Biokinetics and effects of barium sulfate nanoparticles
Particle and Fibre Toxicology
Biokinetics and effects of barium sulfate nanoparticles
Nagarjun Konduru 1
Jana Keller 0
Lan Ma-Hock 0
Sibylle Grters 0
Robert Landsiedel 0
Thomas C Donaghey 1
Joseph D Brain 1
Wendel Wohlleben 0
Ramon M Molina 1
0 Experimental Toxicology and Ecology, BASF SE, GV/TB - Z470 , Carl-Bosch-Strae 38, Ludwigshafen 67056 , Germany
1 Department of Environmental Health, Molecular and Integrative Physiological Sciences Program, Harvard School of Public Health , 665 Huntington Avenue, Boston, MA 02115 , USA
Background: Nanoparticulate barium sulfate has potential novel applications and wide use in the polymer and paint industries. A short-term inhalation study on barium sulfate nanoparticles (BaSO4 NPs) was previously published [Part Fibre Toxicol 11:16, 2014]. We performed comprehensive biokinetic studies of 131BaSO4 NPs administered via different routes and of acute and subchronic pulmonary responses to instilled or inhaled BaSO4 in rats. Methods: We compared the tissue distribution of 131Ba over 28 days after intratracheal (IT) instillation, and over 7 days after gavage and intravenous (IV) injection of 131BaSO4. Rats were exposed to 50 mg/m3 BaSO4 aerosol for 4 or 13 weeks (6 h/day, 5 consecutive days/week), and then gross and histopathologic, blood and bronchoalveolar lavage (BAL) fluid analyses were performed. BAL fluid from instilled rats was also analyzed. Results: Inhaled BaSO4 NPs showed no toxicity after 4-week exposure, but a slight neutrophil increase in BAL after 13-week exposure was observed. Lung burden of inhaled BaSO4 NPs after 4-week exposure (0.84 0.18 mg/lung) decreased by 95% over 34 days. Instilled BaSO4 NPs caused dose-dependent inflammatory responses in the lungs. Instilled BaSO4 NPs (0.28 mg/lung) was cleared with a half-life of 9.6 days. Translocated 131Ba from the lungs was predominantly found in the bone (29%). Only 0.15% of gavaged dose was detected in all organs at 7 days. IV-injected 131BaSO4 NPs were predominantly localized in the liver, spleen, lungs and bone at 2 hours, but redistributed from the liver to bone over time. Fecal excretion was the dominant elimination pathway for all three routes of exposure.
Lung absorption; Bioavailability; Particokinetics; Particle dissolution; Translocation; Inhalation
Barium sulfate nanoparticles (BaSO4 NPs) are used
as fillers in coatings (e.g. in motor vehicles) due to their
mechanical, optical and chemical properties. Recently,
BaSO4 NPs have also been used in orthopedic medicine,
diagnostic imaging and other applications [1-5]. It has
been reported that pellethane, a polyurethane elastomer,
when incorporated with BaSO4 NPs exhibited
antimicrobial properties in vitro . Exposure to aerosolized
BaSO4 NPs may occur during their production, shipping,
handling, incorporation into final products, and the use
and disposal of those products. Chronic exposure to
high levels of micron-scale BaSO4 sulfate may induce
pneumoconiosis (baritosis) in miners [7-9].
Barium sulfate is considered a member of the poorly
soluble particles (PSP) or poorly soluble low toxicity
(PSLT) particle groups, as are cerium dioxide (CeO2)
and titanium dioxide (TiO2) [10-12]. These biodurable
nanomaterials are usually poorly absorbed after oral and
inhalation exposure [13-15]. Particokinetics of
nanoparticles are influenced by particle size and route of
exposure . Poorly soluble particles may also differ in
clearance and biological effects compared to soluble
particles [12,17-19]. It is not established whether the
biokinetics of inhaled BaSO4 NPs are similar to other PSLT
NPs. Therefore, it is of interest whether the biokinetics
of inhaled BaSO4 NPs are different from other PSLT NPs.
Previous studies have described the lung clearance of
intratracheally instilled micron-sized radioactive BaSO4
and showed that the particle size influences lung clearance
of Ba [20,21]. A subchronic inhalation study in rats
showed a neutrophil increase in bronchoalveolar lavage
(BAL) with micron-scale TiO2 but not with BaSO4 at
comparable overload lung burdens (~10 mg Ba) [12,17].
The difference was attributed to the lower surface area of
BaSO4 than TiO2. Toxicity of nanoparticles is influenced
by particle physicochemical properties [16,22-24]. The
biological responses to small particles differ from bigger
particles of the same composition [25,26]. Furthermore, a
short-term inhalation study on BaSO4 NPs has been
reported recently [27,28]. Rats were exposed (nose-only) to
50 mg/m3 BaSO4 (NM-220) for 6 hours/day for 5 days. It
was found that the lung burden of BaSO4 at the end of
exposure was 1.1 mg/lung which decreased to 0.24 mg/lung
within 21 days. This short-term exposure to BaSO4 did
not elicit significant pulmonary or systemic responses
consistent with previous reports in various in vitro and
in vivo test systems . The mechanisms underlying the
lower toxicity and rapid lung clearance of BaSO4 NPs are
not fully understood. For example, more research is
needed to quantify the components of clearance
attributable to intact particles versus particle dissolution and
clearance of barium ions. Thus, there is continuing
interest in the biokinetics and effects of BaSO4 NPs, especially
after pulmonary exposure. A two-year inhalation study of
BaSO4 and CeO2 has been initiated in collaboration
between the German Federal Ministry for the Environment
German government and BASF (Ludwigshafen, Germany).
The project is within the Organization for Economic
Cooperation and Development (OECD) sponsorship
program and the European Union Project NANoREG
(a European approach to the regulatory testing of
The data presented here were used in designing this
long-term inhalation study. Our objective was to
characterize the pulmonary and systemic effects of inhaled
BaSO4 NPs after short-term and subchronic exposure. In
addition, we report here a comprehensive study on the
biokinetics of 131Ba after intratracheal instillation (IT),
intravenous injection (IV) and gavage administration of
radiolabeled 131BaSO4 NPs. These studies are important
in assessment of risks from exposure to BaSO4 NPs.
