Size-dependent in vivo toxicity of PEG-coated gold nanoparticles.

Abstract

Gold nanoparticle toxicity research is currently leading towards the in vivo experiment. Most toxicology data show that the surface chemistry and physical dimensions of gold nanoparticles play an important role in toxicity. Here, we present the in vivo toxicity of 5, 10, 30, and 60 nm PEG-coated gold nanoparticles in mice.
Animal survival, weight, hematology, morphology, organ index, and biochemistry were characterized at a concentration of 4000 μg/kg over 28 days.
The PEG-coated gold particles did not cause an obvious decrease in body weight or appreciable toxicity even after their breakdown in vivo. Biodistribution results show that 5 nm and 10 nm particles accumulated in the liver and that 30 nm particles accumulated in the spleen, while the 60 nm particles did not accumulate to an appreciable extent in either organ. Transmission electron microscopic observations showed that the 5, 10, 30, and 60 nm particles located in the blood and bone marrow cells, and that the 5 and 60 nm particles aggregated preferentially in the blood cells. The increase in spleen index and thymus index shows that the immune system can be affected by these small nanoparticles. The 10 nm gold particles induced an increase in white blood cells, while the 5 nm and 30 nm particles induced a decrease in white blood cells and red blood cells. The biochemistry results show that the 10 nm and 60 nm PEG-coated gold nanoparticles caused a significant increase in alanine transaminase and aspartate transaminase levels, indicating slight damage to the liver.
The toxicity of PEG-coated gold particles is complex, and it cannot be concluded that the smaller particles have greater toxicity. The toxicity of the 10 nm and 60 nm particles was obviously higher than that of the 5 nm and 30 nm particles. The metabolism of these particles and protection of the liver will be more important issues for medical applications of gold-based nanomaterials in future.

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International Journal of Nanomedicine 2011:6 2071–2081
International Journal of Nanomedicine
Size-dependent in vivo toxicity of PEG-coated
gold nanoparticles
Xiao-Dong Zhang
Di Wu
Xiu Shen
Pei-Xun Liu
Na Yang
Bin Zhao
Hao Zhang
Yuan-Ming Sun
Liang-An Zhang
Fei-Yue Fan
Institute of Radiation Medicine,
Chinese Academy of Medical Sciences
and Peking Union Medical College,
Tianjin Key Laboratory of Molecular
Nuclear Medicine, Tianjin, People’s
Republic of China
Correspondence: Xiao-Dong Zhang;
Fei-Yue Fan
Institute of Radiation Medicine, Chinese
Academy of Medical Sciences and Peking
Union Medical College, Tianjin 300192,
People’s Republic of China
Tel/fax +86 22 8568 3173
Email xiaodongzhang@yahoo.cn;
feiyfan@gmail.com
Background: Gold nanoparticle toxicity research is currently leading towards the in vivo
experiment. Most toxicology data show that the surface chemistry and physical dimensions of
gold nanoparticles play an important role in toxicity. Here, we present the in vivo toxicity of
5, 10, 30, and 60 nm PEG-coated gold nanoparticles in mice.
Methods: Animal survival, weight, hematology, morphology, organ index, and biochemistry
were characterized at a concentration of 4000 µg/kg over 28 days.
Results: The PEG-coated gold particles did not cause an obvious decrease in body weight or
appreciable toxicity even after their breakdown in vivo. Biodistribution results show that 5 nm
and 10 nm particles accumulated in the liver and that 30 nm particles accumulated in the spleen,
while the 60 nm particles did not accumulate to an appreciable extent in either organ. Transmis-
sion electron microscopic observations showed that the 5, 10, 30, and 60 nm particles located in
the blood and bone marrow cells, and that the 5 and 60 nm particles aggregated preferentially in
the blood cells. The increase in spleen index and thymus index shows that the immune system
can be affected by these small nanoparticles. The 10 nm gold particles induced an increase in
white blood cells, while the 5 nm and 30 nm particles induced a decrease in white blood cells
and red blood cells. The biochemistry results show that the 10 nm and 60 nm PEG-coated gold
nanoparticles caused a significant increase in alanine transaminase and aspartate transaminase
levels, indicating slight damage to the liver.
Conclusion: The toxicity of PEG-coated gold particles is complex, and it cannot be concluded
that the smaller particles have greater toxicity. The toxicity of the 10 nm and 60 nm particles was
obviously higher than that of the 5 nm and 30 nm particles. The metabolism of these particles
and protection of the liver will be more important issues for medical applications of gold-based
nanomaterials in future.
