Comparative toxicity and biodistribution assessments in rats following subchronic oral exposure to copper nanoparticles and microparticles
Lee et al. Particle and Fibre Toxicology
Comparative toxicity and biodistribution assessments in rats following subchronic oral exposure to copper nanoparticles and microparticles
In-Chul Lee 0
Je-Won Ko 0
Sung-Hyeuk Park 0
Na-Rae Shin 0
In-Sik Shin 0
Changjong Moon 0
Hyoung-Chin Kim 1
Jong-Choon Kim 0
0 College of Veterinary Medicine BK21 Plus Project Team, Chonnam National University , Gwangju 61186 , Republic of Korea
1 Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology , ChungBuk 28116 , Republic of Korea
Background: Copper nanoparticles (Cu NPs) have great potential in electronics and biomedical fields because of their efficient thermodynamic and anti-microbial properties. However, their potential toxic effects and kinetic data following repeated exposure are still unclear. Results: The solubility of Cu NPs and Cu MPs was 84.5 and 17.2 %, respectively, in an acidic milieu; however, they scarcely dissolved in vehicle or intestinal milieus. The specific surface area of Cu NPs and Cu MPs was determined to be 14.7 and 0.16 m2/g, respectively. Cu NPs exhibited a dose-dependent increase of Cu content in the blood and tested organs, with particularly high levels of Cu in the liver, kidney, and spleen. Only for liver and kidney increased Cu levels were found in Cu MPs-treated rats. Cu NPs caused a dose-related increase in Cu levels in urine, whereas Cu MPs did not affect the urine Cu levels. Extremely high levels of Cu were detected in the feces of Cu MPs-treated rats, whereas much lower levels were detected in the feces of Cu NPs-treated rats. A comparative in vivo toxicity study showed that Cu NPs caused damages to red blood cells, thymus, spleen, liver, and kidney at ≥200 mg/kg/days, but Cu MPs did not cause any adverse effects even at the highest dose. Conclusions: Overall, the in vivo repeated dose toxicity study of Cu NPs and Cu MPs demonstrated that large surface area and high solubility in physiological milieus could directly influence the toxicological responses and biodistribution of Cu particles when administered orally. Under these experimental conditions, the no-observedadverse-effect levels of Cu NPs and Cu MPs were determined to be 100 and ≥400 mg/kg/day, respectively.
Copper nanoparticles; Copper microparticles; Comparative toxicity; Biodistribution
Nanomaterials are defined as materials that have at least
one dimension in the 1 to 100 nm range, which were
generated by accidentally or engineering. The engineered
nanoparticles (NPs) can be utilized in an
applicationspecific manner by modifying their size, surface
properties, and shape. Thus, in recent years, remarkable
progress has been made in the area of nanotechnology
as evident from its widespread use in textile, electronics,
cosmetics, and foods . The physicochemical
properties of NPs may determinate toxicological behavior in
vivo by making them interact with biological systems
and be absorbed more than bulk chemicals via various
routes [2, 3]. Hence, NPs tend to exhibit quite different
toxicological profiles in vivo compared to the larger
particles [4–7]. Physiological conditions also influence
the interaction between biological systems and NPs and
they can determine the fate and biosafety of NPs .
Recently, increased usage of NPs raises concerns on
their health risks and environmental effects [9–11].
Copper (Cu) is an essential element required for
normal physiological functioning, including drug/xenobiotic
metabolism, carbohydrate metabolism, and the
antioxidant defense system [12, 13]. The general population is
exposed to Cu through inhalation, consumption of food
and water, and dermal contact with air, water, and soil
that contains Cu. The toxicity of Cu and its compounds
has been studied for decades. A report of the available
data has been given in the “Toxicological Profile for
Copper” from the Agency for Toxic Substances and
Disease Registry of the U.S. Public Health Service .
When intake of Cu exceeds the range of biological
tolerance, it can cause adverse effects, including damage to
liver, kidney, immune system, and gastrointestinal
distress . Although the toxic effects of Cu and its
compounds have been studied, several studies reported
gaps concerning the risk caused by Cu in the form of
NPs [7, 15].
Among the various types of nanomaterials,
metalbased NPs are used in the manufacture of hundreds of
commercial products, and their industrial and consumer
applications are expected to increase the chances of their
exposure to the public [10, 11]. In particular, Cu-based
NPs are widely used in a variety of established and
emerging technologies, including catalysts, solar energy
conversion, and antimicrobial agents, because of their
distinct thermophysical properties and antimicrobial
activities [16–20]. Despites widespread applications and
the growing presence of Cu-containing nanoproducts,
there is only limited information on the potential risks
of exposure to Cu-based NPs compared to other NPs
[17, 21]. To date, many reports regarding the in vitro
toxicity of Cu-based NPs are available. Cu-based NPs
induce cytotoxic effects associated with increase of
reactive oxygen species in various cell lines, including
human laryngeal epithelial cells and human alveolar
type-I epithelial cells [22–25]. Cu NPs showed a higher
toxicity than their oxide nanoparticles (CuO NPs) in
HL60 cells . CuO NPs are highly toxic compared to
carbon nanotubes and other metal oxide NPs .