Physicochemical characterization of NM-220 and the
reproduced batch of BaSO4 nanoparticles
Barium sulfate NPs (NM-220) used in all IT instillation,
gavage and IV injection studies were obtained from BASF
SE (Ludwigshafen, Germany). This sample was a reference
material for the Nanomaterial Testing Sponsorship
Program of the OECD. The characterization of this original
batch was published recently . Since the chronic
inhalation study requires large amounts (>100 kg), BaSO4 NPs
were reproduced at a different production plant using the
same synthesis protocol. This reproduced batch was
characterized by the same methods and was used for the
4-week and 13-week inhalation studies. All
physicochemical endpoints are summarized in Additional file 1:
Table S1 (online Supporting Information), which includes
the previously published characterization of NM-220 for
comparison . Transmission and scanning electron
micrographs show that BaSO4 NPs in both batches were
nonspherical globular with no fiber, rod or platelet
impurities. The primary particle size was 25 nm for both
batches (Figure 1A and 1B). The NPs form larger spherical
agglomerates (215 m diameter) in the as-produced
powder (Additional file 1: Table S1). This agglomerate
structure was confirmed by porosimetry which showed
dominant pore sizes of 30 nm and 5 m for both batches
(Additional file 1: Figure S1A). X-ray diffraction (XRD)
analysis showed that the particle mineralogy was
orthorhombic barite (Additional file 1: Figure S1B) for both
batches. Photocatalytic activity of both batches was
extremely low as shown by the absence of methylene blue
degradation (Additional file 1: Figure S2).
The shape, particle size distribution, primary particle
diameter, state of agglomeration (powder), crystalline
phase, specific surface area, surface charge, photocatalytic
activity, and dispersability in water and in Dulbeccos
modified Eagle/fetal calf serum (DMEM/FCS) media were
similar in both batches. Analyses by several methods (EM,
minimal pore size, specific surface area) indicate that the
two batches have similar primary particle sizes. The
properties that were determined by surface chemistry such as
dispersability, charge/zeta potential and photocatalytic
reactivity were also similar (Additional file 1: Figure S2).
However, significant differences were observed in
crystallite size (36 nm for NM-220 vs. 23 nm for reproduced
batch). The reproduced material also had an
intermediate pore size of 200 nm (agglomerate structure)
which was absent in the original NM-220 material. XPS
analyses showed significantly less carbon atoms exposed
on the surface of the reproduced material (2 vs. 17%).
Additionally, elemental analysis by neutron activation
showed that the NM-220 batch had 599 g Ba/mg
material (59.9 wt%), as expected for relatively pure
BaSO4 (Table 1).
Figure 1 Structural characterization by representative SEM scans of as-produced BaSO4 nanomaterial and after incubation for testing
of persistence. (A) reproduced batch, as-produced powder; (B) NM-220 batch, as-produced powder; (C) pellet of NM-220 after 28d incubation in
PBS; (D) pellet of NM-220 after 28d incubation in PSF; (E) pellet of NM-220 after 1d incubation in 0.1 N HCl.
In physiological simulant fluids, BaSO4 NPs (NM-220)
dissolved only slightly at pH 1 (1% dissolution in 0.1 N
HCl) although its particle shapes changed (Additional
file 1: Table S1, Figure 1E) . Very low (0.1%)
dissolution was observed in phosphate buffered saline (PBS)
or phagolysosomal simulant fluid (PSF, pH 4.5) after
Table 1 Neutron activation analysis of BaSO4 NM-220
Data are mean standard deviation.
28 days incubation (Additional file 1: Table S1). No
morphologic changes were seen in PBS (Figure 1C).
However, the non-spherical BaSO4 NPs lost their structural
features with lowest radius of curvature and
recrystallized to spherical structures in PSF (Figure 1D) . It
was confirmed by selected area electron diffraction that
the crystallinity was retained (data not shown). BaSO4
NPs remained in a low agglomeration state and retained a
significant dispersed fraction (80%) of 1 m diameter in
all simulant buffer conditions. The zeta potential ranged
from 20 mV to 32 mV.
The agglomerate size of BaSO4 NPs in deionized water
suspension employed in IT instillation (0.67 mg/ml),
gavage (10 mg/ml) and IV injection (1 mg/ml) was
assessed using dynamic light scattering (DLS). We found
that BaSO4 NP agglomerate size was influenced by
particle concentration (Table 2): the higher the
concentration, the larger the hydrodynamic diameter. For the
inhalation studies, the particle concentrations and size
Table 2 Dynamic light scattering analysis of BaSO4
Concentration (mg/ml dH2O)
Data are mean standard deviation, n = 3.
dH, hydrodynamic diameter, PdI, polydispersity index, , zeta potential.
distributions are summarized in Table 3. The target
concentration of 50 mg/m3 was achieved and maintained
throughout the inhalation exposures. Particle size
distribution of aerosolized BaSO4 NPs was in the respirable
range for rats.
Pulmonary responses to instilled or inhaled BaSO4
To determine whether BaSO4 NPs elicit toxic or
inflammatory response in rats and to identify a suitable dose
for the IT biokinetic studies, groups of six rats were
IT-instilled with BaSO4 NP suspension (NM-220) at 0, 1,
2, and 5 mg/kg body weight. We found that BaSO4 NPs
caused an acute dose-dependent inflammatory response
evidenced by significant increases in BAL parameters
(Table 4). Neutrophils, myeloperoxidase (MPO) and
lactate dehydrogenase (LDH) levels in bronchoalveolar
lavage (BAL) were elevated 24 hours post-instillation. We
also found that 2 and 5 mg/kg doses caused pulmonary
hemorrhage and edema as indicated by increased BAL
haemoglobin and albumin levels. Based on these data,
we concluded that 1 mg/kg was the maximum safe dose
for the IT biokinetic study, since injury and inflammation
were minimal, yet it was sufficient for gamma detection
of 131Ba in the lungs and other tissues over a period of
To assess pulmonary responses of rats after short-term
and subchronic inhalation of BaSO4 NPs, BAL analysis
was performed one day (4- and 13-week groups) and 35
days (4-week group) after the end of each exposure
protocol. Results for all BAL parameters are presented in
Table 5. After 4 weeks of exposure, neutrophils were
significantly increased compared to concurrent controls
(filtered air-exposed) one day after the end of exposure.
However, these values were within the historical control
range in our previous studies. Rats exposed for 13 weeks
showed significant increases in BAL total cells and
neutrophils compared to control. These neutrophil counts were
significantly lower than those seen in instilled rats (Table 4,
Additional file 1: Figure S3). Cytokine levels of monocyte
chemoattractant protein-1 (MCP-1) and cytokine-induced
neutrophil chemoattractant-1 (CINC-1) were elevated in
both exposure groups (Table 5). The longer 13-week
exposure to BaSO4 NPs induced higher levels of the
cytokine MCP-1 compared to the 4-week exposure. All BAL
parameters elevated at 1 day post-exposure returned to
control levels in the 4-week exposure group at 35 days.
