Copyright © 2010 American Scientific Publishers Journal of

All rights reserved Nanoscience and Nanotechnology

Printed in the United States of America Vol. 10, 8638–8645, 2010

Cytotoxicity of Zinc Oxide Nanoparticles:

Importance of Microenvironment
Sheng-Tao Yang1 , Jia-Hui Liu1 , Jing Wang1 , Yuan Yuan1 , Aoneng Cao2 ,
Haifang Wang1
2
∗ , Yuanfang Liu1
2 , and Yuliang Zhao3
∗
1
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering,
Peking University, Beijing 100871, China
2
Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444, China
3
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Chinese Academy of Sciences,
Beijing 100049, China, and National Center for Nanosciences and Technology of China, Beijing 100190, China

While ZnO particles are widely used Delivered

in manyby Ingenta
fields, to: personal care products, the high
including
toxicity of ZnO nanoparticles Rice University,
has been Fondren
reported and aroused Library
great health concerns. In this study,
the cytotoxicity of ZnO nanoparticles wasIP : evaluated
93.80.35.229and, in particular, the role of microenviron-
ment in their toxicity was investigated.
Mon,Our results
18 Jun show01:22:13
2012 that ZnO nanoparticles are highly toxic to
NIH/3T3 cells, inducing viability loss, membrane leakage and morphology changes. The microenvi-
roment, here the CO2 atmosphere under cell culture condition, promoted the solubilization of ZnO
nanoparticles. Then the released Zn from ZnO nanoparticles induces the cytotoxicity. The impor-
tance of microenvironment on the ZnO nanotoxicity is presented and the implications to future
nanotoxicology studies are discussed.
Keywords: Zinc Oxide, Nanoparticles, Cytotoxicity, Solubility, Microenvironment.
RESEARCH ARTICLE

1. INTRODUCTION is somewhat unexpected and perplexed, because ZnO are

With the rapid development of nanotechnology,
nanomaterials become hopeful and applicable in diverse
areas. While more and more nanomaterials are used as
consumer goods in daily life, great concerns over their
potential toxicity to human beings have been aroused.1–2
Previous studies demonstrate that nanoparticles may cause
adverse health effects, depending on the physical structure,
chemical component and surface functional groups.1–4
Although oxidative stress is defined as a most possible
cause of nanotoxicity,3 the fundamental understanding of
the specific nanotoxicity is still remained unclear.
Among a large number of nanoparticles (NPs), ZnO
NPs have drawn serious concerns from the nano commu-
nity and the public, because they are closely entering the
human’s environment, not only widely used as sunscreens,
antibacterial reagents, rubber additives and pigments,5–8 but
also applicable in many new areas, such as sensors, pho-
tocatalysis, solar cells, transparent electrodes, electrolumi-
nescent devices and ultraviolet laser diodes.9–15 In fact,
ZnO NPs have been found toxic to microorganisms,16–20
cells,21–25 plants26
27 and animals.28–30 However, the toxicity
∗
Authors to whom correspondence should be addressed.
always consumed in a low content as the non-toxic additive
in the skin care products.31
Herein, we investigated the toxicity of ZnO NPs in vitro,
and meanwhile the solubilization of ZnO NPs was mon-
itored in the cell culture system. Our results demon-
strate that ZnO NPs are highly toxic to NIH/3T3 cells,
inducing viability loss, membrane damage and morphol-
ogy changes. The solubilization of ZnO NPs should be
regarded as a crucial step involving the nanotoxicity pro-
cess, where the CO2 atmosphere in the microenviroment
plays a key role. The influence of microenvironment on
the ZnO nanotoxicity is presented and the implications to
future nanotoxicology studies are discussed.
2. EXPERIMENTAL DETAILS
2.1. Materials
ZnO NPs were purchased from Merk Corp., Germany.
Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-
ltetrazolium bromide (MTT) and Pluronic® F-127
were obtained from Sigma Corp., USA. Fetal bovine
serum, L-glutamine and pyridoxine hydrochloride were
obtained from Hyclone, USA. Penicillin G sodium and