However, only a few studies have explored in vivo toxicity;
these studies revealed biochemical and histological
alterations related to liver, kidney, and spleen after a single
or short-term exposure of Cu NPs [7, 15, 28, 29]. To our
knowledge, there has been no report regarding the
potential effects and biodistribution of Cu NPs following
long-term exposure. Therefore, for further practical
applications, it is necessary to evaluate the in vivo
toxicity of Cu NPs and their biodistribution after
subchronic exposure for the purpose of risk assessment.
Herein, we investigated the in vivo toxicity of Cu NPs
and Cu microparticles (Cu MPs) following 28-day
repeated oral dose in rats by evaluating biochemical,
hematological, and histopathological parameters. The
oral route was used because gastrointestinal exposure to
nanomaterials has the potential for wide public exposure
to higher doses and more frequent ingestion [30, 31].
Further, we investigated absorption, tissue distribution,
and excretion to elucidate the main accumulation sites
and elimination routes. In this work, we report for the
first time, to the best of our knowledge, the in vivo
toxicity and biodistribution of Cu NPs by conducting a
repeated dose toxicity study.
Results and discussion
Physiochemical characterization of Cu NPs and Cu MPs
The physiochemical characteristics of Cu NPs and Cu
MPs are summarized in Table 1. The morphology and
actual size of Cu NPs and Cu MPs were characterized by
transmission electron microscopy (TEM) and scanning
electron microscopy (SEM). The morphology of NPs or
MPs was spherical, and the actual size of individual
particles was found to be 32.7 ± 10.45 nm (300 counts) and
25.3 ± 6.64 μm (100 counts), respectively (Fig. 1a–d).
The purities of Cu NPs and Cu MPs were determined as
98.15 and 99.06 %, respectively, using energy dispersive
X-ray spectroscopy (EDX) analysis on the same images
(data not shown). The specific surface area of Cu NPs
and Cu MPs was measured as 14.7 and 0.16 m2/g,
respectively using the Brunauer-Emmett-Teller (BET)
method. Suspension stability and surface charge reflect
the interaction of NPs with physiological milieus because
of their large surface area/volume ratio [32, 33]. The zeta
potential of the Cu NPs was 25.5 ± 0.8 mV for pH 1.5,
1.32 ± 1.2 mV for pH 6.8, and -6.2 ± 0.2 mV for pH 7.8.
Dynamic light scattering (DLS) measurements revealed
that Cu NPs agglomerate in intestinal (pH 7.8) and
vehicle (pH 6.8) milieus with the hydrodynamic
Table 1 Physiochemical characterization
Hydrodynamic size (nm)b
Surface area (m2/g)c
Zeta potential (mV)d
−6.2 ± 0.2
diameters of 334.8 ± 128.26 and 516.4 ± 116.9 nm,
respectively. However, in an acidic milieu (pH 1.5), the
hydrodynamic diameter of Cu NPs was not determined
because Cu NPs rapidly dissolved in acidic conditions
and particle numbers were not enough to determine
hydrodynamic diameter. These findings suggest the
tendency of Cu NPs to aggregate/agglomerate in
suspension or the intestinal milieu. However, the NP form
presents a greater specific surface area compared with
the same compositional particles of micro size scale that
may cause different biological responses and modulate
their fate in biological systems.
Dissolution of Cu NPs and Cu MPs in physiological
The dissolution of NPs in gastrointestinal fluids may
help predict uptake and blood concentrations. The
release of toxic ions has the potential to influence the
toxicity of NPs [34–36]. Most NPs are rarely soluble at
normal physiological conditions; however, their
dissolution can be accelerated in acidic conditions [37, 38].
We evaluated the solubility of Cu NPs and Cu MPs
(5 mg/mL each) under simulated gastric pH (pH 1.5),
vehicle pH (pH 6.8), and intestinal pH (pH 7.8) after a
24 h incubation. Cu NPs in an acidic milieu showed
bluish color changes within 10 min (Fig. 1e). After
monitoring for up to 24 h, Cu NPs showed 84.5 % solubility
in an acidic milieu, whereas only minimal dissolution
was observed in vehicle (0.65 %) and intestinal (0.09 %)
conditions (Fig. 1f ). In contrast, Cu MPs showed much
lower solubility compared to Cu NPs, even in an acidic
milieu (pH 1.5, 17.2; pH 6.8, 0.04; and pH 7.8, 0.03 %). It
has been reported that Cu NPs have high solubility in an
acidic milieu with high positive charge at zeta potential
analysis [15, 39]. Compared with Cu MPs, the large
specific surface area of Cu NPs can lead to high reactivity
and drastic interaction with hydrogen ions (H+) in a
gastric milieu . Because the pH of gastric fluid is
between 1.5 and 2.0, Cu NPs may be dissolved in the
stomach, and dissociated Cu ions can be absorbed into
the systemic circulation when administered orally. The
dissociation of Cu NPs after gastric emptying is
prohibited in the basic milieu of the small intestine. Thus, the
residence time in the stomach may influence the
dissolution of Cu NPs. Cu NPs quickly dissolved in gastric
milieus, and the undissolved NPs showed a delayed
retention in the stomach after 24 h in mice exposed to
Cu NPs, which may lead to durative interaction and
persistent Cu ion generation in vivo .