No morphological changes were detected by
histopathology in the lungs (Additional file 1: Figure S4) and
extrapulmonary organs. Other parameters such as body
weights, micronucleus test of erythrocytes in peripheral
blood, showed no significant change. Rats exposed for 13
weeks showed significantly higher gamma glutamyl
transferase (GGT) and alkaline phosphatase (ALP) levels than
their corresponding controls (Table 5).
In vivo clearance and translocation of 131BaSO4
nanoparticles after IT instillation in rats
The clearance of 131BaSO4 NPs from the lungs
postinstillation is shown in Figure 2A. Approximately 47% of
the total dose was cleared from the lungs by day 7 and
84% by day 28. A linear regression on the natural
logarithm of the lung 131BaSO4 levels (% dose) over time was
performed (y = e-0.003011x , R2 = 0.96, p = <0.0001). The
estimated clearance half-life was 9.6 days. Extrapulmonary
translocation of 131Ba is shown in Figure 2B. A significant
fraction of 131Ba radioactivity was found in the bones
(29% of dose) and lower fractions in all other tissues
combined (7%). The rest of the 131Ba was excreted mostly in
the feces (30%) and to a lesser extent in the urine (3.9%)
(Figure 3). The complete distribution data of 131Ba after
instillation of 131BaSO4 are summarized in Additional
file 1: Table S2.
Fate of 131BaSO4 nanoparticles after oral administration
The tissue distribution of 131Ba activity following oral
administration is summarized in Figure 4 and listed in
Additional file 1: Table S3. Nearly 100% of the
administered dose was measured in the stomach at 5 minutes
post-gavage (Figure 4A). At 7 days, very low percentages
of the total dose were detected in blood, bone and bone
marrow (<0.1%) (Figure 4B). Gavaged 131BaSO4 NPs were
mostly cleared from the GI tract and eliminated in the
Table 3 Aerosol concentrations and particle size distributions of BaSO4 NM-220
Duration of Targeted Measured MMAD Particle count Particle count
exposure concentrations (mg/m3) concentrations (mg/m3) (m)/GSD mean concentration (particle/cm3) median diameter (nm)
Tissue distribution of 131BaSO4 nanoparticles after
intravenous injection in rats
At 2 hours after intravenous injection of 131BaSO4 NPs,
the blood levels of 131Ba were less than 0.5% of the
administered dose (Figure 6). The complete distribution
data at various time point post-injection are summarized
in Additional file 1: Table S4. The tissue distribution was
typical of circulating particles that are taken up in organs
comprising the mononuclear phagocyte system with
access to the circulation . Notably, 131BaSO4 NPs were
predominantly localized in the liver, spleen, bone and
BaSO4 lung burden (mg)
Total cells (million)
1 day 0.649 0.20 0.562 0.12 0.610 0.20
35 days 0.580 0.11 0.454 0.12 ND
1 day 0.007 0.003 0.021 0.010* 0.016 0.006
35 days 0.019 0.008 0.032 0.024 ND
Total protein (mg/L)
1 day 60 4 77 13* 52 10
35 days 81 23 59 13 ND
1 day 37 17 40 10 41 7
35 days 42 12 37 12 ND
1 day 0.83 0.16 0.83 0.20 0.51 0.10
35 days 0.70 0.09 0.70 0.12 ND
1 day 14.0 0.0 54.7 14.3* 24.2 8.4
35 days 17.3 2.6 14.7 1.7 ND
1 day 104.2 26.7 158.7 22.4* 93.7 18.7
35 days 158.8 38.1 167.6 41.1 ND
Data are mean SD, n = 5/group. Control rats were exposed to filtered air.
ND, not determined.
*p 0.05, BaSO4-exposed vs. control; #p 0.05, 13-week vs. 4-week exposure.
Neutrophils counts were significantly much lower compared to data from rats instilled with 1.4 mg BaSO4 (5 mg/kg BaSO4) (Table 4).
Figure 2 Clearance and translocation of 131BaSO4 NPs following IT instillation. (A) Lung clearance of 131BaSO4 over time. The clearance half
life was approximately 9.6 days. By 28 days, 84% of dose has been cleared from the lungs. (B) Translocated 131Ba from the lungs gradually accumulated
in other organs. By 28 days, 29% of the instilled 131Ba dose was retained in the bone and 7% in all the other organs. Data are mean standard error of
the mean, n = 5 per group.
Figure 3 Cumulative fecal and urinary excretion of 131Ba
following IT instillation. Elimination of 131Ba was mainly via the
feces. By 28 days post-dosing, 30% of the instilled dose was excreted in
the feces (A) and only 4.4% in the urine (B). Data are mean standard
error of the mean, n = 5 per group.
bone marrow. Interestingly, a significant fraction was also
measured in the lungs. This may represent the larger
agglomerates that may be lodged within pulmonary
capillaries. Over the period of 7 days after IV administration,
131Ba in the liver significantly decreased and redistributed
into lungs, bone, and bone marrow (Figure 6). 131Ba
activity in the lungs also significantly decreased over time
(Figure 6). By day 7, a significant fraction of 131Ba
radioactivity was found in the bones (46%). The
cumulative fecal and urinary excretions of 131Ba are shown in
Additional file 1: Figure S5. The cumulative fecal excretion
was 17% while only 4% of the total injected dose was
excreted in the urine over a period of 7 days (Additional
file 1: Figure S5B).
Barium tissue concentration - influence of route of
We examined how the route of exposure affects tissue
barium concentrations after dosing with 131BaSO4 NPs.
Using the measured specific activity of 131BaSO4 NPs and
each tissue 131Ba concentration, we estimated Ba
concentration in ng Ba/g tissue. The Ba concentrations at 7 days
post-dosing are shown in Table 6. These data demonstrate
that IT instillation resulted in significantly higher tissue
concentrations than gavage, especially in the bone. Barium
tissue levels ranged from very low to not detectable
postgavage despite dosing the animals with a higher mass dose
(1 v. 5 mg/kg). As expected, IV injection resulted in higher
Ba concentrations in most tissues compared to IT and
Lung and lymph node barium analysis after inhalation
exposure to BaSO4 nanoparticles
The amounts of BaSO4 in the lungs and lymph nodes
were estimated by measuring Ba with ICP-MS. Inhalation
Figure 4 Tissue distribution of 131Ba following gavage. A. Immediately post-gavage, 100% of dose was recovered in the stomach. B. At 7 days
post-gavage, 131Ba was negligible in all tissues. Very low percentages of the dose were detected in bone and bone marrow. Data are mean standard
error of the mean, n = 5 per group.
exposure to 50 mg/m3 resulted in an equivalent BaSO4
lung burden of 0.84 0.18 mg at 1 day after the end of
a 4-week exposure. Lung BaSO4 burden decreased by
95% (0.84 0.18 to 0.04 mg) on day 1 versus day 35
after exposure. After 13 weeks of exposure, the lung,
Figure 5 Cumulative fecal and urinary excretion of 131Ba
post-gavage. A. Elimination of 131Ba was nearly 100% via the feces.