8638 J. Nanosci. Nanotechnol. 2010, Vol. 10, No. 12 1533-4880/2010/10/8638/008 doi:10.1166/jnn.2010.2491

Yang et al. Cytotoxicity of Zinc Oxide Nanoparticles: Importance of Microenvironment
streptomycin sulfate were purchased from Amresco,
USA. Lactate dehydrogenase (LDH) kits were purchased
from Nanjing Jiancheng Biotechnology Institute, China.
FluoZin-3AM was obtained from Invitrogen Corp., USA.
Other chemicals were of analytical grade.
2.2. Characterization of ZnO NPs
The shape and morphology of ZnO NPs were investigated
by transmission electron microscopy (TEM, JEM-200CX,
JEOL, Japan) and the purity was analyzed by X-ray flu-
orescence (XRF, S4-Explorer, Bruker, Germany). X-ray
diffraction (XRD, D/MAX 2000, Rigaku, Japan) anal-
ysis was adopted to reveal the crystalline phase and
particle size. Brunauer-Emmett-Teller (BET, ASAP2010,
Micromeritics, USA) technique was adopted to gain the
specific surface area (SSA) and particle size. ZnO NPs
were dispersed in phosphate buffered saline (PBS) solu-
tion to prepare stock dispersion (concentration 10 mg/mL).
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Stock dispersions were sonicated for 15 min in an ultra-
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sound bath (40 W) to break up aggregates and diluted to
Fondren
IP : 93.80.35.229
the final exposure concentrations with the culture medium
just prior to the cell exposure.
Mon, 18 Jun 2012
2.3. Cell Culture and Morphology
Mouse fibroblast cells NIH/3T3 were obtained from
Professor D. Ma of Genetic Institute at Peking University.
They were cultured in DMEM culture medium [DMEM
with 10% (v/v) fetal bovine serum, 0.1 mg/mL penicillin
G sodium, 0.1 mg/mL streptomycin sulfate, L-glutamine,
and pyridoxine hydrochloride] at 37  C in a humidified
atmosphere of 5% CO2 /95% air.
NIH/3T3 cells were plated in the 96-well plates (1 × 10 4
cells per well) and incubated for 24 h. Then, ZnO NPs
were introduced to cells with a concentration of 120 M or
240 M zinc-equivalent in culture medium. Cells cultured
in the medium without adding ZnO NPs were taken as the
control. The morphology was recorded under microscope
at 24 h post exposure.
2.4. Cell Viability
The cell viability was evaluated by measuring the ability
of mitochondrial reduction of the MTT to formazan by
succinic dehydrogenase (MTT assay). NIH/3T3 cells were
plated in the 96-well plates (1 × 104 cells per well)
and incubated for 24 h. Then, ZnO NPs were intro-
duced to cells with a concentration of 12, 120, 240,
600 and 1200 M zinc-equivalent in culture medium,
respectively. Cells cultured in the medium without adding
ZnO NPs were taken as the control. Twenty-four hours
later, cells were washed with 200 L PBS. Then, 20 L
MTT (5 mg/mL in PBS) together with 180 L of cul-
ture medium was added to each well and incubated for
another 4 h. After centrifugation (1000 rpm × 5 min), the
J. Nanosci. Nanotechnol. 10, 8638–8645, 2010
supernatants were discarded and the dark blue formazan
crystals formed were dissolved with 200 L dimethyl sul-
foxide. The optical density (OD) of formazan at 550 nm
was recorded on a Microplate Reader (Varioskan Flash,
Thermo Electron, Finland). The relative cell viability (%)
is expressed as a percentage of ODtest /ODcontrol , where
ODtest is the optical density of the cells exposed to ZnO
NPs, and ODcontrol is the optical density of the control
sample.
In the time-dependent study, the cells were exposed to
ZnO NPs at a concentration of 240 M zinc-equivalent
for 1, 2, 3, 4, 5 and 24 h. The viability was assayed by
MTT method described above.
2.5. LDH Leakage Assay
LDH test-kit was used to assess LDH leakage from cell.
NIH/3T3 cells were plated in the 96-well plates (1 × 104