Absorption, distribution, and excretion of Cu
The dissolution of NPs in physiological conditions and
the physicochemical features of NPs are both likely to
influence the absorption and biological response of NPs
when administered orally [3, 35, 40]. The Cu levels in
blood reflect the absorption of Cu following oral
exposure of Cu NPs or Cu MPs. Cu levels in the blood of Cu
NPs-treated rats showed a dose-dependent increase and
were 4-fold higher than that of the vehicle control group
(3.33 ± 0.89 μg/g vs. 0.83 ± 0.21 μg/g). In contrast, oral
exposure to Cu MPs did not lead to an increase in blood
Cu levels, which were not different from the vehicle
control group (1.16 ± 0.30 μg/g vs. 0.83 ± 0.21 μg/g) (Fig. 2a).
Exposure to nano-Cu (75 mg/kg) markedly elevates
serum Cu level (3.5-fold higher) compared to a minimal
increase in Cu from the same mass of micro-Cu exposed
mice at 72 h after single oral dose . Consistent with
the results of a previous report, Cu NPs-treated rats
showed 2.9-fold higher levels of Cu than that in rats
exposed to a corresponding high dose of Cu MPs at 24 h
after the last administration. With a delayed retention in
the stomach, the higher levels of Cu in the blood of rats
treated with Cu NPs compared to Cu MPs indicate that
more Cu ions were dissociated from Cu NPs and
absorbed into systemic circulation. Moreover, high and
rapid dissolution of Cu NPs in the gastric milieu
suggests that Cu NPs may be mainly absorbed as ionic
forms rather than nanoparticulate states. Absorbed Cu
ions could enter into systemic circulation and be
distributed in various tissues. Generally, exposure of human
body to Cu NPs can occur through different routes (e.g.,
inhalation, ingestion, injection or physical contact).
Absorbed NPs may interact with biomolecules such as
proteins, nucleic acids, lipids, and even biological
metabolites . Of particular importance is the absorption of
proteins on the surface of NPs and form the NP-protein
complexes, which referred to as the NP-protein corona.
The protein corona alters the size and interfacial
Fig. 1 (See legend on next page.)
(See figure on previous page.)
Fig. 1 Morphology and dissolution of copper nanoparticles (Cu NPs) and copper microparticles (Cu MPs). Transmission electron microscopy (TEM)
and scanning electron microscopy (SEM) images of (a, b) Cu NPs and (c, d) Cu MPs show generally spherical shape. e The color changes of
artificial gastric fluid (AGF; pH 1.5), vehicle (1 % hydroxypropyl methylcellulose, HPMC; pH 6.5), and deionized water (pH 7.8) solution before
(top, t = 0) or after mixing with the Cu NPs and Cu MPs for 10 min (middle, t = 10 min) and 24 h (bottom, t = 24 h). f Dissolution ratios of Cu NPs
and Cu MPs in AGF, vehicle (pH 6.5), and deionized water (pH 7.8) milieus, which simulated in vivo physiological conditions. Cu NPs and Cu MPs
(5 mg/mL) were suspended in above solutions and percentage of dissolution was measured using inductively coupled plasma mass spectrometry
after 24 h of incubation (t = 24 h)
composition of NPs, giving it a new biological identity,
which was determine the agglomeration, uptake,
translocation, accumulation, as well as toxicological response
. Thus, further study to explanation for interaction
between Cu NPs and proteins is needed.
In vivo biodistribution of Cu NPs can provide essential
information regarding their accumulation sites. Cu levels
in the tested organs of Cu NPs-exposed groups showed
a dose-dependent increase when compared with that in
the vehicle control group (Fig. 2b–g). The main organs
with accumulation of Cu were liver (109-fold higher,
2038.1 ± 758.36 μg/g vs. 18.7 ± 5.79 μg/g), kidney
(34fold higher, 950.0 ± 197.58 μg/g vs. 27.7 ± 8.62 μg/g), and
spleen (38-fold higher, 171.7 ± 23.64 μg/g vs. 4.5 ±
1.59 μg/g). Cu levels in nano-Cu treated mice showed a
significant increase in the kidney, whereas micro-Cu did
not elevate Cu levels in the kidney after a single oral
dose (70 mg/kg) . Feng et al.  reported that liver,
Fig. 2 Absorption, biodistribution, and excretion of copper nanoparticles (Cu NPs) and copper microparticles (Cu MPs) following 4 weeks-repeated
oral dose. a Systemic absorption of Cu was determined in blood Cu contents. b–g Biodistribution of Cu was determined in liver, kidney, spleen, lung,
heart, and brain. The contents of Cu in (h) feces and (i) urine were used to determine excretion of Cu. The bar graphs present Cu contents of Cu
NPs- and Cu MPs-treated rats at dose levels of 0, 100, 200, and 400 mg/kg/day, respectively (Cu NPs, black bars; Cu MPs, white bars). Values are
presented as mean ± SD (n = 10). *, ** P < 0.05, P < 0.01 versus vehicle control
spleen, lung, and kidney appear to be the major organs for
accumulation of Cu sulfide nanoplates after intravenous
injection. Thus, the biodistribution of Cu indicated that
Cu from Cu NPs was mainly distributed in the liver,
kidney, and spleen. Further, accumulated Cu in these organs
can be a toxic reservoir based on their toxic potential.
However, equivalent dose levels of Cu MPs are not only
lower than those of Cu NPs, but also showed no
doseresponse increase in the tested organs, except in the liver
(83.1 ± 20.94 μg/g) and kidney (46.7 ± 10.14 μg/g). This
low distribution was due to the minimal absorption rate
of Cu MPs.