B. By 7 days post-gavage, only 0.02% of the administered dose was
excreted in the urine. Data are mean standard error of the mean,
n = 5 per group.
tracheobronchial and mediastinal lymph node burdens
were 1.73 0.85 mg, 5.92 6.52 g, 2.72 3.38 g BaSO4,
Our studies examined the effects of short-term (4-week)
and subchronic (13-week) inhalation exposure and a
single IT instillation of BaSO4 NPs in rats. We also
performed comprehensive biokinetic studies of 141Ba when
141BaSO4 NPs were administered in rats via different
routes. Four weeks of inhalation of 50 mg/m3 BaSO4
resulted in no pulmonary toxicity by 35 days post-exposure.
BAL parameters were comparable to control values after
the post-exposure period. Delayed onset of adverse effects
beyond this post-exposure period is unknown.
Histopathologic examination performed in 4-week exposed
animals showed no morphological changes in lungs and
extrapulmonary organs (e.g. brain, heart, liver, spleen,
kidneys). These results are consistent with our previous
short-term inhalation study that tested a variety of
nanomaterials including BaSO4 NPs . A 13-week exposure
elicited a slight inflammatory response in rat lungs. The
long-term effects of inhalation exposure to BaSO4 NPs are
being evaluated in an ongoing two-year study. Our
instillation data showed a moderate dose-dependent
inflammatory response to BaSO4 NPs at 24 hours. Lung burdens at
1 day after 4 or 13 weeks of inhalation exposure were
0.84 0.18 and 1.73 0.85 mg BaSO4/lung, respectively.
At the 5 mg/kg instilled dose (1.4 mg BaSO4 lung burden)
the neutrophil response was significantly higher than at
24 hours after the last inhalation exposure. The difference
in neutrophil response may be due to the differences in
dose rate, particle distribution, particle clearance,
agglomerate surface properties and gender between the two
studies. That the two exposure methods yield different
responses is also consistent with previous reports [32,33]. A
Figure 6 Tissue distribution of 131Ba post-IV injection. Two hours post-injection, 58% of the injected dose was recovered in the liver and lower
percentages in the spleen, bone, bone marrow and the lungs. Over time, 131Ba levels in the liver and lungs decreased with accompanying increases in
bone and bone marrow. Data are mean standard error of the mean, n = 5 per group. *P <0.05, decrease over time, #P <0.05, increase over time,
Table 6 Comparison of tissue barium concentrations at 7 days after dosing
Route (dose) IT Instillation (1 mg/kg) Gavage (5 mg/kg)
ng/g SE ng/g SE
69074.5 4993.8 < 0.01
2271.9 124.6* 0.079 0.014
1018.3 71.4* 0.13 0.12
119.3 23.8 < 0.01
113.1 16.3 < 0.01
106.6 31.2 < 0.01
43.2 6.7 < 0.01
37.5 1.5 < 0.01
30.3 18.6* 0.016 0.016
7.8 2.7 < 0.01
3.6 1.2 < 0.01
3.6 1.2 < 0.01
3.5 0.8 < 0.01
3.1 0.5 < 0.01
3.0 0.8 < 0.01
2.5 0.5 < 0.01
2.5 1.0 < 0.01
0.7 0.7 < 0.01
study by Baisch et al. reported a higher inflammatory
response to a similar deposited dose of TiO2 NPs when
delivered via IT instillation rather than whole body
inhalation . It is clear that although IT instillation is a
reliable method for administering a precise dose to the lungs,
it does not model inhalation exposure. Particle
distribution and dose rate are different between these two
exposure methods. However, IT instillation is useful in
biokinetic studies that require precise dosing and timing
especially for radioactive materials such as 131BaSO4 NPs.
Our use of radiolabelled NPs provided a very sensitive
method that measured only 131Ba from the nanoparticles
and excluded background Ba from other sources, such as
food and water. The sensitivity of 131Ba detection also
avoided the use of high BaSO4 doses while allowing us to
measure very low levels in tissues.
Pulmonary clearance kinetics post-inhalation was
similar to the previous 5-day inhalation study . We
observed a 95% clearance of Ba from the lungs in
34 days. This is consistent with the previously observed
77% clearance over 21 days . The lung burden of
BaSO4 after 13-week exposure to a high concentration
of BaSO4 was also similar to those of rats exposed
to lower concentrations of TiO2 and CeO2 [28,34]. This
shows that clearance of BaSO4 is much faster than these
other two nanomaterials. But the similar lung burdens
from exposure to TiO2 and CeO2 resulted in greater
inflammatory responses [10,28]. Our data suggest that the
low toxicity of inhaled BaSO4 is inherent to the
nanomaterial as well as its relatively faster clearance.
The biokinetic data based on radioactive 131BaSO4
showed a fast clearance of 131Ba from the lungs. We
observed that 50% of the initial dose was cleared from the
lungs after 9.6 days. By 28 days only 16% of the initial
dose was retained in the lungs. This clearance rate also
roughly correlated with that obtained from our
inhalation experiment. Based on a linear regression on the
natural logarithm of lung 131Ba levels (% of instilled dose)
over time, the extrapolated clearance of instilled 131BaSO4
dose at 35 days is 92%. The lung burden post-instillation
was 0.28 0.004 which was lower than lung burden after
4-week inhalation (0.84 0.18 mg BaSO4). Despite this
difference in initial lung burden, the clearance rate of
BaSO4 NPs was not different between the two exposure
The lung clearance of BaSO4 NPs was similar to that
shown for micron-sized radiolabeled BaSO4 where 17%
of radiolabeled barium remained at 22 days
postinstillation in rat lungs [20,21]. The fate of the 16% of
131Ba remaining in the lungs at the end of our
observation needs longer-term studies. Since lung epithelial
injury may alter the fate of instilled NPs, we chose a dose
that would not cause significant injury that might affect
the outcome of our IT biokinetic study. Our data
suggest that 131Ba from instilled 131BaSO4 NPs was
cleared from the entire animal mainly via the
gastrointestinal route. The excreted fraction in the feces might
include contributions from both the mucociliary and
biliary clearance pathways. Although lung clearance of
131BaSO4 NPs is relatively fast and only 16% of the
administered dose remained 4 weeks post-instillation, a
substantial fraction (37%) was retained elsewhere in the body.
