Bioavailability, distribution and clearance of tracheally-instilled and gavaged uncoated or silica-coated zinc oxide nanoparticles
Particle and Fibre Toxicology
Bioavailability, distribution and clearance of tracheally-instilled and gavaged uncoated or silica-coated zinc oxide nanoparticles
Nagarjun V Konduru 0
Kimberly M Murdaugh 0
Georgios A Sotiriou
Thomas C Donaghey
Joseph D Brain
Ramon M Molina
0 Equal contributors Center for Nanotechnology and Nanotoxicology, Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, School of Public Health, Harvard University , 665 Huntington Avenue, Boston, MA 02115 , USA
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.
Zinc oxide; Nanoparticles; Pharmacokinetics; Bioavailability; Silica coating; Nanotoxicology
Zinc oxide nanoparticles (ZnO NPs) are widely used in
consumer products, including ceramics, cosmetics,
plastics, sealants, toners and foods . 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 . ZnO NPs filter
both UV-A and UV-B radiation but remain transparent
in the visible spectrum . For this reason, ZnO NPs
are commonly added to sunscreens  and other
cosmetic products. Furthermore, advanced technologies
have made the large-scale production of ZnO NPs
possible . 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
. 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
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.  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 . 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
. Surface characteristics of NPs, such as their chemical
and molecular structure, influence their
pharmacokinetic behavior . 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 .
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 . Furthermore, coating magnetic
NPs with amorphous silica enhances particle stability
and reduces its cytotoxicity in a human bronchial
epithelium cell line model . Amorphous SiO2 is
generally considered relatively biologically inert ,
and is commonly used in cosmetic and personal care
products, and as a negative control in some
nanoparticle toxicity screening assays . However, Napierska
et al. demonstrated the size-dependent cytotoxic
effects of amorphous silica in vitro . 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 . Moreover, SiO2-coated nanoceria
induced minimal lung injury and inflammation . It
has also been demonstrated that SiO2 coating improves
nanoparticle biocompatibility in vitro for a variety of
nanomaterials, including Ag , Y2O3 , and ZnO . 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 . 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 . 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 .
Studies examining nanoparticle structure-pharmacokinetic
relationships have established that plasma protein binding
profiles correlate with circulation half-lives .
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
Synthesis and characterization of ZnO and SiO2-coated
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].
Flamemade nanoparticles typically exhibit a lognormal size
distribution with a geometric standard deviation of g =
1.45 . To create the SiO2-coated ZnO nanorods, a
nanothin (~4.6 2.5 nm) amorphous SiO2 layer
encapsulated the ZnO core  (Figure 1B). The amorphous
nature of the silica coating was verified by X-ray
diffraction (XRD) and electron microscopy analyses . The
average crystal size of uncoated and SiO2-coated NPs
were 29 and 28 nm, respectively . Their specific
surface areas (SSA) were 41 m2/g (uncoated) and 55 m2/g
(SiO2-coated) . 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 . 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 . 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 , which includes the pH conditions in the
airways/alveoli and small and large intestines. The
postirradiation hydrodynamic diameter and zeta potential in
water suspension were similar to those of pristine NPs
used in the lung toxicity/inflammation experiments.
Pulmonary responses to intratracheally instilled ZnO and
We compared the pulmonary responses to uncoated
versus SiO2-coated ZnO NPs at 24 hours after IT instillation
in rats. Groups of 46 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
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
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.
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).
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.
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
Figure 3 Lung clearance of 65Zn post-IT instillation of 65ZnO
and SiO2-coated 65ZnO 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).
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, 2 3 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.
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).
Pharmacokinetics of gavaged uncoated or SiO2-coated
Absorption of 65Zn from the gut was studied at 5 minutes
and 7 days post-gavage of uncoated or SiO2-coated 65ZnO
Figure 4 Extrapulmonary distribution of 65Zn post-IT instillation
of 65ZnO and SiO2-coated 65ZnO 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.
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).
Table 3 Tissue distribution of 65Zn at 28 days after
intratracheal instillation of 65ZnO or SiO2-coated 65ZnO
NPs in rats
Table 4 Distribution of 65Zn 7 days after gavage
administration of 65ZnO or SiO2-coated 65ZnO NPs in rats
Nanoparticles can be released into the workplace
environment during production and handling of
nanomaterials . For example, studies have shown that ZnO NPs
were released during an abrasion test of commercially
available two-pack polyurethane coatings with ZnO NPs
. 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
Sicoated 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  and cellular
toxicity . 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 . 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 . 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
Figure 5 Fecal and urinary excretion of 65Zn post-IT instillation of 65ZnO and SiO2-coated 65ZnO 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).
. However, it has also been shown that the lung
injury and inflammatory responses to amorphous silica are
transient . 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 .
For example, the adsorption of endogenous proteins
like albumin to the surface of charged NPs increases
their hydrodynamic diameter and alters their
translocation rate . 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  while zwitterionic or neutral organic
coatings have been shown to prevent adsorption of
serum proteins . A recent study also showed that
nanoparticle protein corona can alter their uptake by
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
Figure 6 Tissue distribution of 65Zn post-gavage of 65ZnO and SiO2-coated 65ZnO 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 65Zn post-gavage of 65ZnO and SiO2-coated 65ZnO 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).
by translocation across the alveolar epithelium into the
blood circulation . Since ZnO NPs have been shown
to dissolve in culture medium and in endosomes , it
is not surprising that lung clearance of 65ZnO NPs was
rapid compared to that of poorly soluble NPs of cerium
oxide  and titanium dioxide . 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 . As
shown previously, the silica coating reduced the
dissolution of ZnO NPs in culture medium , 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
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 .
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
pancreatobiliary secretion, and to a lesser extent via mucociliary
clearance of instilled NPs . 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%) .
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 . 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) .
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
5100 nm crossing the air-blood barrier and reaching
blood circulation and extrapulmonary organs can
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 , and is required for protein
synthesis in osteoblasts . 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 .
Because it is an essential trace element, zinc is routinely
added to animal food products and fertilizer . Due to
its antimicrobial properties, there is increasing interest
in adding ZnO to polymers in food packaging and
preservative films to prevent bacterial growth . 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 . 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
. However, no effect on tissue distribution was
observed. This is in contrast to our findings at 7 days
postgavage 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) . 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 . 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
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
postgavage. For both routes of administration, the SiO2
coating enhanced the transport of 65Zn to the thoracic
Synthesis of ZnO and SiO2-coated ZnO NPs
The synthesis of these NPs was reported in detail
elsewhere . 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 K, = 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 2 = 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 150C
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
neutronactivated at the Massachusetts Institute of Technology
(MIT) Nuclear Reactor Laboratory (Cambridge, MA).
Samples were irradiated with a thermal neutron flux of
5 1013 n/cm s 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
SiO2coated 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 () by DLS
using a Zetasizer Nano-ZS (Malvern Instruments,
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
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 4C 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 4C 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
SiO2coated 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 (024 hours, 23 days, 67 days,
910 days, 1314 days, 2021 days, and 2728 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 01 day, 23 days, and 67 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).
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 .
NVK, KMM, RMM, and JDB designed and performed the lung toxicity and
pharmacokinetic studies. TCD performed statistical analyses. PD and GAS
synthesized and characterized the NPs. This manuscript was written by NVK,
RMM, and KMM and revised by JDB, GS, PD and RMM. All authors read,
corrected and approved the manuscript.
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.
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