Keywords: gold nanoparticles, in vivo, toxicity, size
Introduction
Gold-based nanomaterials have been focused on in diverse biomedical applications
due to their unique surface chemistry and optical properties.1,2 Because of their
strong and size-tunable surface plasmon resonance, fluorescence, and easy surface
functionalization, gold-based nanomaterials have been widely used in biosensors,
cancer cell imaging, photothermal therapy, and drug delivery.3–9 Recently, gold
nanoparticles have been suggested as a novel radiosensitizer technique in radiotherapy,
because their strong photoelectric absorption and secondary electron caused by gamma
or X-ray irradiation can accelerate DNA strand breakage.10–12 Therefore, it was neces-
sary to investigate the toxicity of these materials.
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The toxicity of gold-based nanomaterials has been
reported in recent years.13–18 In general, their toxicity depends
on particle size and surface coating. The biosafety of metallic
gold is well known. Gold was used in vivo in the 1950s, but
functionalized gold particles showed obvious cytotoxicity.19
Recently, the cytotoxicity of gold nanoparticles in human
cells was studied in detail, and the results showed that
these nanoparticles were nontoxic up to 250 mM, while
ionic gold showed obvious cytotoxicity at 25 mM.20 Similar
results were also reported recently using gold nanoparticles
in radiotherapy experiments in vitro.21–28 However, in vitro
systems cannot replicate the complexity of an in vivo
system or provide meaningful data about the response of a
physiological system to an agent. A case in point is carbon
nanotubes.29 Manna et al29 found them to be toxic in vitro,
whereas Schipper et al found no significant toxicity in vivo.30
Similarly, Sayes et al found that in vivo toxicology results for
fullerenes were very different from in vitro data.31
Therefore, the in vivo toxicity of gold nanoparticles is
important. Very recently, size-dependent organ distribution
of gold nanoparticles has been investigated, and the results
showed that small gold nanoparticles of 5–15 nm had a wider
organ distribution than that of larger gold nanoparticles of
50–100 nm, with the liver and spleen being the dominant
targeted organs.32–37 Meanwhile, it has been found that gold
nanoparticles with a long blood circulation time can accumu-
late in the liver and spleen, and have obvious effects on gene
expression.38–40 Furthermore, the toxicity and biodistribution
of polyethylene glycol (PEG)-coated gold nanoparticles have
also been investigated, and the results showed that 20 nm
gold nanoparticles coated with TA-terminated PEG5000 were
the most stable, and had the lowest toxicity among the 20,
40, and 80 nm gold nanoparticles coated with TA-terminated
PEG5000.36,38,41,42 However, the PEG-coated gold nanoparticles
had a very low zeta potential which was significantly different
from that of naked gold nanoparticles. For example, it has
been reported that 13.5 nm PEG-coated gold nanoparticles
can damage the liver and that 100 nm particles can be cleared
partially.36,38 Therefore, it is desirable to clarify what size of
PEG-coated gold particle is safe.
In previous work, we evaluated the in vitro and in vivo
toxicity of naked gold nanoparticles.43,44 Here, we investigate
the in vivo toxicity of 5, 10, 30, and 60 nm PEG-coated gold
nanoparticles by evaluating biodistribution, blood chemistry,
biochemistry, and characteristics on transmission electron
microscopy. The outcome of this research will determine
which size would be suitable for photothermal therapy
and radiotherapy. Furthermore, the relevant hematology
parameters of organs were analyzed.
Materials and methods
Fabrication of gold nanoparticles
The gold nanoparticles were fabricated following the
classical method devised by Turkevich et al.45 A 100 mL
aliquot of 0.01% chloroauric acid (HAuCl4⋅4H2O) solution
was refluxed, and 0.8, 1.3, and 5 mL of 1% sodium citrate
solution was then added to the boiling solution. Reduction
of gold ions by the citrate ions was complete after 5 minutes,
and the solution was then boiled for a further 30 minutes
and left to cool to room temperature. This method yielded
spherical particles with an average diameter of about 10, 30,
and 60 nm. The small-sized gold nanoparticles of 5 nm were
reduced by NaBH4. Subsequently, PEG-SH (Sigma-Aldrich,
St Louis, MO) 1 mg was mixed with the gold nanoparticles
and stirred for 1 hour to modify the surface of the gold
nanoparticles covalently with PEG. The resulting PEG-coated
gold nanoparticles were collected by centrifugation at 16,000
rpm for 30 minutes and washed twice with distilled water.
The PEG-coated gold nanoparticle solution was stored at
4°C in order to prevent aggregation. Although the mean size
varied slightly for each preparation, the size distribution was
always found to be less than 20% of the standard deviation
shown in Figure 1.