The tissue distribution of 131Ba following IT instillation
showed a significant translocation to bone, consistent with
other heavy earth alkaline metals like calcium and
strontium  as well as to the thoracic lymph nodes. Whether
the 131Ba measured in these extrapulmonary organs was
131BaSO4 NPs or ionic 131Ba could not be ascertained in
this study. A previous study showed that IT-instilled ionic
barium cleared much more rapidly than BaSO4 particles
. We have also demonstrated that ionic cerium was
more toxic and was cleared more rapidly than CeO2
The clearance of inhaled BaSO4 NPs was fast as
evidenced by the decrease in BaSO4 lung burden over time.
Only 5% of retained BaSO4 in the lungs (4-week-exposure)
remained 35 days after the end of exposure. The relatively
high bioavailability of inhaled or instilled BaSO4 does not
correlate with its very low dissolution rate in
phagolysosomal simulant fluid, a proposed model of macrophage
dissolution/clearance of particles . This strongly
suggests that PSF does not fully simulate the complex kinetic
processes of lung transport and clearance, especially the
mechanism of particle dissolution within macrophage
phagolysosomes. Our cell-free in vitro dissolution studies
showed very low dissolution in PSF even after 28 days.
However, we observed that the non-spherical BaSO4 NPs
lost their feature of lowest radius of curvature and
later recrystallized over this period. Interestingly, it has
been shown that the NP surface charge and interactive
properties may vary with the local radius of curvature
. The regions of the particle surface with different
curvature become charged at differing pH values of the
surrounding solution . Previous studies showed that
non-spherical nanomaterial may exhibit different toxicity
from that of spherically shaped nanomaterial of the same
composition due to the varying local charge density .
Likewise, quartz and vitreous silica NPs, with irregular
surfaces and sharp edges were more toxic than spherical silica
. The significance of the noted structural changes of
BaSO4 NPs in vitro remains to be studied in the
phagolysosomal compartment of lung macrophages. How these
structural changes relate to cytotoxicity is likewise yet to
Whole-body exposure of rats to NP aerosols results in
not only pulmonary deposition but also in ingestion of
NPs due to the grooming behavior of rats. This ingestion
can complicate the pattern of bioavailability from
wholebody inhalation exposures. However, for animal welfare
considerations, whole-body is more convenient than
noseonly exposure for long-term inhalation studies. For some
applications of BaSO4, the gastrointestinal tract is also a
common route for human exposures. Thus, we
investigated the fate of orally administered 131BaSO4 NPs. Since
the GI transit time is generally less than one day, it is less
likely for nanoparticles to remain in the GI tract for a
prolonged period of time. Even particles adherent to or
ingested by columnar epithelial cells are eliminated rapidly
since the epithelium sloughs off and regenerates
constantly. Our data showed that 88% of the dose was
eliminated in the feces within 24 hours, and almost 100%
by 7 days post-gavage. Since very low radioactivity was
detected in other organs, we conclude that neither 131BaSO4
NPs nor 131Ba ions significantly crossed the intestinal
barrier. This low oral bioavailability correlates with our
observation of very low dissolution of BaSO4 NPs in simulated
gastric and intestinal fluid (Additional file 1: Table S1).
This indicates that there is negligible contribution from fur
deposition and ingestion during inhalation exposure. It also
means that barium detected in extrapulmonary organs after
inhalation translocates from the lungs to the blood.
As we and others have shown, a small fraction of
inhaled nanoparticles may translocate into the systemic
circulation . Although our study focused on normal
lungs, when they are compromised by injury or
inflammation increased rates of NP translocation may occur.
Therefore, we evaluated the biokinetics and tissue
distribution of intravenously injected 131BaSO4 NPs to
elucidate their fate in the circulation. When we sacrificed
animals at 2 hours post-IV injection of 131BaSO4 NPs,
we found very low radioactivity in the blood. Since the
first time point we examined was at 2 hours, we could
not determine the vascular clearance rate. Previously,
we have shown that clearance half-lives of circulating
particles are on the order of minutes even for
nanoparticulates such as gold colloid . Initially, a significant
hepatic accumulation of 131Ba was observed but liver
retention was decreased by 7 days. This likely reflects
rapid ingestion of 131BaSO4 NPs by the abundant
hepatic macrophages (Kupffer cells) and possibly
subsequent dissolution followed by release of barium ions
into the blood. The decrease was accompanied by
increasing accumulation in bone similar to that observed
following IT instillation. Despite the significant uptake
of Ba in the bone, no evidence of genotoxicity in the
bone marrow was noted. We found no micronucleus
formation in peripherial blood cells that originate from
hematopoiesis in the bone marrow.
Our data show that inhaled BaSO4 NPs elicited minimal
pulmonary response and no systemic effects. Equivalent
lung burdens of CeO2 and TiO2 elicit more pulmonary
response than BaSO4 . This difference might be due
to its lower inherent toxicity and also to its faster lung
clearance. The mechanism of this faster clearance needs
further investigation. There is no direct correlation
between abiotic in vitro dissolution of BaSO4 in several
cell-free biological simulation fluids and actual in vivo
biopersistence and bioavailability of barium from BaSO4
NPs. Our data suggest that cell-free in vitro assays either
lack crucial constituents or do not adequately simulate
the processes that facilitate particle dissolution and
increase bioavailability. The Ba in BaSO4 from the lungs
translocates to many tissues, especially the bones. The
comparison of pulmonary versus ingestion routes of
exposure provides a quantitative measure of relative doses
to a variety of non-pulmonary tissues. From our data, it
is evident that the bioavailability of Ba from ingestion of
BaSO4 NPs is very low and that no significant
contribution from ingestion should occur during whole-body
inhalation studies in rats.
Our study underscores the high Ba bioavailability and
clearance of BaSO4 NPs deposited in the lungs. Unlike
CeO2 and TiO2, BaSO4 NPs are retained to a lesser
extent in the lungs after inhalation. Even at lung burdens
similar to CeO2 and TiO2, BaSO4 NPs cause lower
pulmonary toxicity. Barium sulfate exhibits lower toxicity
and biopersistence in the lungs compared to poorly
soluble CeO2 and TiO2.