The excretion of Cu was consistent with the absorption
and distribution patterns of Cu NPs or Cu MPs. The levels
of Cu in urine from the Cu NPs-treated group showed a
significant increase with clear dose-response when
compared to that in the vehicle control group (12.8-fold
higher, 6.31 ± 1.59 μg/g vs. 0.49 ± 0.21 μg/g) (Fig. 2h). In
contrast, only trace Cu levels were observed in the urine
of the Cu MPs-treated groups (0.60 ± 0.19 μg/g vs. 0.49 ±
0.21 μg/g). Cu levels in the feces of Cu NPs treated rats
showed clear dose-response when compared to that in the
vehicle control group (115-fold higher, 26.2 ± 8.41 mg/g
vs. 0.2 ± 0.09 mg/g). Cu MPs treated rats showed
extremely high levels of Cu in the feces, which were 2.8-fold
higher than that in Cu NPs treated-rats (73.7 ± 16.98 mg/g
vs. 26.2 ± 8.41 mg/g) (Fig. 2i). Ingested Cu ions are mainly
metabolized in the liver, and the major excretory route is
via liver/bile . The dissociation of Cu NPs after gastric
emptying is prohibited in the basic milieu of the small
intestine, and then unabsorbed NPs are excreted as feces
. Thus, extremely high levels of Cu in feces suggest
that most of the absorbed Cu, dissociated from Cu NPs,
or unabsorbed Cu NPs were predominantly excreted
through feces; small amounts were excreted via the
kidney/urine route. Most of the unabsorbed Cu MPs were
also eliminated from gastro-intestinal tracts via the feces.
Clinical signs, body weights, and food consumption
Manifestations of toxicity, including anorexia, diarrhea,
lethargy, and body weight loss, were observed in rats
treated with Cu NPs ; these manifestations were
similar to the effects of excessive Cu compound treatment
[45–47]. In this study, treatment-related clinical signs
observed in the high dose group of Cu NPs were consistent
with toxic manifestations observed in a previous study.
The body weight and the amount of food consumption in
the high dose group of Cu NPs decreased significantly
during the test period (Fig. 3a and b). In contrast, only test
article-colored feces were observed in the high dose group
of Cu MPs. The body weight and food consumption of the
Cu MPs-treated groups showed no significant changes,
even at the high dose, compared to that in the vehicle
control group (Fig. 3c and d).
Urinalysis, serum biochemistry, and hematology
It has been reported that oral exposure to Cu NPs cause
imbalance of acid and base by interacting with H+,
resulting in metabolic alkalosis . In a chronic
metabolic alkalosis state, bicarbonate excretion ceased and
led to a state of paradoxical aciduria . The decreased
urine pH observed in the high dose group of Cu NPs
may be due to chronic metabolic alkalosis caused by
sub-chronic exposure to Cu NPs (Additional file 1: Table
S1). Other urinalysis parameters, including urine protein
(PRO), occult blood (OB), leukocytes (LEU), specific
gravity (SG), ketone body (KET), and nitrite (NIT), were
increased significantly in the high dose group of Cu NPs.
Hematological findings revealed that repeated exposure
to Cu NPs resulted in red blood cell (RBC) destruction,
which was characterized by a reduction of RBC,
hemoglobin (HB), hematocrit (HCT), mean corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH),
and mean corpuscular hemoglobin concentration
(MCHC), as well as an increase in reticulocytes (RET)
(Table 2). This interpretation was well supported by
increased yellow pigmentation in the spleen. Chronic Cu
intoxication causes hemolytic anemia with diverse
hematological changes, including decreased RBC, HB,
HCT, MCV, MCH and white blood cells (WBC) in
rodents [46, 49, 50], which were consistent with the
results of this study. In addition, the changes in the
hematology indicate a microcytic anemia that generally
observed with iron deficiency. Elevated levels of Cu
levels have been shown to competitively inhibit iron
absorption and utilization and to be correlated with
diminution in serum iron levels [51, 52]. In the
differential WBC count, a dose-dependent decrease in the
percentage of lymphocytes (LYM) implied that Cu NPs
might have adverse effects on the immune system, which
was well correlated with the reduction of cellularity seen
in the thymus and spleen (Fig. 4). The increased
percentages of neutrophils (NEU) and monocytes
(MON) were thought to be related to the inflammatory
response of the affected organs and the decreased LYM
percentage (Table 2). These results indicated that Cu
NPs might affect red blood cells and immune organs
(spleen and thymus). As reported previously, Cu NPs
caused liver and kidney damages with biochemical
alterations, including increased aspartate aminotransferase
(AST), alanine aminotransferase (ALT), total bilirubin
(TBIL), blood urea nitrogen (BUN), and creatinine
(CRE) [28, 29]. With obvious changes in urinalysis
parameters, Cu NPs treated rats showed a dose-related
response in the increment of serum BUN, CRE, AST,
ALT, TBIL, alkaline phosphatase (ALP), and lactate
dehydrogenase (LDH), as well as the decrease of
triglyceride (TG) and total protein (TP) with electrolytes
disturbance (Table 3). These findings on the consequence
Fig. 3 Body weight changes and food consumption of copper nanoparticles (Cu NPs)- and copper microparticles (Cu MPs)-treated rats following
4 weeks-repeated oral dose. Repeated oral dose toxicity of Cu NPs and Cu MPs was assessed by determining (a, c) Body weight changes and
(b, d) food consumption of Cu NPs- and Cu MPs-treated rats. Values are presented as mean ± SD (n = 10). *, ** P < 0.05, P < 0.01 versus vehicle control
of Cu NPs exposure proved that Cu NPs cause
substantial damage to the liver and kidney. Collectively, Cu
dissociated from Cu NPs mainly distributed into liver,
kidney, and spleen, which caused obvious functional and
structural damage. However, the parameters of
urinalysis, serum biochemistry, and hematology were not
affected by repeated exposure to Cu MPs (Additional file
1: Table S1, Tables 2 and 3).