The zeta potential for the gold nanoparticles was
determined using a Nano Zetasizer particle analyzer (Malvern
Instruments, Worcestershire, UK). Data in the phase analysis
were acquired in the light scattering mode at 25°C, and sample
solutions were prepared by diluting the gold nanoparticles in
10 mM phosphate-buffered saline solution (pH 7.0). The zeta
potential of the 5, 10, 30, and 60 nm naked gold particles was
−22.1, −28.23, −26.7, and −12.27 mV, respectively. Due to
citric acid coating and the negative surface charge, PEG-SH
coating decreased the zeta potential to −2.96, −1.55, −1.97,
and −1.65 mV, respectively.
The size and morphology of the gold nanoparticles were
analyzed by transmission electron microscopy using a Hitachi
HF-2000 field emission high-resolution transmission electron
microscope operating at 200 kV. The optical absorption
spectra in a wavelength range of 200–850 nm was measured
in a 5 mL glass cuvette using a DU800 spectrometer.
In vivo study design
The animals are purchased, maintained, and handled accord-
ing to protocols approved by the Institute of Radiation
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Medicine, Chinese Academy of Medical Sciences. Male
mice were obtained from the Institute of Radiation Medicine
laboratory at 11 weeks of age and were housed on a 12-hour
light/12-hour dark cycle, and had access to food and water
ad libitum. In the size-dependent toxicology experiment,
30 mice were randomly divided into five groups (six mice in
each group) comprising one control group and four experi-
mental groups for administration of different sizes of gold
nanoparticles. The mice received an intraperitoneal injection
of approximately 200 µL of gold nanoparticle solution at a
dose of 4000 µg/kg, based on the previously identified toxic
dose in vivo.44 The mice were weighed following injection
and assessed for behavioral changes every day for 28 days.
Hematology, biochemistry, and sample
collection
Using a standard blood collection technique, blood was drawn
from the saphenous vein into a potassium ethylenediamine
tetra-acetic acid collection tube for hematology analysis.
Analysis of standard hematological parameters was performed.
For blood analysis, 300 µL of blood was collected from the
mice. At 28 days, the mice were sacrificed by isoflurane anes-
thetic and exsanguinated with phosphate-buffered saline using
an angiocatheter. One mouse from each treatment group was
fixed with 10% buffered formalin following exsanguination.
The liver, kidneys, spleen, heart, lungs, thyroid, lymph nodes,
and bone marrow were then collected and weighed.
The spleen and thymus indices (Sx) can be expressed as:

S
Weightof experimental organ
Weightof experimental anima
x
mg
=
(
l
l ( )g
)
Transmission electron microscopic
analysis
Bone marrow and blood cells were obtained after 28 days of
administration of a nanoparticle dose of 4000 µg/kg. For the
transmission electron microscopy analysis, the solution was
centrifuged and the pellet fixed with 2.5% glutaraldehyde
in 0.03 M potassium phosphate buffer (pH 7.4). The cells
were postfixed with 1% osmium tetroxide in 0.1 M sodium
cacodylate buffer, and 0.5% uranyl acetate in 0.05 M
maleate buffer. Cells are then dehydrated in a graded series
of ethanol and embedded in Epon resin. Ultrathin sections
were cut and transferred onto 200 mesh uncoated copper
grids, stained with uranyl acetate, counterstained with lead
citrate, and observed using an Hitachi HF-2000 field emission
high-resolution transmission electron microscope operating
at 200 kV.
Results and discussion
Body weight and biodistribution
Figure 2 shows the variations in body weight of mice treated
with different-sized gold nanoparticles. It can be seen that
PEG-coated nanoparticles at a dose of 4000 µg/kg did not
0.0
400500600
Wavelength (nm)
700
5 nm
10 nm
30 nm
60 nm
0.2
0.4
Absorption (au)
0.6
0.8
1.0
Size (nm)
PEG-SHGold NPs
Zeta (mV)
60 ± 12.1 −1.65
30 ± 4.3−1.97
10 ± 1.6−1.55
5 ± 1.1−2.96
50 nm50 nm100 nm100 nm
Figure 1 Transmission electron micrographs and optical absorption of 5, 10, 30, and 60 nm PEG-coated gold particles.
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cause any deaths, and that body weight was slightly higher
than in the control group during a 1-day observation period.
The body weight of mice which received the 5 nm particles
was slightly different from the other groups. During the
study period, treatment with gold nanoparticles for 28 days
did not cause obvious adverse effects on growth because
no statistically significant differences in weight gain were
observed between the gold nanoparticle-treated mice and
the control mice. Furthermore, no abnormal clinical signs
or behaviors were detected in either the controls and treated
groups. Considered overall, treatment with gold nanoparticles
did not induce any apparent toxicity in the mice. Necropsy
at the end of the experiment did not show any macroscopic
changes in the organs in any of the four treatment groups.