cells per well) and incubated for 24 h. Then, ZnO NPs
to:
were introduced to the cells with a concentration of 12,
Library
120, 240, 600 and 1200 M zinc-equivalent. A centrifu-
gation (1000 rpm × 5 min) was performed at 24 h post
01:22:13
exposure and 30 L of the supernatants was taken out
for the LDH assay on a Microplate Reader (Varioskan
Flash, Thermo Electron, Finland) at 440 nm. Pyruvic acid
was used as the standard reference. The LDH leakage was
shown as LDH activity (U/L) in the supernatants.
RESEARCH ARTICLE
2.6. Ultrastructural Analysis
To estimate the uptake of ZnO particles, thin-sections
of cells were observed under TEM. NIH/3T3 cells were
plated in the 6-well plates (2 × 105 cells per well) and
incubated for 24 h. Then, ZnO NPs were introduced to the
cells with a final concentration of 240 M zinc-equivalent.
Cells without ZnO NPs exposure were taken as the con-
trol. At 0.5, 2 and 24 h post exposure, the cells were
washed with ice-cold PBS for four times. After centrifu-
gation (3000 rpm × 5 min), cells were collected, prefixed
with 3% glutaraldehyde, post-fixed in 1% osmium tetrox-
ide, dehydrated in a graded alcohol series, and embedded
in epoxy resin. Then the specimens were cut with an ultra-
microtome. Thin-sections poststained with uranyl acetate
and lead citrate were inspected with TEM.
2.7. Intracellular Zinc Detection
For the intracellular Zn2+ content measurements, FluoZin-
3AM was dissolved in dimethyl sulfoxide with 10% (w/v)
Pluronic® F-127 at a concentration of 2 mM and was
finally diluted to 2 M by the cell culture medium.
NIH/3T3 cells were plated in the 96-well plates (1 × 104
cells per well) and incubated for 24 h. The FluoZin-
3AM solution was added to cells (100 L/well) and incu-
bated for 1 h. After that, the cells were washed by PBS
and then exposed to culture medium with or without
8639
 Cytotoxicity of Zinc Oxide Nanoparticles: Importance of Microenvironment
240 M zinc-equivalent ZnO NPs for 0.5 h. Relative flu-
orescence intensity, reflecting the intracellular Zn content,
was recorded on the Microplate Reader. The fluorescent
images were recorded under microscope.
2.8. Solubility of ZnO NPs
The solubility of ZnO NPs in DMEM culture medium
(cell-free) under 5% CO2 /95% air was measured by induc-
tively coupled plasma-atomic emission spectrometry (ICP-
AES, Profile, Leeman, USA). Briefly, ZnO NPs were
suspended in the culture medium to different concentra-
tions (0, 12, 60, 120, 180, 240, 600 and 1200 M zinc-
equivalent). The mixtures were cultured at 37  C under
5% CO2 /95% air for 24 h. After that, the centrifuga-
tion (12000 rpm × 10 min) was performed to remove the
insoluble residues from the mixtures and the supernatants
were collected for the Zn content analysis. To prepare
ICP-AES sample, the supernatants were digested usingby Ingenta to:
Delivered
HNO3 /H2 O2 (v/v:1/1) at 100  C for 15 min and diluted
Rice University,by Fondren
2% HNO3 . IP : 93.80.35.229
In dynamic solubilization study, ZnO NPs Mon,were
18 sus-
Jun 2012
pended in the culture medium (cell-free) to a concentra-
tion of 1200 M zinc-equivalent. Then, the mixtures were
incubated at 37  C under 5% CO2 /95% air and normal
air, respectively. At certain time points (0, 2, 5, 10, 15,
30 min and 1, 2, 4, 8, 12, 24 h), the mixtures were taken
out from incubator and centrifuged (12000 rpm × 10 min).
RESEARCH ARTICLE
The supernatants were collected, digested and analyzed by
ICP-AES. Similarly, the solubility of water insoluble MgO
and CuO was measured after 24 h incubation.
2.9. Cytotoxicity of Zn2+ Released from ZnO NPs
The influence of soluble Zn on the cell viability was inves-
tigated by MTT assay. ZnO NPs with a concentration of