Physicochemical characterization of BaSO4 nanoparticles
BaSO4 NPs (NM-220) used for IT instillation studies were
obtained from BASF SE (Ludwigshafen, Germany). It was a
reference material for the Nanomaterial Testing
Sponsorship Program of the Organization for Economic
Cooperation and Development (OECD). The characterization
of the original batch distributed as NM-220 was
published recently . The reproduced BaSO4 used in
inhalation studies was characterized by the same methods (See
Animals for intratracheal instillation, gavage and
intravenous injection studies
The protocols used in this study were approved by
the Harvard Medical Area Animal Care and Use
Committee. Male Wistar rats (8 weeks old) were obtained
from Charles River Laboratories (Wilmington, MA) and
were housed in standard microisolator cages under
controlled conditions of temperature, humidity, and light at
the Harvard Center for Comparative Medicine. They
were fed commercial chow (PicoLab Rodent Diet 5053,
Framingham, MA) and reverse-osmosis purified water
was provided ad libitum. The animals were acclimatized
in the facility for 7 days before the start of experiments.
Preparation of BaSO4 suspension for animal dosing
Suspensions of BaSO4 NPs were prepared at appropriate
concentrations in sterile polyethylene tubes. The critical
dispersion sonication energy (DSEcr) required to achieve
the lowest reported particle agglomeration was used as
previously reported . Suspensions in sample tubes
were sonicated with a Branson Sonifier S-450A (Branson
Ultrasonics, Danbury, CT) fitted with a cup sonicator
at 242 J/ml, the critical dispersive energy shown to
maximally disperse these particles in water  while
immersed in running cold water to minimize heating of the
particles. The hydrodynamic diameter (dH), polydispersity
index (PdI), and zeta potential () of each suspension were
measured by dynamic light scattering using a Zetasizer
Nano-ZS (Malvern Instruments, Worcestershire, UK).
Pulmonary responses to intratracheally instilled BaSO4
nanoparticles Bronchoalveolar lavage and analyses
This experiment was performed to determine a particle
dose for pulmonary particle instillation that does not cause
significant injury or inflammation. Twenty rats (mean wt
standard deviation, 280 15 g) were IT-instilled with
BaSO4 suspension at 1, 2 and 5 mg/kg dose (5 rats per
dose) to determine the acute pulmonary effects of BaSO4
particles. The nanoparticle concentrations were 0.67, 1.33,
and 3.33 mg/ml for the 1, 2, and 5 mg/kg dose,
respectively. Rats instilled with an equivalent volume of sterile
distilled water served as controls. The volume dose was
1.5 ml/kg. The particle suspensions were delivered to
the lungs through the trachea, as described earlier .
Twenty-four hours later, the rats were anesthetized and
euthanized via exsanguination. The trachea was exposed
and cannulated. The lungs were then lavaged 12 times
with 3 mL of Ca- and Mg-free 0.9% sterile PBS. The cells
of all washes were separated from the supernatant by
centrifugation (350 g at 4C for 10 min). Total cell count
and hemoglobin measurements were made from the cell
pellets. After smearing and staining the cells, a differential
cell count was performed. The supernatant of the two first
washes was clarified via centrifugation (14,500 g at 4C
for 30 min), and used for spectrophotometric assays for
lactate dehydrogenase (LDH), myeloperoxidase (MPO)
Neutron activation of BaSO4 nanoparticles for
Barium sulfate NM-220 particles were neutron activated
at the MIT Nuclear Reactor Laboratory (Cambridge, MA)
with a thermal neutron flux of 5 1013 n/cm2s for 24
hours. The process generated 131Ba, which decays with a
half life of 10.5 days and emits multiple gamma rays with
varying energies. The specific activity was 2.6 Ci 131Ba
per mg BaSO4 NPs.
Pharmacokinetics of tracheally-instilled, gavaged or
intravenously-injected 131BaSO4 nanoparticles
Fifty rats (mean wt standard deviation, 270 12 g) were
used for this study. Neutron-activated 131BaSO4 NPs were
suspended in sterile distilled water at 0.67 mg/ml for
intratracheal instillation (IT), 10 mg/ml for gavage, and 1 mg/ml
for intravenous (IV) injection. The mass and volume doses
were 1) IT - 1 mg/kg (1.5 ml/kg), 2) gavage - 5 mg/kg
(0.5 ml/kg), and 3) IV - 1 mg/kg (1 ml/kg). The particle
suspensions were dispersed as described earlier. Aliquots
of each suspension were measured in a WIZARD gamma
counter (PerkinElmer, Inc., Waltham, MA) to estimate
each rats 131Ba dose. Gamma energies at 200270 KeV
were utilized for 131Ba quantitation. Each rat was
anesthetized with isoflurane (Piramal Healthcare, Bethlehem, PA)
during particle administration. After dosing, each rat was
placed in a metabolic cage with food and water ad libitum.
Twenty-four-hour samples of urine and feces were
collected at 01, 23, 67, 910, 1314, 2021, and 2728
days after dosing.
The 131BaSO4 NP suspension was delivered to the lungs
through the trachea as described earlier. For gavage,
131BaSO4 NPs were delivered into the stomach via the
esophagus. IV injection was done using the penile vein in
similarly anesthetized animals. Five rats from the IT group
were humanely killed at each time point: 5 minutes and 2,
7, 14 and 28 days post-dosing. Analysis of rats at 5 minutes
post-instillation was performed to get an accurate measure
of the initial deposited dose. Equal numbers of rats (5 per
timepoint) were analyzed at 5 minutes and 7 days
postgavage, and at 2 hours, 2 days, and 7 days post-IV
injection. At each time point, rats were anesthetized and blood
collected from the abdominal aorta. Plasma and red blood
cells were separated by centrifugation. The lungs, brain,
heart, spleen, kidneys, gastrointestinal tract, liver, testes,
and samples of skeletal muscle, bone marrow, skin, and
femoral bone were collected and placed in pre-weighed
tubes. Sample weight was recorded and radioactivity
(200270 KeV) was measured in a WIZARD gamma
counter (PerkinElmer, Inc., Waltham, MA). Disintegrations per
minute were calculated from the counts per minute and
the counter efficiency. The limit of detection for 131Ba was
0.05 nCi. All radioactivity data were adjusted for physical
decay over the entire observation period. Data were
expressed as Ci/g and as a percentage of the administered
dose retained in each organ. Total radioactivity in organs
and tissues not measured in their entirety was computed
using the following estimates of tissue percentage of total
body weight: skeletal muscle, 40%; bone marrow, 3.2%;
peripheral blood, 7%; skin, 19%; and bone, 6% [44,45].