To demonstrate the biodistribution of the PEG-coated
gold nanoparticles, gold concentrations in the heart, liver,
spleen, and kidney after 28 days are shown in Figure 3. It
can be seen that concentrations of the 5, 10, 30, and 60 nm
particles in the heart were 214, 18, 8.6, and 37 µg/kg,
respectively, with the 5 nm particles showing the highest
concentration. These gold concentrations are markedly
higher than previous biodistribution results.42 For the liver,
the gold concentrations achieved by the 5, 10, 30, and 60 nm
particles were 1797, 2898, 21, and 432 µg/kg, respectively.
It was found that the 5 nm and 10 nm particles had a very
high concentration in the liver, which is in good agreement
with previous work.42 For the spleen, the gold concentrations
achieved by the 5, 10, 30, and 60 nm particles were 628, 138,
3363, and 721 µg/kg, respectively, with the 30 nm PEG-
coated gold particles showing the highest gold concentration.
Finally, we showed that the gold concentrations achieved
by 5, 10, 30, and 60 nm particles in the kidney were 390,
48, 151, and 14 µg/kg, respectively, with the 5 nm particles
showing the highest gold concentration. The 5 nm particles
had the widest distribution, and the 60 nm particles showed
the lowest concentration in all organs examined.
It is well known that the size and surface-capping of gold
nanoparticles play an important role in their biodistribution in
mice.36–41 It has been shown that naked gold can accumulate
in the liver and spleen, and that the spleen and liver are the
dominant target organs.32,33 Small-sized particles have a wider
distribution, and larger-sized particles preferentially accu-
mulate in the liver and spleen.34,35 Furthermore, the different
surface charge on gold nanoparticles can influence their distri-
bution. PEG-coated gold nanoparticles have a neutral charge,
so their dynamic behavior in mice should be different from
that of naked and alternatively coated gold nanoparticles.36–38
Therefore, we can conclude that the liver is the dominant target
organ for 5 nm and 10 nm PEG-coated gold particles, and that
the spleen is the dominant target organ for the 30 nm particles.
14
1357911 13
Control
5 nm
10 nm
30 nm
60 nm
15
Day after injection
Body weight (g)
1719 2123252729 31
16
18
20
22
24
26
28
30
32
34
36
38
40
Figure 2 Body weight changes in mice for the 5, 10, 30, and 60 nm PEG-coated gold particles at a dose of 4000 µg/kg. The body weight of the treated mice was measured
every 2 days. Each point represents the mean ± standard deviation of six mice. Data were analyzed using the Student’s t-test and the differences between the different doses
and control group for each organ were not significant (P . 0.05).
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The 60 nm PEG-coated gold particles, similar to previously
reported 100 nm gold particles, have a low distribution in all
organs,36 and our results show that 60 nm gold particles can
be metabolized partially by the liver and kidney. However, the
smaller-sized 5 nm and 10 nm gold nanoparticles showed high
accumulation and were difficult to metabolize. Indeed, previ-
ous biodistribution and pharmacokinetic studies of PEGylated
gold nanoparticles have demonstrated high colloidal stability,
nonaggregation, and nontoxicity for 20 nm PEG-coated gold
nanoparticles.41 Other recent work carried out by Cho et al
showed that 13 nm PEG-coated gold nanoparticles could
induce acute inflammation and apoptosis in the liver, and were
found to accumulate in the liver and spleen for up to 7 days
after tail vein injection and to have a long blood circulation
time.38 The results reported by Cho et al demonstrated organ
micropathology, indicating that gold nanoparticles potentially
induce long-term organ damage and toxicity.38
Morphology of gold nanoparticles
in blood and bone marrow cells
Figure 4 shows transmission electron microscopic images of
5, 10, 30, and 60 nm gold particles in blood and bone marrow
cells after intraperitoneal injection at a dose of 4000 µg/kg
for 28 days. The gold nanoparticles were found as mono-
dispersed particles within the bone marrow cells. The 5 nm
particles were found in bone marrow cells, with no significant
decrease in size, demonstrating that the small particles did
not undergo any obvious breakdown. The 10, 30, and 60 nm
particles could be found easily in both the intracellular and
extracellular environment, which indicates that these gold
particles have a long time retention in the bone marrow,
and is in good agreement with previous work.36,38 However,
in the blood cells, the morphology of the gold particles is
different from that in bone marrow cells. The 5 nm nano-
particles showed good aggregation and hybridization with
macromolecules, and could form 10–20 nm compounds.