12, 120, 240, 600 and 1200 M zinc-equivalent were incu-
bated in culture medium (cell-free) at 37  C under 5%
CO2 /95% air for 24 h. The insoluble precipitate was dis-
carded by centrifugation at 12000 rpm for 10 min. The
supernatants were collected and introduced to NIH/3T3
cells (1 × 104 cells per well in 96-well plate). Twenty-
four hours later, cells were washed with 200 L PBS and
analyzed by MTT method. For comparison, the cytotox-
icity of Zn(Ac)2 at a concentration of 12, 120, 240, 600
and 1200 M zinc-equivalent was assayed in the same
manner.
2.10. Statistical Analysis
Data have been represented as the mean ±SD (stan-
dard error) from at least four individual experiments.
Significance has been calculated using Student’s t-test.
Difference in means was considered significant if
p < 005.
8640
Yang et al.
3. RESULTS
3.1. Characterization of ZnO NPs
The TEM image shows that ZnO NPs are mostly rod shape
(Fig. 1(a)). The average diameter is 30 ± 10 nm and the
average length is 100 ± 40 nm. In addition, the diame-
ter and length distributions were counted to present the
size of ZnO nanoparticles more accurately. As shown in
Figures 1(c and d), the peak value for diameter is 25 nm
and the peak value for length is 90 nm according to the
size distribution. The XRF analysis suggests the purity of
ZnO NPs is higher than 99.9 wt%. The BET analysis indi-
cates a high specific surface area of 12.88 m2 /g. The XRD
spectra demonstrate that the ZnO NPs are zincite phase
crystal (Fig. 1(b)). No extra peak is observed, indicating
the high purity of ZnO NPs. The mean crystallite diame-
ter calculated from XRD spectra is 40 nm, well consistent
with that from the TEM observation and BET analysis.
Library of ZnO NPs
3.2. Cytotoxicity
The01:22:13
results of the MTT assay, as a measure of cell
viability/proliferate following the exposure to ZnO NPs,
are given in Figure 2(a). Their cytotoxicity increases
with increasing particle concentration. No cytotoxicity is
observed at the concentration of 120 M zinc-equivalent
and lower. But a dramatic loss of cell viability occurs at
the concentration of 240 M zinc-equivalent. The dramatic
cell viability loss is in agreement with the LDH assays,
where significant increase of LDH leakages is observed
at the concentration of 240 M zinc-equivalent or higher
(Fig. 2(b)). In addition, the cytotoxicity of ZnO NPs is
also time-dependent (Fig. 2(c)). Half of the cell viability
is lost after 4 h exposure to 240 M zinc-equivalent ZnO
NPs. The cell morphology of cells exposed to 120 M
and 240 M zinc-equivalent ZnO nanoparticles for 24 h
was also recorded to directly show the toxicity (Fig. 2(d)).
While the cells exposed to 120 M zinc-equivalent ZnO
NPs remain the typical morphology, the cells exposed to
240 M zinc-equivalent ZnO NPs shrink and the mem-
brane is damaged.
3.3. Cellular Uptake of ZnO NPs and Zinc Ions
The TEM investigation of cell sections shows no ZnO par-
ticulate is found inside the cytoplasmic vesicles or other
cellular parts after cells were exposed to ZnO NPs for 0.5,
2 and 24 h (Figs. 3(a and b)). There are very few phago-
cytotic vesicles observed at 0.5 h. And even in those vehi-
cles, no particulate is distinguishable. Similar results were
achieved for the 2 h samples (data are not shown). In the
24 h sample, no ZnO NPs are observed, either. The cell
structure is totally destroyed. The cell membrane breaks
and no clear cell nucleus presents.
In the contrast to the absence of ZnO NPs in cells, it
was found that the intracellular Zn level increased upon
J. Nanosci. Nanotechnol. 10, 8638–8645, 2010
Yang et al. Cytotoxicity of Zinc Oxide Nanoparticles: Importance of Microenvironment