Animals for inhalation studies
Protocols for the inhalation studies were approved by the
local authorizing agency in Landesuntersuchungsamt
Koblenz, Germany. Animals were housed in an
AAALACaccredited facility in accordance with the German Animal
Welfare Act and the effective European Council Directive.
Female Wistar Han rats were obtained at 5 or 7 weeks of
age from Charles River Laboratories (Sulzfeld, Germany).
The animals were maintained in groups of up to 5 animals
in a polysulfon cage (H-Temp [PSU], TECNIPLAST,
Germany) with a floor area of about 2065 cm2 with access
to wooden gnawing blocks, GLP certified diet (Kliba
laboratory diet, Provimi Kliba SA, Kaiseraugst, Basel
Switzerland) and water ad libitum. Animal rooms were
kept under controlled conditions (20 - 24C
temperature, 30-70% relative humidity, 15 air changes per hour,
12-hour light/dark cycle). To adapt to the exposure
conditions, the animals were acclimatized to exposure
conditions over two days (3 and 6 hours, respectively). Up to
two animals per wire cage type DK III (BECKER & Co.,
Castrop-Rauxel, Germany) were exposed in the
wholebody exposure chamber.
Study design - inhalation exposure for four and thirteen
Thirty female rats (in groups of five) were whole-body
exposed to 50 mg/m3 BaSO4 NPs for 6 hours per day on five
consecutive days for 4 weeks (15 rats). Another cohort of
15 rats was exposed for 13 weeks. Body weights were
recorded before and every week throughout the duration of
the experiments. After 4 weeks of exposure, one group
was examined and another after a post-exposure period of
35 days. The short-term inhalation study with 4 weeks of
exposure was performed according to the OECD
Principles of Good Laboratory Practice (GLP) , according to
OECD Guidelines for Testing of Chemicals, Section 4:
Health Effects, No. 412 . This study provides
information on biokinetics and effects of BaSO4 NPs required for
the design of the long-term inhalation study. Barium
burden in lungs was measured at three time points to
determine the retention half-life. BAL analysis and
histopathology of the lungs were performed. In addition,
systemic effects were investigated with histopathology of
extrapulmonary organs, examination of blood and systemic
genotoxicity by micronucleus test (MNT). Based on the
result of the short-term study with 4 weeks of exposure,
the long-term study was started at the same concentration
of 50 mg/m3 BaSO4. The long-term inhalation study is
performed according to OECD Guidelines for Testing of
Chemicals, Section 4: Health Effects, No. 453 .
The animals were exposed while in wire cages that were
located in a stainless-steel whole-body inhalation chamber
(V = 2.8 m3 or V = 1.4 m3). The aerosols were passed into
the inhalation chambers with the supply air and were
removed by an exhaust air system with 20 air changes per
hour. For the control animals, the exhaust air system was
adjusted in such a way that the amount of exhaust air was
lower than the filtered clean, supply air (positive pressure)
to ensure that no laboratory room air reaches the control
animals. For the BaSO4-exposed rats, the amount of
exhaust air was higher than the supply air (negative
pressure) to prevent contamination of the laboratory as a
result of potential leakages from the inhalation chambers.
Aerosol generation and monitoring
BaSO4 aerosols were produced by dry dispersion of
powder pellets with a brush dust generator using compressed
air at 1.5 m3/h (developed by the Technical University of
Karlsruhe in cooperation with BASF, Germany). The dust
aerosol was diluted by conditioned air passed into the
whole-body inhalation chambers. The control group was
exposed to conditioned clean air. The desired
concentrations were achieved by varying the feeding speed of the
powder pellet or by varying the rotation speed of the
brush. Based on a comprehensive technical trial,
atmospheric concentrations within the chambers were found to
be homogenous (Table 3). Nevertheless, exposure cages
were rotated within each chamber daily for the 4-week,
and weekly for the 13-week group.
Generated aerosols were continuously monitored by
scattered light photometers (VisGuard, Sigrist). Particle
concentrations in the inhalation chambers were analyzed
by gravimetric measurement of air filter samples. Particle
size distribution was determined gravimetrically by
cascade impactor analysis using eight stages Marple Personal
Cascade Impactor (Sierra-Anderson, USA). In addition, a
light-scattering aerosol spectrometer (WELAS 2000, Palas,
Karlsruhe, Germany) was used to measure particle sizes
from 0.24 to 10 m. To measure particles in the
submicrometer range, a scanning mobility particle sizer
(SMPS 5.400, Grimm Aerosoltechnik, Ainring, Germany)
was used. The sampling procedures and measurements to
characterize the generated aerosols were previously
Pulmonary responses to inhaled BaSO4
nanoparticles - Bronchoalveloar lavage and analysis
Five animals per group were examined. After euthanasia,
the lungs were lavaged twice in situ with 22 mL/kg body
weight (4 to 5 ml) of normal saline. The recovered
volume ranged from 8 to 10 ml per animal. Aliquots of
BAL were used for determinations of total protein
concentration, total cell count, differential cell count and
enzyme activities. In the 4 week-exposure group and its
control, BAL analysis was performed twice (1 and 35
days after the end of exposure) but only at 1 day
postexposure in the 13-week exposure group. Lavaged lung
tissue and aliquots of the BAL fluid (1 ml) were stored
at 80C and used for determination of barium content.
Total BAL cell counts were determined with an Advia 120
(Siemens Diagnostics, Fernwald, Germany) hematology
analyzer. Differential cell counts were made on
Wrightstained cytocentrifuge slide preparations. Using a Hitachi
917 (Roche Diagnostics, Mannheim, Germany) reaction
rate analyzer, levels of BAL total protein and activities of
lactate dehydrogenase (LDH), alkaline phosphatase (ALP),
-glutamyltransferase (GGT) and
N-acetyl--glucosaminidase (NAG) were measured. Inflammatory cytokines
(MCP-1, IL-8/CINC-1, M-CSF, osteopontin) in BAL
were measured using ELISA test kits as described
Tissue analysis of barium content
Ba levels were measured in the lungs and lung-associated
lymph nodes of exposed animals and controls. The
lavaged lungs and aliquots of BAL of five animals per
group were used. Barium content in the 4 week-exposure
group lungs was measured three times (1, 2 and 35 days
after the end of exposure) but only once (1 day
postexposure) in the 13-week exposure group. Each tissue
sample was dried and sulfuric acid was added. The sample
was then ashed and acid was vaporized at 500C for 15
min. Sulfuric and nitric acid were added to the residue.