A similar phenomenon was observed for the 10 and 60 nm
gold particles but not the 30 nm particles. The gold nano-
particles could be found in the blood cells easily, because
of surface chemistry and endocytosis. It is noteworthy that
the gold nanoparticles could still be found in the blood cells
28 days after administration, which indicates that the gold
nanoparticles have a long blood circulation time, and is
again in good agreement with previous reports.38–40 It has
been suggested that interaction between protein and the
gold nanoparticles may be closely related to the toxicity of
0
5 nm
Au concentration (µg/kg)
10 nm 30 nm60 nm
50
100
150
200
250
0
−500
5 nm
Au concentration (µg/kg)
10 nm30 nm
Kidney (µg/kg)
60 nm
500
1500
1000
2000
2500
3000
3500
0
5 nm
Au concentration (µg/kg)
10 nm30 nm60 nm
500
1500
1000
2000
2500
3000
4000
3500
0
5 nm
Au concentration (µg/kg)
10 nm 30 nm 60 nm
50
150
100
200
250
300
350
400
450
Liver (µg/kg) Heart (µg/kg)
Spleen (µg/kg)
Figure 3 Biodistribution of body weight in mice for 5, 10, 30, and 60 nm PEG-coated gold particles at the dose of 4000 µg/kg after 28 days of treatment. Spleen and liver
were the main target accumulation organs. The 5 nm particles had a wide distribution in live, heart, kidney, while the 10 and 30 nm particles preferentially stayed in the liver
and spleen, respectively.
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the latter.27 The surface of a PEG-coated gold nanoparticle
probably has a low zeta potential, and cellular uptake of these
nanoparticles is different from that of naked gold nanopar-
ticles. The PEG-coated gold nanoparticles can also interact
with the cell by endocytosis and a conformational change in
the protein, although PEG-coated gold particles have a low
charge in the surface.
Endocytosis plays an important role in the interaction
between gold nanoparticles and cells, and the PEG-
coated gold nanoparticles are different from naked gold
nanoparticles. Recent quantitative evaluation revealed that
the surface coating on gold nanoparticles could modulate
endocytotic uptake pathways and cellular trafficking of the
nanoparticles. The naked gold nanoparticles were shown
to be taken up by macropinocytosis as well as by clathrin-
mediated and caveolin-mediated endocytosis.46–48 The
difference is due to interactions with different proteins or
lipids, related to mechanisms of uptake and endocytosis.
Increased transcytosis of PEG-coated gold nanoparticles
has been observed and a significant number of PEG-coated
gold nanoparticles were measured.49 Thus, further study is
necessary to understand how the mechanism of endocytosis
and the protein adsorption process changes the protein
conformation and the tendency of 5 nm and 60 nm PEG-
coated gold particles to aggregate.
Organ indices, hematology,
and biochemistry results
Table 1 gives the organ weights of mice for the different nano-
particle sizes, and illustrates the effect of the PEG-coated gold
nanoparticles on the organs. It can be seen that the weights of
the heart, liver, spleen, lung, and kidneys showed no obvious
variation at the dose of 4000 µg/kg. However, the weight of the
thymus increased with increasing particle size, but no statisti-
cally significant differences were found in these data. To dem-
onstrate the immune reaction in the organs further, the organ
indices for the thymus and spleen are shown in Figure 5. The
average values of the thymus and spleen indices in the control
5 nm
Bone marrow cell
Blood cell
100 nm100 nm 100 nm100 nm
100 nm 50 nm 100 nm50 nm
10 nm30 nm60 nm
5 nm 10 nm30 nm 60 nm
Figure 4 Transmission electron micrographs of 5, 10, 30, and 60 nm PEG-coated gold particles in bone marrow and blood cells 28 days after intraperitoneal injection at a
dose of 4000 µg/kg. In the bone marrow cells, the gold particles can be found as monodispersed particles, and aggregations of 10 nm and 60 nm particles are found in the
blood cells.
Table 1 Weights for the liver, lung, spleen, kidneys, heart, and thymus for 5, 10, 30, and 60 nm PEG-coated gold particles at a dose
of 4000 µg/kg after 28 days
Dose (μg/kg)
Heart (g)
Liver (g)
Spleen (g)
Lung (g)
Kidneys (g)
Thymus (g)
Control 5 nm 10 nm30 nm 60 nm
0.137 ± 0.019
1.496 ± 0.203
0.125 ± 0.034
0.183 ± 0.004
0.301 ± 0.037
0.008 ± 0.011
0.149 ± 0.013
1.248 ± 0.102
0.126 ± 0.010
0.192 ± 0.018
0.335 ± 0.033
0.099 ± 0.015
0.139 ± 0.017
1.748 ± 0.326
0.143 ± 0.038
0.199 ± 0.008
0.373 ± 0.100
0.095 ± 0.010
0.173 ± 0.019
2.520 ± 0.544
0.196 ± 0.073
0.195 ± 0.028
0.456 ± 0.019
0.104 ± 0.013
0.122 ± 0.008
1.787 ± 0.222
0.229 ± 0.055
0.176 ± 0.014
0.330 ± 0.035
0.095 ± 0.025
Notes: Data were analyzed by the Student’s t-test and the differences between the different doses for each organ were not significant (P . 0.05 vs controls). All values
represent the mean ± standard deviation of six mice.
