-
phages become larger and multi-sudden shaped laden by studied
particles with more phagosomes or enlarged organelles including
mitochondria, lysosomes, smooth and rough endoplasmic reticular.
Fig. 3. Histopathological pictures of the lungs from mice intratracheally instilled with PBS (A and B), 1.0 mg/kg NDs-4 (C and D) and 1 mg/kg NDs-50 (E and F) at 1 day post-
exposure. The normal lung architecture in graphs A, C and E (magnification =200×) indicates that exposures to NDs do not produce detectable pulmonary toxicity. B, D and F
(magnification =1000×) are the magnifications of the defined areas in A, C and E, respectively. The black arrows in D and F point to the entrapped ND aggregates, which have
shining black color and uniform small size.
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Alveolar macrophage is a type of macrophage found in the pulmonary
alveolus, bigger than neutrophil, near the pneumocytes, but separated
from the wall. Besides, some NDs-4 and NDs-50 are observed in the
interstitial sites (Fig. S3 in SM). Notably, at 28 days post-exposure,
some macrophages engulfing NDs-4 or NDs-50 merged with mucus
are observed in the respiratory bronchiole and alveolar area (Figs. 4
Fig. 4. Histopathological pictures of the lungs from mice intratracheally instilled with 1.0 mg/kg NDs-4 at 28 days post-exposure. B, D and F (magnification =400×) are
magnifications of the defined areas in A (magnification =40×). C, E and G (magnification =1000×) are the magnifications of the defined areas in B, D and F, respectively. The black
arrows in C, E and G point to the ND loaded macrophages in the bronchia.
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and 5). Liver and spleen tissues of mice exposed to both kinds of NDs
did not present any abnormity and no NDs were found in the liver and
spleen in all exposed mice (data not shown).
In order to reveal the structure of NDs and their location in cells,
ultrastructuralobservationoflungtissueswasperformed.ManyNDs-50
are clearly seen in the cytoplasm (Fig. 6). Facetted structure of NDs-50
Fig. 5. Histopathological pictures of the lungs from mice intratracheally instilled with 1.0 mg/kg NDs-50 at 28 days post-exposure. B, D and F (magnification =400×) are the
magnifications of the defined areas in A (magnification =40×). C, E and G (magnification =1000×) are the magnifications of the defined areas in B, D and F, respectively. The black
arrows in C, E and G point to the ND loaded macrophages in the bronchia.
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remains intact in phagosomes, indicating the non-biodegradable
property of ND during the transportation process.
3.6. HRTEM images of NDs in digested organs
It is difficult to clearly identify ND particle, especially NDs-4, in
tissue sections owing to the very small particle size and weak contrast
in the thick tissue sections. To further confirm the existence of NDs in
the lungs, we chemically digested the tissues into solution to visualize
NDs clearly with HRTEM. NDs are stable against the oxidation
reaction. Fig. 7 shows the high-resolution images of NDs-4 and NDs-
50 in the digested solution of the lung at 28 days post-exposure to
1.0 mg/kg NDs. The lattice fringes for the two particles in the images
correspond to the (111) planes of the diamond, by which the
existence of NDs is evidenced. There were no detectable NDs in the
liver and spleen even at a much higher dose of 20 mg/kg (data not
shown).
3.7. Lung tissue GSH and MDA levels
The oxidative stress aroused by NDs in the main organ, the lung,
was measured to reveal the possible toxicological mechanism. As
shown in Table 3, the levels of reduced GSH in the lungs of all exposed
groupsdonotchangestatistically.SimilartoGSH,theMDAlevels ofall
the ND treated groups change slightly, but show no significant
difference from that of the PBS controls (Table 4).
4. Discussion
4.1. Pulmonary toxicity of NDs
Intratracheal instillation, as a convenient, effective and practical
way, is widely adopted in the pulmonary toxicological study. The
present study shows that the two ND products, regardless of their
sources and sizes, do not induce any obvious pulmonary toxicity.
Lung index is widely used to evaluate pulmonary toxicity. One day
after the mice were exposed to NDs-4, their lung indices significantly
increased. However, as the time was prolonged to 7 days, the lung
indices returned to the normal levels, suggesting gradual recovery of
the lung cells. Such index changes may be attributed to the operation
of intratracheal instillation.
Fig. 6. TEMimagesofthelungsectionfrommouseintratracheallyinstilledwith1.0 mg/kg
NDs-50 and sacrificed at 1 day post-exposure. The black arrows point to the NDs-50
entrapped in phagosomes. B is the magnification of the defined areas in A.
Fig. 7. HRTEM images of NDs-4 and NDs-50 in the digested solution of the lungs from
mice intratracheally instilled with 1.0 mg/kg NDs-4 (A) and 1.0 mg/kg NDs-50 (B) at
28 days post-exposure.
Table 3
Lung GSH levels of mice after exposure to NDs and PBS at different time points (mean±
SD, n=4).