(a) (b)

(c) (d)

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Mon, 18 Jun 2012 01:22:13

RESEARCH ARTICLE
Fig. 1. Characterization of ZnO NPs. (a) TEM image, (b) XRD spectrum, (c) The diameter distribution, (d) The length distribution.

the exposure to ZnO NPs. Figure 3(c) directly shows the
presence of intracellular Zn compared with the cell con-
trol sample at 0.5 h post exposure, by using a fluorescent
Zn probe FluoZin-3AM . The increase of Zn can also be
quantitatively demonstrated by measuring the intensity of
fluorescence (Fig. 3(d)). Our results suggest that the uptake
of Zn is a quick process and almost reaches the saturation
within 0.5 h.
3.4. Solubility of ZnO NPs
The solubility of ZnO NPs in DMEM culture medium was
investigated using ICP-AES (Fig. 4). The concentrations
of soluble Zn are exactly the same as those of added ZnO
NPs when the concentration of added ZnO NPs is less
than 246 M zinc-equivalent. When the content of added
ZnO NPs surpasses 246 M zinc-equivalent, the concen-
tration of soluble Zn in culture medium keeps at about
246 M. Hence, the solubility of ZnO NPs is 246 M
zinc-equivalent in cell-free DMEM culture medium under
the normal cell culture condition.
The solubilization processes of ZnO NPs in DMEM cul-
ture medium were also monitored under both cell culture
condition and normal air. As shown in Figure 4(b), the
zinc content in the supernatant nearly reaches the satura-
tion around 0.5 h, indicating their fast dynamic dissolution
process. But, under normal air, the solubility of ZnO NPs
keeps around 20 M zinc-equivalent.
3.5. Cytotoxicity of Zn2+ Released from ZnO
Nanoparticles
In order to verify that the toxicity from the released zinc,
the supernatants (containing soluble zinc) of the cultured
mixture of ZnO NPs in DMEM culture medium were col-
lected and used to test their toxicity to the cells. As shown
in Figure 5, there is no sign of toxicity when the initial
ZnO concentration is 120 M zinc-equivalent and lower.
When the initial ZnO concentration increases to 240 M
zinc-equivalent and higher, the cytotoxicity becomes very
pronounced. A very similar toxicity profile of zinc ions
is achieved, where 240 M zinc-equivalent is the critical
concentration of cytotoxicity, too.
4. DISCUSSION
In this study, we find that ZnO NPs are highly toxic to
NIH/3T3 cells. The release of zinc into the cell culture
medium with the help of cell culture atmosphere CO2
should be regarded as an important step toward the cyto-
toxicity of ZnO NPs.

J. Nanosci. Nanotechnol. 10, 8638–8645, 2010 8641

 Cytotoxicity of Zinc Oxide Nanoparticles: Importance of Microenvironment Yang et al.

(a) (b)

(c) (d)

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Fig. 2. Cytotoxicity of ZnO NPs to NIH/3T3 cells. (a) The dose-dependent viability loss after 24 h exposure, (b) The dose-dependent LDH leakage
after 24 h exposure, (c) The time-dependent viability loss after the exposure to 240 M zinc-equivalent ZnO NPs, (d) The cell morphology after the
exposure to 120 M zinc-equivalent (top) and 240 M zinc-equivalent (bottom) ZnO NPs for 24 h.

Our cytotoxicity data collectively show the high toxi-
city of ZnO NPs to NIH/3T3 cells. The viability assays
suggest the cytotoxicity of ZnO NPs be significantly pro-
moted at 240 M zinc-equivalent, which is consistent with
the previous report.23 The LDH leakage and morphology
change are compatible with the viability assays. The ultra-
structure analyses clearly show the collapse of the cell
structures after the exposure to ZnO NPs. Although our
results together with data in the literature demonstrate the
high toxicity of ZnO nanoparticles.21–25 However, the high
toxicity is still somewhat unexpected and perplexed, espe-
cially considering that micronised ZnO powder is com-
monly used as the safe additives in sunscreen and other
skin care products.31 Such contradictory results obviously
require further research and clearer explanation.
We find that the solubility is a pivotal factor determin-
ing the cytotoxicity of ZnO NPs. The TEM investigation
suggests the absence of intracellular ZnO NPs, exclud-
ing the toxicity from the ZnO particulates uptake. On the
other hand, the increase of intracellular Zn level implies
that soluble Zn might be the real toxicant form of ZnO
NPs. The dissolved part of ZnO NPs could induce similar
toxicity to cells just as Zn2+ and ZnO NPs did, which fur-
ther points out that the soluble Zn should be the toxicant
of ZnO NPs. In fact, the underlying mechanism of ZnO
nanotoxicity has already aroused great interest and debate.
There are two major speculations on the mechanism. One
was proposed by Brunner et al., who speculated that the
cytotoxicity of ZnO NPs came from the high solubility of
ZnO.23 But they also figured out that the slight water sol-
ubility of ZnO (4.2 g/g) recorded in the handbook is too
low to induce toxicity.23
32 Another speculation is based
on that the toxicity is attributed to the oxidative stress
induced by ZnO NPs.22
24
33 Actually, Zn2+ has already
been found to induce serious oxidative damages in vitro.34
However, it is hard to draw such a conclusion without con-
ducting the oxidative stress measurements directly in the
exposed cells. Recently, Xia et al. combined both hypothe-
ses together, suggesting both particle solubility and oxida-
tive damage should be responsible for the cytotoxicity of
ZnO NPs.35 Despite the unclear mechanism of zinc toxi-
city, our results and current data in literature suggest that
the solubilization should be regarded as a crucial step pro-
ducing the cytotoxicity of ZnO NPs.23
35
The solubility results clearly show that ZnO NPs can
be dissolved under the cell culture condition, much higher
than the reported solubility in handbook.32 Taking the