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group were 2.3 and 3.3, respectively. The spleen indices of the
mice treated with the 5, 10, 30, and 60 nm particles changed to
3.7, 4.1, 3.3, and 4.3, respectively, at a gold nanoparticle dose
of 4000 µg/kg. The thymus indices for the mice treated with the
5, 10, 30, and 60 nm particles changed to 2.2, 2.7, 3.3, and 2.5,
respectively, at a gold nanoparticle dose of 4000 µg/kg. There
were still no statistically significant differences between the
treated groups and the control group, showing that injections of
PEG-coated gold nanoparticles of different sizes do not cause
obvious immune effects in either the thymus or spleen. The
thymus index increased for all sizes, except for 10 nm. The
spleen index increased for all particle sizes. Combining the
variations of body weight and biodistribution, it can be seen
that the PEG-coated gold nanoparticles had only a slight influ-
ence on the mice compared with the naked gold nanoparticles.
Moreover, the results imply that the spleen is one of the target
organs for PEG-coated gold nanoparticles, which is in good
agreement with previous work.32–42,50 The liver and spleen have
been described as the two dominant organs for biodistribution
and metabolism of gold nanoparticles.32–42
To quantify the toxicity of the PEG-coated gold
nanoparticles, the next important step is assessment of
standard hematology and biochemistry, which is very useful
for toxicity.50–53 We selected standard hematology markers
for analysis, ie, platelets, hematocrit, hemoglobin, red blood
cells, white blood cells, mean corpuscular volume, mean
corpuscular hemoglobin, and mean corpuscular hemoglobin
concentration. Nanoparticle size-dependent hematology
results are presented in Figure 6. The white blood cells
in mice treated with 5 nm and 30 nm particles decreased
significantly, while white blood cells from mice treated with
10 nm particles increased at a dose of 4000 µg/kg. Similarly,
red blood cells from mice treated with 5 and 30 nm particles
decreased significantly, while the red blood cells from
mice treated with 10 nm and 60 nm particles increased. In
addition, platelets, hematocrit, and hemoglobin from mice
treated with 5 and 10 nm particles also increased, but this
does not indicate a size-dependent trend associated with
treatment. The mean corpuscular volume, platelets, mean
corpuscular hemoglobin, and mean corpuscular hemoglobin
concentration changed, but no statistically significant
difference was found.
White blood cells are sensitive to physiological responses
in mice. The rise in white blood cells seen in mice treated with
2.5
Con
Spleen index (mg/g)
5 nm10 nm30 nm60 nm
3.0
3.5
4.0
4.5
5.0
5.5
Spleen index (mg/g)
Con
Thymus index (mg/g)
5 nm10 nm30 nm 60 nm
1.5
2.0
2.5
3.0
3.5
*
4.0
Thymus index (mg/g)
Figure 5 Size-dependent spleen and thymus indices of mice were calculated 28 days after 4000 µg/kg intraperitoneal injections.
Notes: All values are reported as the mean ± standard deviation. Data were analyzed using Student’s t-test. *Represents a significant difference from the control group
(P , 0.05).
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10 and 60 nm particles indicate an inflammatory response,
and the decrease in white blood cells seen in mice treated
with 5 nm and 30 nm particles may be associated with infec-
tion. Red blood cells are derived from hemopoietic stem
cells in the bone marrow. Following a series of maturation
steps, and directed largely by the hormone erythropoietin,
red blood cells enucleate and enter the circulatory system.
Thus, any variation in red blood cell levels can be related
to the hematopoietic system. The increase in red blood cells
found in mice treated with 10 and 60 nm particles indicates
that particles of this size have an effect on the hematopoietic
system. The four sizes of gold nanoparticles used in this study
could cause different reactions in the mice. The 10 and 60 nm
particles caused a more serious toxic response than the 5 nm
and 30 nm particles.