Dose group
(mg/kg)
Lung GSH level (mg/g pr., ±SD)
1 days 7 days14 days28 days
PBS
NDs-50
52.92±4.57
51.59±14.56
55.21±8.92
52.39±3.72
57.22±7.02
55.28±4.06
54.32±2.41
53.95±5.20
64.12±10.92
62.55±5.31
54.63±12.56
51.03±6.74
47.93±3.61
53.54±1.74
48.89±3.88
48.16±7.69
47.67±2.23
61.09±11.24
47.70±4.29
44.25±1.48
0.1
1.0
0.1
1.0
NDs-4
Table 4
Lung MDA levels of mice after exposure to NDs and PBS at different time points (mean±
SD, n=4).
Dose group
(mg/kg)
Lung MDA level (nmol/mg pr., ±SD)
1 day7 days 14 days28 days
PBS
NDs-50
2.937±0.716
2.797±0.386
3.498±0.357
2.865±0.667
3.770±0.872
2.735±0.579
3.171±0.279
2.737±0.565
2.533±0.457
2.675±0.626
3.186±0.400
2.946±0.664
2.434±0.305
2.604±0.120
2.801±0.391
3.849±0.538
2.415±0.460
2.719±0.618
2.692±0.608
2.488±0.423
0.1
1.0
0.1
1.0
NDs-4
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Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for
removing phosphate groups from many types of molecules, including
nucleotides, proteins and alkaloids. For pulmonary toxicity evalua-
tion, ALP activity is a measurement of Type II alveolar epithelial cell
secretory activity, and the increased ALP activity in serum is
considered to be an indicator of the toxicity to Type II cell. LDH is a
cytoplasmic enzyme that catalyzes the conversion of lactate to
pyruvate. It is ubiquitous and enriched in the liver, lung, kidneys
and so on. The activity of LDH in these tissues is much higher than that
in the serum. Once damage or necrosis happens in tissues, LDH
releases and makes the level of LDH in serum increase. In the
pulmonarytoxicity study, the levelof LDH is often used as an indicator
of the injury of alveolar microphage (AM). In this work, high level of
LDH activity manifests the serious pulmonary injury aroused by NDs
since the other tissues were not affected by the NDs. Our biochemical
results reveal that within the dose range taken, NDs do not injure the
Type II cell but produce a slight impairment to some AMs during the
experiment period. However, these adverse effects on the lung do not
induce any clinical signs in mice.
Oxidative stress is taken as an important pathway of toxicity of
nanomaterials [26]. GSH and MDA levels were chosen to reflect the
oxidative stress of the lung in this study. When oxidative stress is
aroused by the tested materials, reactive oxygen species increase and
then trigger lipid oxidation. As a result, MDA is formed. GSH, an
antioxidant, helps protect cells from reactive oxygen species such as
free radicals and peroxides. Therefore, the increase of MDA level and
the decrease of GSH level in the lung usually indicate oxidative
damage to the lung. The reduced GSH level and MDA level in the lung
remain unchanged in all groups in this study. Namely, there is no
observed oxidative damage to the lung.
The histopathological analyses of lung tissues reveal that pulmo-
nary exposure to NDs does not produce any observed adverse effects
in mice compared with the PBS controls. It is evidenced by the normal
lung architecture in the exposed animals during 1 day to 3 months
post-exposure (see Figs. 3 and S4 in SM).
Many toxicology studies of carbon based nanomaterials in the
lungs have been published in recent years. Pulmonary exposure to
SWNTs in rats and mice produced inflammation and epithelioid
granulomas [27–30]. Slight lung toxicity effects were observed in rats
intratracheally instilled with fullerene (C60) and fullerenol (C60
(OH)24) [31]. Besides, intratracheal instillation of carbon black
nanoparticles (14 nm and 56 nm) could exacerbate lung inflamma-
tion related to bacterial endotoxin [32]. Compared with the results of
these carbon materials, NDs possess very low pulmonary toxicity.
Fullerene and CNTs mainly consist of carbon with sp2hybrid orbital,
whereas NDs are constructed by the very stable sp3hybrid carbon.
NDs were shown to be chemically inert when entered into human
being and animals. Undoubtedly, the size and shape of carbon
nanomaterials also play very important roles in their toxicity. Besides,
pulmonary toxicity is closely related to the accumulation of particles
in the lung. CNTs with the length of several hundred nanometers to
microns are too long and stiff to be taken up by one macrophage and
eliminated normally from the lung [33]. This might be part of the
reason that CNTs induce granulomas in rodents' lungs whereas other
carbon nanoparticles do not. On the other hand, fullerene, which is
smaller than NDs, could move from the lung into blood circulation,
causing potential toxic effects [34]. However, in this work, we did not
find the translocation of NDs from the lung into the blood.
4.2. Translocation of NDs in the lung
In our experiment, most of the NDs entering the bronchiole and
alveolar area were engulfed by macrophages and epithelia at 1 day
post-exposure. It is easy to find ND loaded cells in the bronchia and
alveoli during the period of 1 day to 28 days post-exposure (Figs. 4
and 5, and S2 and S3 in SM). However, it becomes much more difficult
to discern NDs at 90 days post-exposure (Fig. S4 in SM). We therefore
inferthat NDsleave thelungs after theirprettylongtime deposition in
the lungs.