Histopathology and organ weight changes
Our findings confirmed previous studies showing that a
single or short-term oral exposure of Cu NPs induces
severe damage to the kidney and liver [7, 15, 28, 29]. The
major histopathological findings, including mononuclear
cell infiltration, dilated sinusoid, degenerated or
binucleated hepatocytes in the liver, dilated tubules, cell
debris or pink or purple-colored casts in tubules,
degenerated tubular cells, and inflammatory cell infiltration in
the kidney, were observed in the rats treated with Cu
NPs (Table 4 and Fig. 5). Excessive Cu intake results in
impairment of both cellular and humoral immune
responses . Recently, Cu (II) chloride causes
apoptosis of splenocytes and thymocytes, especially CD4+ T
cell death [54, 55]. Exposure to nano-Cu caused
dwindling of splenic units and reduction of lymphocytes .
In rats treated with the high dose of Cu NPs, the spleen
exhibited atrophic white pulp, decreased number of
follicles and cellularity, and yellow pigmentation, and the
thymus displayed disrupted demarcation of
medulla/cortex, decreased cellularity, and cytoplasmic vacuolation,
which was consistent with the previous studies (Fig. 4).
In particular, an apparent atrophic change of follicles (B
cell area) and periarteriolar lymphoid sheath (T cell area)
in the spleen and decreased cellularity in the cortex of the
thymus were in agreement with the hematological findings
of our study and the results of previous studies [7, 54, 55].
The changes in organ weight included increased kidney
weight and decreased liver, spleen, and thymus weights in
the high dose group of Cu NPs (Additional file 1: Table
S2). These findings were of toxicological significance,
because they were well supported by correlated biochemical,
hematological, and histopathological changes. In contrast,
Fig. 4 Histopathological changes in spleen and thymus of rats treated with copper nanoparticles (Cu NPs) and copper microparticles (Cu MPs)
following 4 weeks-repeated oral dose. The rats treated with Cu NPs at 400 mg/kg/day showed moderate to severe degree of atrophic white
pulp, decreased number of follicles and cellularity, and yellow pigmentation in spleen, disrupted demarcation of medulla/cortex, decreased
cellularity of medulla/cortex, and cytoplasmic vacuolation in thymus. There were no changes in spleen and thymus from rats treated with
Cu MPs. Hematoxylin and eosin stain
Cu MPs-treated rats did not show obvious changes in
histopathology and organ weights even at the high dose
(Figs. 4, 5, and Additional file 1: Table S3). The remarkable
reduction in prostate and seminal vesicle weights was
observed at the high dose of Cu NPs. Chattopadhyay et al.
 demonstrated that male rats treated with copper
chloride at 2 mg/kg/day intraperitoneally for 26 days
displayed adverse effects on testicular spermatogenesis and
development of reproductive organs. Test
substancerelated stress in the toxicity study causes a decrease in the
weights of reproductive organs, including epididymides,
seminal vesicles, and prostates, but not in the testes .
Thus, further study is needed to determine the potential
reproductive/developmental toxicity of Cu NPs because it
is unclear whether decreased reproductive organ weights
are related to the anti-androgenic effects of Cu dissociated
from Cu NPs or to the stress-response phenomenon
during the toxicity study.
Exposure to Cu NPs can occur through various routes
(e.g., inhalation, ingestion, injection or physical contact).