Finally, we investigated the biochemical effects of
5, 10, 30, and 60 nm PEG-coated particles, as shown in
Figure 7, including for alanine transaminase, aspartate
transaminase, blood urea nitrogen, globulin, creatinine, total
protein, albumin, and total bilirubin. It was found that alanine
transaminase and aspartate transaminase in mice treated
with the 10 and 60 nm particles increased significantly,
Con
WBC (109/L)
4
6
8
10
12
14
16
5 nm 10 nm30 nm
*
*
*
*
*
*
*
*
60 nm
Con5 nm 10 nm30 nm 60 nm Con 5 nm10 nm 30 nm 60 nmCon 5 nm10 nm30 nm60 nm Con5 nm10 nm30 nm60 nm
Con5 nm10 nm30 nm60 nm Con 5 nm10 nm 30 nm60 nm Con5 nm10 nm30 nm 60 nm
550
500
450
400
350
550
500
450
400
350
600
650
700
750
A
PLT (109/L)
E
30
50
45
40
35
HCT (L/L)
F
40
46
48
44
42
MCV (fl)
G
MCHC (g/L)
H
RBC (1012/L)
8.0
8.5
9.0
9.5
10.0
10.5
B
HGB (g/L)
160
180
200
220
240
260
C
MCH (pg/cell)
16
18
20
22
24
26
28
30
32
D
Figure 6 Size-dependent hematology results from mice treated with PEG-coated gold nanoparticles and control group 28 days after intraperitoneal injection at the dose of
4000 µg/kg. These results show mean and standard deviations of (A) white blood cells, (B) red blood cell, (C) hemoglobin, (D) mean corpuscular hemoglobin, (E) platelet,
(F) hematocrit, (G) mean corpuscular volume, and (H) mean corpuscular hemoglobin concentration.
Notes: Bars represent mean ± standard deviation. Data were analyzed using the Student’s t-test. *Represents a significant difference from the control group (P , 0.05).
Abbreviations: WBC, white blood cells; RBC, red blood cell; HGB, hemoglobin; MCH, mean corpuscular hemoglobin; PLT, platelet; HCT, hematocrit; MCV, mean
corpuscular volume; MCHC, mean corpuscular hemoglobin concentration.
Con
ALT (U/L)
60
80
100
120
140
5 nm10 nm30 nm 60 nm
*
*
*
*
*
Con 5 nm10 nm30 nm 60 nm
Con5 nm10 nm30 nm60 nm
Con5 nm10 nm 30 nm60 nm
Con5 nm10 nm30 nm60 nm
Con
5 nm
10 nm 30 nm60 nm
Con 5 nm 10 nm30 nm 60 nm
Con 5 nm 10 nm30 nm 60 nm
14
12
10
8
0.8
0.6
0.4
0.2
0.0
1.0
1.2
1.4
1.6
1.8
A
CREA (µM)
E
44
64
62
58
56
60
54
52
46
48
50
TP (g/L)
F
26
32
34
36
30
28
ALB (g/L)
G
TBIL (µM)
H
AST (U/L)
120
140
160
180
200
B
BUN (mmol/L)
7
8
9
10
11
12
C
GLOB (g/L)
16
18
20
22
24
26
28
30
D
Figure 7 Size-dependent biochemical results for mice treated with PEG-coated gold nanoparticles and control group 28 days after intraperitoneal injection at a dose of
4000 µg/kg. These results show mean and standard deviations of (A) alanine transaminase, (B) aspartate transaminase, (C) blood urea nitrogen, (D) globulin, (E) creatinine,
(F) total protein, (G) albumin, and (H) total bilirubin.
Notes: Bars represent mean ± standard deviation. Data were analyzed by Student’s t-test. *Represents significant difference from the control group (P , 0.05).
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen, GLOB, globulin, CREA creatinine; TP, total protein; ALB, albumin;
TBIL, total bilirubin.
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International Journal of Nanomedicine 2011:6
and globulin is decreased, indicating that the liver was
damaged after injection of the nanoparticles. Creatinine
levels in mice treated with the 60 nm particles decreased
sharply, indicating kidney damage in the mice. Total protein
and albumin in mice treated with the 10 and 60 nm particles
also decreased after PEG-coated gold injection, while total
bilirubin and blood urea nitrogen showed no significant
change for any size of particles. These results show clearly
that the 10 nm particles are highly toxic to the liver and that
the 60 nm particles are toxic to both the kidney and liver.
However, the 5 nm and 30 nm particles did not cause any
significant liver and kidney damage.
Alanine transaminase and aspartate transaminase
are mainly distributed in liver cells, and their levels rise
with necrosis of liver cells. The levels of these enzymes
correspond well with the extent of liver cell damage, and
are commonly used indicators of liver function. These two
enzymes are distributed differently among the liver cells.
Alanine transaminase mainly exists in the cytoplasm of liver
cells, whereas aspartate transaminase mainly exists in the
cytoplasm and mitochondria of liver cells. Therefore, the
increase in alanine transaminase in our mice treated with 10
and 60 nm gold nanoparticles indicates damage to liver cells.