Then a mixture of nitric acid, sulfuric acid and perchloric
acid ( 2:1:1 v/v/v) was added and the solution was heated
to oxidize organic matter. After evaporation, the residue
was dissolved in concentrated sulfuric acid. The resulting
solution was analyzed for 137Ba content by inductively
coupled plasma mass spectrometry (ICP-MS) using
Agilent 7500C (Agilent, Frankfurt, Germany). The limit of
detection for Ba is 0.3 g per tissue sample.
Necropsy and histopathology
After 4 weeks of exposure, necropsy and histopathology
were performed on selected rats at 1 day and 34 days after
the end of exposure. Gross and histopathological
examination of the lungs and extrapulmonary organs were
performed on ten rats per group. The animals were
euthanized by cutting the abdominal aorta and vena cava under
sodium pentobarbital anesthesia. According to OECD no.
412, the following organs were weighed: adrenal glands,
brain, heart, ovaries, uterus with cervix, kidney, liver, lungs,
spleen, thymus, thyroid glands. The lungs were IT-instilled
with neutral buffered 10% formalin at 30 cm water
pressure. All other organs were fixed in the same fixative. The
organs and tissues were trimmed, paraffin embedded and
sectioned according to RITA trimming guides for
inhalation studies [51-53]. Paraffin sections were stained with
hematoxylin and eosin. Extrapulmonary organs and the
respiratory tract, comprised of the nasal cavity (four levels),
larynx (three levels), trachea (transverse and longitudinal
with carina), lungs (five lobes), and mediastinal and
Pharmacokinetic and single instillation studies
All BAL parameters and tissue 131Ba distribution data
were analyzed using multivariate analysis of variance
(MANOVA) followed by Bonferroni (Dunn) post hoc tests
using SAS Statistical Analysis software (SAS Institute,
Cary, NC). Lung clearance data were analyzed by linear
regression of the natural logarithm of the lung 131BaSO4
levels (% dose) over time using R Program v. 3.1.0 (The R
Foundation for Statistical Computing, Vienna, Austria).
Body weight differences were compared between
BaSO4-exposed and control groups using Dunnetts test.
Bronchoalveolar lavage cytology, enzyme and cell
mediator data were analyzed by non-parametric one-way
analysis of variance using the Kruskal-Wallis test
(twosided). If the resulting p value was equal or less than
0.05, a pair-wise comparison of each test group with the
control group was performed using the Wilcoxon test
or the MannWhitney U-test. Comparison of organ
weights was performed by nonparametric one-way
analysis using the two-sided KruskalWallis test, followed
by a two-sided Wilcoxon test for the hypothesis of equal
Additional file 1: Online Supporting Information. Table S1.
Physicochemical characterization of BaSO4 nanoparticles. Table S2.
Distribution of recovered 131Ba post-intratracheal instillation of 131BaSO4
nanoparticles. Table S3. Distribution of recovered 131Ba post-gavage of
131BaSO4 nanoparticles. Table S4. Distribution of recovered 131Ba
post-intravenous injection of 131BaSO4 nanoparticles. Figure S1. Structure
of BaSO4 NM-220. A) Pore size distribution by Hg intrusion. B) Crystallinity
by XRD (black line), with assignment of peaks to reference spectrum of
bulk BaSO4 orthorombic (red). There were no unexpected peaks.
Figure S2. Photocatalytic reactivity of A) NM-220 batch, B) reproduced
batch: shown are UVvis absorption spectra at 0, 2, 6 and 22 h incubation
of Methylene Blue with BaSO4, irradiated with 1 mW/cm2 UV (350 nm) as
specified in DIN 52980:200810, adapted for dispersed surfaces . The
blue curves are spectra of samples kept in the dark, the red-yellow curves
are spectra of irradiated samples. The evaluation is compatible with zero
degradation of the dye. Figure S3. Total lavaged neutrophils at 1 day
post-instillation (A) or after the end of 4 and 13 weeks inhalation
exposure of BaSO4 NPs (B). The x-axis represents the lung burden of barium
at the end of 4-week (0.8 mg) or 13-week inhalation exposure (1.7 mg) (B).
The neutrophil counts were significantly higher in instilled (A) than
aerosol-exposed rats (B). Figure S4. Microscopic appearance of lungs after 4
weeks of inhalation exposure. Lung section of control animal (A) and animal
exposed to 50 mg/m3 BaSO4 (B). Figure S5. Cumulative fecal and urinary
excretion of 131Ba post-IV injection. Elimination of 131Ba was 17% via the
feces (A) and only 4.4% of dose via the urine (B).
ALP: Alkaline phosphatase; BAL: Bronchoalveolar lavage; BALF: Bronchoalveolar
lavage fluid; CINC: Cytokine-induced neutrophil chemoattractant; DLS: Dynamic
light scattering; FaSSIF: Fasted state simulated intestinal fluid; GGT:
-Glutamyltranspeptidase; GSD: Geometric standard deviation; ICP-MS: Inductively coupled
plasma mass spectrometry; IL: Interleukin; LDH: Lactate dehydrogenase;
M-CSF: Macrophage colony stimulating factor; MCP: Monocyte chemoattractant
protein; MMAD: Mass median aerodynamic diameter; MPO: Myeloperoxidase;
NAG: N-Acetyl--glucosaminidase; NM: Nanomaterial; OPN: Osteopontin;
PBS: Phosphate buffered saline; PMN: Polymorphonuclear; PSF: Phagolysosomal
simulant fluid; PSLT: Poorly soluble low toxicity; SEM: Scanning electron
microscopy; SMPS: Scanning mobility particle sizer; TEM: Transmission electron
microscopy; TGA: Thermogravimetric analysis.
JK, LM, SG, RL and WW are employees of BASF SE, a company that produces
and markets nanomaterials. NVK was a BASF fellow for the duration of the
study. All other authors declare that they have no competing interests.
RL, RMM and JDB designed the project and evaluated the experimental
results. RMM, NVK, TCD and JDB carried out the biokinetic studies with
radioactive barium sulfate and pulmonary toxicity experiments after
intratracheal instillation. JK, LM, SG, RL and WW performed the inhalation
toxicity studies and physico-chemical characterizations of barium sulfate
nanoparticles. NVK and JK drafted the manuscript. All authors read, revised
and approved the manuscript.
This study was funded by BASF SE (Ludwigshafen, Germany) and by the
National Institute of Environmental Health Sciences (ES000002). Nagarjun
Konduru was supported with a BASF Fellowship. We thank Thomas Bork for
technical assistance with neutron activation, the inhalation and pathology
team of BASF for their technical support, Christa Watson for technical help
with DLS analysis and Melissa Curran for her editorial assistance.
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