When administered orally, high solubility in the acidic
milieu implies that Cu NPs can be dissociated into Cu
ions in gastric pH conditions. Further, higher Cu levels
in blood and tissues in rats treated with Cu NPs than Cu
MPs indicate that absorbed Cu ions were distributed via
circulation and accumulated in various tissues, which
can be a toxic reservoir. Consistent with the above
results, the toxicological study revealed that Cu NPs
were more toxic than MPs of the same chemical
composition at the same mass. The dissolution of Cu NPs
may have an important role in their toxicity [7, 15]. Cu
ion overload caused by excessive Cu NPs administration
can cause damage to their accumulation sites, especially
liver, kidney, and spleen [15, 43]. Additionally, the
toxicity of CuO NPs was largely explained by soluble Cu
ions . Thus, the differences in dissolution play a
crucial role in the gap of toxicological responses between
Cu NPs and Cu MPs. In addition, the biopersistence of
NPs influences long-term toxicity and is considered to
be an important parameter needed for the risk
assessment of NPs . Therefore, further studies will be
necessary to investigate whether the toxic responses of Cu
NPs observed in this study are transient or persistent
Table 4 Histological changes in male rats treated with Cu NPs and MPs following 28 days-repeated oral dose
Mononuclear cell infiltration
Cell debris in tubules
Purple-colored casts in tubules
Degenerated tubular cells
Inflammatory cell infiltration
Decreased number of follicles
Decreased cellularity in medulla/cortex
Disrupted demarcation of medullar/cortex
a-, normal; +, mild; ++, moderate; +++, severe; (), number of case
Test chemicals and preparation of test chemicals
Cu NPs (CAS No. 7440-50-8; 99.8 % purity) and Cu MPs
(99 % purity) were purchased from SkySpring
Nanomaterials (Houston, TX, USA) and Sigma-Aldrich (St. Louis,
MO, USA), respectively. The information of particle size
(measured by TEM) of Cu NPs and Cu MPs was 25 and
14–25 μm, respectively. Hydroxypropylmethylcellulose
(HPMC, suspending vehicle) was purchased from
Sigma-Aldrich. All other chemicals were of the highest grade
commercially available. Test chemicals were dispersed
into 1 % HPMC solution (w/v) with Milli-Q water. Particle
suspensions were made fresh every day and prepared by
ultrasonic dispersion (VCX130, Vibra Cell Sonics &
Materials, Newtown, CT, USA) on ice for 20 min (130 W,
20 kHz, pulse 59/1) in agreement with recommendations
by Taurozzi et al. .
Physicochemical characterization and solubility of Cu NPs
and Cu MPs
The primary size and morphology were measured by
TEM (JEM-2100 F, JEOL, Tokyo, Japan) operating at
150 kV and SEM (Zeiss EVO-MA10, Carl Zeiss SMT,
Cambridge, UK) operating at 15 kV. The purity of NPs
and MPs was determined by EDX analysis on the same
images from TEM (JEM-2100 F TEM equipped with
X-MaxN 150 mm2 silicon drift detector; Oxford
Instruments, UK). The samples for TEM were deposited
on carbon-coated nickel grids and were air-dried
overnight before analysis. The average size was obtained by
measuring at least 100 particles using an image analyzer
program (JEOL). The samples for SEM were dispersed
on double-sided adhesive carbon tape onto an aluminum
SEM stub, and then dusted to release loose particles.
The specific surface area of NP and MP powder was
measured by the nitrogen (N2) absorption based on the
multipoint BET method using an ASAP2020
(Micromeritics, Norcross, GA, USA). In the solubility study, Cu
NPs and Cu MPs were incubated under three
physiological conditions: acidic conditions using artificial
gastric fluid (AGF, pH 1.5), vehicle (1 % HPMC, pH 6.5),
and basic conditions using deionized water (pH 7.8) for
24 h. AGF was prepared according to the previously
described method . In brief, 1.0 g NaCl (Affymetrix,
Santa Clara, CA, USA) and 1.6 g pepsin (Sigma-Aldrich)
were dissolved in 500 mL of DW and the pH of AGF
was adjusted to 1.5 using 2 N HCl (Sigma-Aldrich).
Deionized water (pH 7.8) was used to simulate the basic
Fig. 5 Histopathological changes in liver and kidney of rats treated with copper nanoparticles (Cu NPs) and copper microparticles (Cu MPs)
following 4 weeks-repeated oral dose. Liver from rats treated with Cu NPs at 400 mg/kg/day showed mononuclear cell infiltration (closed arrows),
dilated sinusoid (open arrows), degenerated hepatocytes (vacuolation; open arrowheads), and binucleated hepatocytes (closed arrowheads). The rats
treated Cu MPs at 400 mg/kg/day showed only dilated sinusoid. Kidneys from rats treated with Cu NPs showed dilated tubules (closed arrows), cell
debris in tubules (closed arrowheads), pink- or purple-colored cast in tubules (open arrows), degenerated tubular cells (open arrowheads), and
inflammatory cell infiltration (asterisks). The rats treated with Cu MPs did not show changes in kidney structure. Hematoxylin and eosin stain
condition. Cu NPs and Cu MPs (5 mg/mL) were
incubated in the above solutions for 24 h. NP- or MP-free
supernatants were collected by three rounds of
centrifugation at 150,000 × g for 30 min . The samples
weighing about 1 g were placed in 55 mL microwave
digestion vessels and digested with 10 mL of
concentrated nitric acid and 1 mL of 30 % H2O2 overnight. The
samples were heated in a microwave digestion system
(ETHOS One; Milestone, Sorisole, Italy). The microwave
digestion system condition was 40 °C for 1 min, 100 °C
for 20 min, and 170 °C for 2 h to remove the remaining
nitric acid. Afterward, the samples were allowed to cool.
After the samples were completely digested and
colorless, the remaining solutions were diluted with 2 % nitric
acid. The degree of ionization was evaluated by
determining Cu63. Cu analysis of each sample was carried out
using an ICP-MS method (NexION 300X, Perkin Elmer,
Waltham, MA, USA). Cu standard solutions for ICP-MS
calibration were prepared at concentrations of 5, 10, 50,
and 100 ng/g. The fraction of solubilized Cu ions was
calculated and expressed as a percentage by dividing the
mass of Cu ions by the initial mass of Cu in Cu NPs or
Cu MPs. The hydrodynamic diameter and zeta potential
of NPs was measured by the DLS method using
ELS8000 (Otsuka Electronics, Tokyo, Japan) equipped with a
633 nm laser under above simulated physiological
Animal handling and environmental conditions
Male Sprague–Dawley rats aged 7 weeks were obtained
from a specific pathogen-free colony at Samtako Co.