8642 J. Nanosci. Nanotechnol. 10, 8638–8645, 2010

Yang et al. Cytotoxicity of Zinc Oxide Nanoparticles: Importance of Microenvironment

(a) (b)

(c) (d)

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Fig. 3. Uptake of ZnO NPs and Zn2+ by NIH/3T3 cells. (a) TEM image of cells exposed to ZnO NPs for 0.5 h. N indicates the nuclear and V

RESEARCH ARTICLE
indicates the vesicle, (b) TEM image of cells exposed to ZnO NPs for 24 h, (c) The fluorescent image of cells exposed to ZnO NPs for 0.5 h, (d) The
quantification of the intracellular Zn level post the exposure to ZnO NPs.

microenvironment into consideration, the promoted solubi-
lization of ZnO nanoparticles can be understood by simple
chemistry.36 There is continuous supply of CO2 under the
normal cell culture condition. The pH of culture medium
(pH 7.5) is buffered by the balance between HCO− 3 and
H2 CO3 (dissolved CO2 in H2 O) under the atmosphere of
5% CO2 /95% air (Eq. (1)). The H+ drives the solubiliza-
tion of ZnO NPs towards Zn2+ (Eq. (2)). The presence of
CO2 provides a continuous H+ supply, thus driving the bal-
ance toward Zn2+ . Besides, fewer OH− and more HCO− 3 NPs can not penetrate the skin, mostly staying on the sur-
under CO2 give soluble zinc less opportunity to deposit
again. In contrast, under the normal air, the solubilization
of ZnO NPs is restricted by the limited H+ .
+
CO2g + H2 Oaq ↔ H2 CO3 aq ↔ Haq + HCO−
3aq
+
↔ 2Haq + CO2−
3 aq
+
2Haq + ZnOs → Zn2+
aq + H2 Oaq
The solubilization mechanism discussed above suggests
the importance of microenvironment parameters in toxicol-
ogy study of ZnO NPs. Taking the blood stream in humans
and animals as an example, the H+ concentration, HCO− 3
concentration and the content of CO2 are quite close to
those in the cell culture condition.37 Thus, ZnO NPs is
expected to be dissolved very quickly, with the help of
CO2 in blood, after entering the blood stream and present
the toxicity of Zn2+ . Similar results are also expected in
pulmonary studies, where lung is the major breath organ
to exhale CO2 . Indeed, the serious pulmonary toxicity of
ZnO NPs was observed and reported.28 The solubilization
is regarded as one of the reasons for the pulmonary tox-
icity. In the contrast, in the skin toxicity study, ZnO NPs
are expected to be low toxic or even nontoxic. Since ZnO
face of skin and being exposed to the normal air.38 Such
microenvironment can be summarized as under a rather
dry and negligible CO2 atmosphere. The ZnO NPs would
hardly dissolve during the skin toxicity assay, therefore,
less toxicity is expected.
(1) The importance of microenvironment might not be
restricted to the ZnO NPs. For examples, water insolu-
(2) ble MgO and CuO have much higher solubility under
cell culture conditions. Also, the depletion of environ-
mental amino acid is regarded as one pathway of carbon
nanotubes (CNTs) toxicity.39 These examples remind us
that the microenvironment parameters, not only restricted
to CO2 , should be carefully investigated in the nanotoxi-
cology studies to pursue the accurate results.

J. Nanosci. Nanotechnol. 10, 8638–8645, 2010 8643

 Cytotoxicity of Zinc Oxide Nanoparticles: Importance of Microenvironment Yang et al.

(a) culture system, in which the ambient CO2 is a determi-

nant factor. Our results not only obviously benefit the
ongoing in-depth toxicity study of ZnO NPs, but also
remind us to pay more attention on the microenvironment
of the experimental system to avoid any inaccurate infor-
mation and gain better understanding of the nanotoxicity
and nanobioactivity. We believe that our finding marks a
notable step toward in the long journey of the nanotoxico-
logical and nanobiological study.

Acknowledgments: We thank Dr. Huaping Li at

University of California, Santa Barbara for his valuable
suggestions and comments. We acknowledge financial sup-
port from the China Natural Science Foundation (No.
(b) 20871010) and the China Ministry of Science and Tech-
nology (973 project No. 2006CB705604).

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Received: 22 January 2009. Accepted: 16 April 2009.

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