The elimination of particles from the lung after inhalation or
intratracheal instillation has received much concern [10,35,36].
Several different pathways were proposed. The most prevalent and
classic approach for the clearance of the invaded insoluble particles
from the alveoli is mediated by macrophages, through phagocytosis of
deposited particles [37]. This is followed by gradual movement of the
macrophages with internalized particles toward the mucociliary
escalator. The insoluble particles merged in the airway mucus are
partly moved by the action of the ciliated epithelial cells, pushing the
mucus along with the nanoparticles to the larynx, where they are
swallowed to the gastro-intestinal tract or excreted through the
mouth by cough within 1–2 days [38,39]. The particles that are not
removed by phagocytosis, might gain access to epithelial and
interstitial sites [36]. Those particles may finally enter the systemic
circulation andthe lymphatic system followingtranscytosisacross the
alveolar epithelial cells [10,40].
According to our results, the clearance of NDs from the alveoli is
predominantly mediated by macrophages. At 1 day post-exposure,
the uptake of both kinds of NDs by macrophages is observed
prominently in the bronchia (Fig. S2 in SM). From 7 to 28 days post-
exposure, we still find many macrophages containing NDs in the
bronchia all the time. (Figs. 4 and 5. Data of 7 days and 14 days post-
exposure are not shown). As most of the particles directly depositing
in the airway can be cleared from the lungs by the continuous
swinging of cilia within 1–2 days [38,39], we speculate that these NDs
are moved upward by macrophages from the alveoli site instead of
depositing in the bronchia directly. In summary, NDs in the alveolar
region could be cleared up through such a way: NDs are phagocytized
by macrophages and these ND loaded macrophages migrate to the
airway, and then expelled by the mucociliary escalator through the
airway. The velocity of such clearance is pretty slow since the NDs are
still observed in the bronchia and alveoli at 28 days post-exposure
(Figs. 4 and 5, and S5 in SM). At 90 days post-exposure, it is very
difficult to find NDs both in the alveoli or bronchia (data not shown).
Although theclearanceof NDs from the lungs is governed mainly by
macrophageuptakeandmucociliarytransport,weobservethatsomeof
NDs-4 and NDs-50 enter the epithelial and interstitial sites (Fig. S3
in SM). But we did not find NDs in liver, spleen and lymphatic node by
histopathological and HRTEM observations, even when mice were
instilled with a much higher dose of 20 mg/kg NDs (data not shown).
The absence of NDs in organs other than the lungs implies that
the translocation of the NDs from the alveolar space to the systemic
circulation is rare, even if it occurs. However, in order to elucidate such
translocation mechanism, the related quantitative data are required in
the further study for the thorough understanding of the process.
For carbon nanomaterials, no definite conclusion about their
systemic translocation after inhalation or instillation has been drawn
todate.Inhaledultrafinecarbonparticleswerereportedtodiffuseinto
the systemic circulation within 5 min [41]. Xu et al. found that C60
(OH)xis able to translocate rapidly into the blood circulation in rats
[42]. Oberdörster et al. also observed the rapid translocation of carbon
nanoparticlestotheliverofratsfollowinginhalation[43].However,no
significant translocation of inhaled carbon particles to the circulation
inhumanswasreportedbyWiebertetal. [44]andMillsetal.[45].Lam
etal.showedthatSWNTsareverydifficulttobeclearedfromthelungs,
although they can translocate from alveolar cavity to interstitial [28].
The conflicting results of extrapulmonary translocation of carbon
nanomaterials may come from the differences in structures, particle
sizes, surface characteristics, labeling materials, measurements and
experimental models reported in different studies [46].
In this work, most of the NDs entering the alveoli are clearly
located in alveolar microphages and epithelia. The clearance of NDs
from the lungs is governed mainly by macrophages and mucociliary
298
Y. Yuan et al. / Diamond & Related Materials 19 (2010) 291–299

Page 9

transport. It seems that entering into the blood circulation and the
lymphatic system is not remarkable for NDs. However, in order to
elucidate such translocation process, the related quantitative data are
required for the thorough understanding of the translocation
mechanism in the future.
5. Conclusions
In conclusion, biochemical assays, ultrastructural and histopatho-
logical evaluationsofthe mouse lungs intratracheally exposedto NDs-4
and NDs-50, within the doses used in this study, do not show the
measureable pulmonary toxicity. The direct imaging of NDs in the
alveoli and bronchia at different time points indicates the translocation
and clearance pathway of NDs: NDs engulfed by macrophages on the
alveolar surface migrate upward, move up with mucus to the trachea
with the assistance of the escalator/mucociliary system, and finally
enter the pharynx. Considering their low pulmonary toxicity and easy
immobilizationwithbiomolecules[18,47–50],wepredictthatNDsmay
havegreatpotentialinthedevelopmentofthepulmonarydrugdelivery
system in the future.
Acknowledgements
We are grateful for the financial support from the China Natural
Science Foundation (no. 20871010) and the China Ministry of Science
and Technology (973 Project no. 2006CB705604 and 2009CB930303).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.diamond.2009.11.022.
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