(Osan, Republic of Korea). The animals were acclimated
for 1 week before starting the experiments. The body
weight of the animals at the beginning of the study was
(220 ± 19 g). Two rats per stainless wire mesh cage were
housed in a room maintained at a temperature of 23 ±
3 °C and a relative humidity of 50 ± 10 % with artificial
lighting from 08:00 to 20:00 and with 13 to 18 air
changes per hour. Rats were provided tap water
sterilized by ultraviolet irradiation and commercial rodent
chow (Samyang Feed, Wonju, Korea) ad libitum. The
Institutional Animal Care and Use Committee of
Chonnam National University approved the protocols for the
animal study (approval number: CNU
IACUC-YB-20141), and the animals were cared for in accordance with
the Guidelines for Animal Experiments of Chonnam
Experimental protocols and dose selection
The study was carried out in compliance with the
Organization for Economic Cooperation and
Development (OECD) test guideline TG407 for the testing of
chemicals . As males are more susceptible to the
toxic effects of Cu NP than females [7, 15], we utilized
male Sprague-Dawley rats for the in vivo toxicity study.
A total of 80 healthy male rats were randomly assigned
to eight experimental groups (n = 10). The test articles
were administered by oral gavage to rats at dose levels of
100, 200, and 400 mg/kg/day, and two vehicle control
groups were received 1 % HPMC alone. The
experimental doses were selected based on the results of a
preliminary dose-range finding study. Three groups of five
male rats were exposed to Cu NPs via oral
administration at doses of 50, 200, and 800 mg/kg/day for 2 weeks.
At 800 mg/kg/day, the male rats displayed obvious
general toxicity, such as suppressed body weight gain,
decreased food intake, and various clinical signs, as well
as death. At 200 mg/kg/day, Cu NPs produced a mild
decrease in body weight gain and food intake. There
were no treatment-related effects on clinical signs, body
weights, or food intake at 50 mg/kg/day. On the basis of
these results, 400 mg/kg/day was used as the high-dose,
and the doses of 200 and 100 mg/kg/day were selected
as mid- and low-doses, respectively, using a scaling
factor of × 2. The dose levels of Cu MPs were also
selected as 100, 200, and 400 mg/kg/day equivalent to
the dose levels of Cu NPs for comparing the toxic effects
and biodistribution. The administration volume (10 mL/
kg body weight) of Cu NPs and Cu MPs was calculated
based on the body weight of the individual animal
measured each week. All animals were observed twice daily
(before and after treatment) throughout the study period
for any clinical signs of toxicity and mortality. The body
weight of each rat and the level of food consumption
were measured prior to the beginning of treatment and
once a week during the experimental period. The
amounts of food were calculated before they were
supplied to the cages, and the remnants were measured the
next day in order to calculate the difference, which was
regarded as daily food consumption (g/rat/day). The
weight gain was calculated by body weight on day 28 –
body weight on day 0. The animals were sacrificed at
24 h (test day 28) after last administration of Cu NPs or
Urinalysis, hematology, and clinical chemistry
To collect urine and feces, six animals per groups were
assigned to a metabolic cage for 6 h during the last week
of the test period (test day 21). Urinalysis was carried
out with fresh urine (2 mL per rats) within 1 h after
collection to determine the urine levels of SG, pH, PRO,
LEU, KET, OB, NIT, glucose, bilirubin, and urobilinogen
by using the Multistix 10SG reagent strips and the
Clinitek Status analyzer (Bayer Healthcare, Leverkusen,
Germany). To collect blood samples, the animals
underwent fasting overnight before scheduled necropsy (test
day 28). During the scheduled necropsy, the blood
samples (approximately 4 mL) were collected from the vena
cava under carbon dioxide anesthesia. Approximately
1 mL of blood was collected in CBC bottles containing
EDTA-2 K and analyzed within 1 h using an automatic
hematology analyzer (Bayer ADVIA 120 Hematology
Analyzer System, Leverkusen, Germany). Samples were
analyzed for RBC (erythrocyte), HB, HCT, MCV, MCH,
MCHC, platelets, WBC (leukocyte) count and the
differential count of WBC. A portion of the blood (about
3 mL) was placed into tubes for serum separation and
incubated at room temperature within 90 min. Serum
samples were collected by centrifugation at 5000 × g for
10 min and evaluated with a blood chemistry
autoanalyzer (Dri-chem 4000i, Fujifilm Co., Tokyo, Japan) for the
following: AST, ALT, ALP, TP, BUN, CRE, TG, TBIL,
glucose, albumin, total cholesterol, chloride, sodium, and
potassium within 3 h after blood collection. After
analysis of urine and blood, remaining urine and blood
samples were stored immediately at −80 °C before Cu
Organ weights and histology
All organs were removed, weighed, and examined for
macroscopically visible lesions. The weights of the
following organs were measured: brain, thymus, heart,
lung, liver, spleen, kidneys, adrenal glands, testes,
seminal vesicles, prostates, and epididymides. The
histopathological evaluation of organs and tissues was
performed by fixing in a 10 % neutral-buffered formalin
solution for 1 week. The tissues were stained with
hematoxylin and eosin for microscopic examination. All
observations were made manually in a blinded manner
using a light microscope with × 5, ×10, ×20, and × 40
objective lenses and a × 100 oil immersion lens.