However, their biodistribution shows that the concentration
reached by 60 nm gold particles in the liver was not as high
as that reached by 10 nm particles, indicate that the liver
damage caused by the 60 nm particles was due to metabolism
of the gold nanoparticles. In contrast, the 5 nm particles also
reached high concentrations in the liver, but liver function
was not significantly affected, showing that the 5 nm particles
did not caused direct damage to the liver. The 5 nm particles
are promising for the treatment of liver cancer without liver
damage, which is very important for further work.
Creatinine is another important indicator of kidney
function. Endogenous human creatinine is a product of
muscle metabolism. In muscle, creatine mainly generates
creatinine slowly through nonenzymatic dehydration, which
is then released into the blood, with excretion in the urine. The
serum creatinine concentration depends on the glomerular
filtration rate. However, serum creatinine is not entirely
consistent with the creatinine clearance rate, and creatinine
clearance is more sensitive than serum creatinine. In early
renal dysfunction (decompensated), creatinine clearance
rate and serum creatinine are normal. When the glomerular
filtration rate rises to above 50% of normal, serum creatinine
begins to rise rapidly. Therefore, when the serum creatinine
is significantly higher than normal, kidney function is
seriously damaged. The decrease in creatinine seen in mice
treated with the 60 nm particles is closely related to kidney
function, although the kidney is not the main target organ
for gold distribution. The 5 nm particles did not induce any
kidney damage, but the 10 nm particles caused some damage
by decreasing total protein and globulin.
In summary, the 60 nm particles induced serious toxicity
and damage to the liver and kidney, but had a relatively low
distribution in the liver, spleen, and kidney. It is proposed that
this toxicity may be due to the metabolism of PEG-coated
gold particles, which is similar to that previously reported
for 100 nm PEG-coated gold particles.36 The toxicity of
the 10 nm particles was also high, due to liver and kidney
damage, as well as an inflammatory response, and large
amounts of gold can accumulate in the liver. The present
results are in good agreement with the results of Cho et al,
showing that 13.5 nm PEG-coated gold particles can induce
serious toxicity.36,38 Our 5 nm and 30 nm particles showed low
toxicity, although accumulation of 5 nm particles did occur in
the liver and kidneys, and the 30 nm particles preferentially
remained in the spleen. Therefore, we suggest that 10 nm
PEG-coated gold particles are not sufficiently safe to be
administered at a concentration of 4000 µg/kg. If we want to
use these particles to encapsulate drugs for use in the clinical
setting, metabolism must be considered. High accumulation
of gold may induce long-term toxicity. The most important
finding for the size-dependent toxicity of PEG-coated gold
nanoparticles are that the 5 nm and 30 nm particles are safe,
but the 10 nm and 60 nm particles are toxic, which is not
consistent with previous in vitro findings, ie, the smaller the
particle, the greater the toxicity.
Conclusion
A size-dependent in vivo toxicity study of PEG-coated
gold nanoparticles in mice was carried out. In the study
animals, survival, weight, hematology, biochemistry, and
morphology were characterized 28 days after administration
of the gold nanoparticles at a concentration of 4000 µg/kg.
Biodistribution studies showed that the spleen and liver are
two dominant target organs. Our results showed that the
5 nm and 10 nm particles mainly accumulate in the liver,
and that the 30 nm particles preferentially accumulate in
the spleen. The 60 nm particles had a wider distribution,
with limited accumulation in the organs. Blood chemistry
revealed that white blood cells were increased in mice treated
with the 10 nm and 60 nm particles, while those treated
with the 5 nm and 30 nm particles showed decreased white
blood cells. Furthermore, alanine transaminase and aspartate
transaminase increased in mice treated with the 10 and 60 nm
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International Journal of Nanomedicine 2011:6
particles, while creatinine decreased with the 60 nm particles,
indicating that the 10 and 60 nm particles caused liver and
kidney damage. The present work clearly shows that 10 and
60 nm PEG-coated gold nanoparticles are not sufficiently
safe, and that the 5 and 30 nm particles have relatively low
toxicity. These conclusions are very important for future
cancer therapy, drug delivery, and diagnosis.
Acknowledgments
The authors would like to thank Professor Warren Chan for
his helpful discussions. This work was supported by the
National Natural Science Foundation of China (Grant No.
81000668, 30970867), the Specialized Research Fund for
the Doctoral Program (SRFDP) of Higher Education State
Education Ministry (Grant No. 200800231058), and the
Subject Development Foundation of Institute of Radiation
Medicine, Chinese Academy of Medical Sciences CAMS.
Disclosure
The authors report no conflicts of interest in this work.
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