In vivo absorption, distribution, and excretion of Cu from
Cu NPs or Cu MPs
After The concentration of Cu in blood, tested organs
(liver, kidney, spleen, heart, lung, and brain), urine and
feces was determined by ICP-MS. Absorption of Cu in
the Cu NP-treated rats was determined by using blood
samples. To evaluate tissue distribution, tissue samples
from the liver (left part of median lobe), spleen (left
half ), kidney (part of left kidney), lung (part of left lobe),
heart (left half ), and brain (left hemisphere) were
obtained and weighed. Excretion of Cu was measured in
urine and feces. The tissues, feces (about 0.3 g) or blood
and urine (about 2 mL) samples were weighed and
digested with concentrated nitric acid and 30 % H2O2
overnight. The samples were heated in a microwave
digestion system (Milestone) at 170 °C to remove the
remaining nitric acid until the samples were completely
digested and till they became colorless. Finally,
remaining solutions were diluted with 2 % nitric acid to
a final acid concentration of 8–12 %. All samples were
analyzed in duplicates for elemental Cu concentration
(Cu63) using ICP-MS methods (Perkin Elmer, MA,
The numerical data were presented as means ± standard
deviations (SD), and all statistical comparisons were
analyzed by one-way analysis of variance (ANOVA) followed
by Dunnett’s multiple comparison test. The urinalysis
data were rank-transformed and analyzed by the
nonparametric Kruskal–Wallis H-test. If a statistically
significant difference was observed between groups, the
Mann–Whitney U-test was used to identify the groups
that were significantly different from the vehicle control
group. A P value of < 0.05 was considered significant.
Statistical analyses were performed using the GraphPad
InStat v.3.0 (GraphPad Software, Inc., CA, USA).
We described the in vivo toxicity and biodistribution of
Cu NPs and Cu MPs following repeated oral exposure.
The greater reactive surface area originating from its
small size can lead to high reactivity, subsequently
inducing rapid dissolution of Cu NPs in an acidic milieu.
Thus, Cu NPs may be readily dissociated into their ionic
forms in stomach compared with micro-size particles of
the same composition. This is demonstrated by the Cu
levels in blood and tested organs after Cu NP or Cu MP
exposure. In vivo repeated dose toxicity study
demonstrated that high surface area and high solubility could
contribute to the toxicological responses of particles by
causing Cu ion overload in their accumulation sites. Cu
NPs affected RBC, liver, kidney, and immune organs
(spleen and thymus), as well as male accessory
reproductive organs at ≥ 200 mg/kg/day, whereas Cu MPs did
not cause obvious changes at ≤ 400 mg/kg/day. Under
these experimental conditions, the
no-observed-adverseeffect level of Cu NPs and Cu MPs was considered to be
100 mg/kg/day and ≥400 mg/kg/day, respectively. In
light of our findings, dissolution in physiological milieus
influences absorption and biodistribution and acts as a
determination factor in the toxic responses of particles
in vivo when administered orally.
Additional file 1: Table S1. Urinalysis findings in male rats treated with
Cu NPs or Cu MPs following 28 days-repeated oral dose. Table S2.
Absolute and relative organ weights in male rats treated with Cu NPs
following 28 days-repeated oral dose. Table S3. Absolute and relative organ
weights in male rats treated with Cu MPs following 28 days-repeated oral
dose. (DOCX 47 kb)
AGF: Artificial gastric fluid; ALP: Alkaline phosphatase; ALT: Alanine
aminotransferase; AST: Aspartate aminotransferase; BET:
Brunauer-EmmettTeller; BUN: Blood urea nitrogen; CRE: Creatinine; Cu MPs: Copper
microparticles; Cu NPs: Copper nanoparticles; CuO NPs: Copper oxide
nanoparticles; DLS: dynamic light scattering; EDX: Energy dispersive X-ray
spectroscopy; H+: Hydrogen ion; HB: Hemoglobin; HCO3−: Bicarbonate;
HCT: Hematocrit; HPMC: Hydroxypropylmethylcellulose; ICP-MS: Inductively
coupled plasma mass spectrometry; LDH: Lactate dehydrogenase;
LEU: Leukocytes; MCH: Mean corpuscular hemoglobin; MCHC: Mean
corpuscular hemoglobin concentration; MCV: Mean corpuscular volume;
NIT: Nitrite; OB: Occult blood; PRO: Protein; RBC: Red blood cell;
RET: Reticulocytes; SD: Standard deviation; SEM: Scanning electron
microscopy; SG: Specific gravity; TBIL: Total bilirubin; TEM: Transmission
electron microscopy; TG: Triglyceride; TP: Total protein;
ICL and JCK designed the experimental approach and wrote the manuscript.
JWK, SHP, and ISS refined the experimental protocols. ICL, NRS, and JHK
performed the animal experiments, physicochemical analyses of particle
suspensions. ICL, JWK, and JHK performed the urinalysis, hematology, and
serum biochemistry. NRS and SHP performed preparation of tissue samples
for ICP-MS analysis. Statistical analyses were done by ICL, JWK, and CM. CM,
HCK, and JCK refined manuscript. All authors reviewed and approved the
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