ZHANG ET AL.VOL. 5
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August 25, 2011
C2011 American Chemical Society
Mechanistic Toxicity Evaluation of
Uncoated and PEGylated Single-Walled
Carbon Nanotubes in Neuronal
Yongbin Zhang,†Yang Xu,‡Zhiguang Li,†Tao Chen,†Susan M. Lantz,†Paul C. Howard,†Merle G. Paule,†
William Slikker, Jr.,†Fumiya Watanabe,‡Thikra Mustafa,‡Alexandru S. Biris,‡,*and Syed F. Ali†,*
University of Arkansas at Little Rock, 2801 S. University Avenue, Little Rock, Arkansas 72204, United States
of the numbers of single tubes formed
by graphene layers, CNTs are classified as
single-walled carbon nanotubes (SWCNTs),
and multiple-walled carbon nanotubes
(MWCNTs). Due to their unique physical,
mechanical, and chemical properties, car-
bon nanotubes have attracted tremendous
interest in electronics, optical communi-
cations, and energy storage in the past
decade.1?5SWCNTs, a material with one-
high aspect ratio (generally a diameter of
around 0.7?3 nm and lengths of up to
several centimeters), elicit different biologi-
cal behavior compared to spherical nano-
particles when introduced in biological
systems. The unique crystalline structure
and morphological characteristics of these
nanostructures have made them excellent
candidates for a number of applications that
tic imaging, drug delivery, and other bio-
relatively flexible andbased on theirunique
morphology have considerable ability to
interact with the membranes of cells and
Recently, we have shown that carbon nano-
tubes have the ability to penetrate a num-
ber of other biological systems ranging
from tissues to plant seeds.10?12Moreover,
their high surface area allows the loading of
multiple molecules along the nanotubes'
side walls; however, the poor solubility of
ical applications for many years. Therefore,
this approach may affect the surface of
arbon nanotubes (CNTs) are hollow
cylinders made of graphene sheets
strategies for surface functionalization of
carbon nanotubes have been developed to
make CNTs more biocompatible. Two ap-
proaches, including covalent and noncova-
lent functionalization, have been successfully
established.13SWCNTs can be coated by
single-strand DNA via π?π stacking be-
tween aromatic DNA base units and the
nanotubes' surface;14however, DNA mol-
ecules noncovalently coated on the surface
of SWCNTs can be cleaved by nucleases in
systems.15Another approach is to functio-
(PEG) through covalent functionalization,
an approach that has been intensively stu-
died and employed in biomedical applica-
tions, since it allows excellent individual
dispersion and good stability of the nano-
tube over a long period of time.16Although
*Address correspondence to
Received for review May 3, 2011
and accepted August 25, 2011.
ABSTRACT We investigated and compared the concentration-dependent cytotoxicity of single-
walled carbon nanotubes (SWCNTs) and SWCNTs functionalized with polyethylene glycol (SWCNT-
PEGs) in neuronal PC12 cells at the biochemical, cellular, and gene expressional levels. SWCNTs
potency than uncoated SWCNTs. Reactive oxygen species (ROS) were generated in both a
concentration- and surface coating-dependent manner after exposure to these nanomaterials,
andmitochondria dysfunctionwere highlyrepresented. Interestingly, alteration ofthegenesis also
surface coating-dependent with a good correlation with the biochemical data. These findings
suggest that surface functionalization of SWCNTs decreases ROS-mediated toxicological response
KEYWORDS: single-walled carbon nanotubes.functionalization.toxicity.
reactive oxygen species.gene expression
ZHANG ET AL.VOL. 5
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SWCNTs and interfere with their optical and electrical
properties, covalently functionalized carbon nano-
tubes show promising applications in a number of
biomedical applications such as gene and drug deliv-
SWCNTs havetremendouspotential forapplications
in biomedicine.18?22For example, a large number of
reports have indicated the ability of SWCNTs to deliver
both in vivo and in vitro drug molecules21and onco-
gene suppressor genes18to tumors and cells. A sig-
nificant finding was recently reported that indicates
that the graphitic structure of the SWCNTs can be
degraded enzymatically23and therefore leaves open
the possibility that nanomaterials may biodegrade
when introduced in various biological systems. How-
ever, the single major limitation was related to their
possible induced side effects, which need to be thor-
oughly addressed and studied.24On the other hand,
evidence forthe toxic potential ofcarbon nanotubes is
controversial. Many publications have reported no
apparent toxicity,19,25?31while there are a number of
and in vivo.38?43The physicochemical characterization
of nanomaterials has been recognized as the funda-
induced toxicological or biological responses and the
unique properties of these nanomaterials.24The major
issues addressed in these toxicological profiles are the
purity, dosage, surface chemistry, dimensions, and
exposure route of carbon nanotubes used in these
studies. Several SWCNT synthesis methods involve the
use of metal catalysts (i.e., Fe, Cu, and Mg), which have
been known to be toxic to cells or organisms under
certain conditions.44Such impurities introduced dur-
be removed from the samples before evaluating the
inherent SWCNT toxicity. Current published reports
An acute and chronic toxicity study using functiona-
lized single-walled carbon nanotubes showed no evi-
dence of toxicity in clinical chemistry and histo-
pathology through intravenous injection of mice, with
SWCNTs accumulating in macrophages of liver and
spleen over 4 months.25By contrast, it has been
reported that carbon nanotubes induced granulomas
and inflammation after i.p. administration in mice,
showing asbestos-like pathogenicity.45A number of
studies have demonstrated that several types of metal
nanoparticles (such as silver, copper, and manganese)
can deplete dopamine and its metabolites 3,4-dihy-
droxyphenylacetic acid and homovanillic acid and
elicit dopaminergic toxicity in PC12 cells.46,47Recent
studies have also shown that nanosized carbon nano-
particles can interact with neurons and ultimately
translocate to the brain.48,49It has been reported that
as scaffolds in neural cells and brain tissues could
prevent ischemic injury of neurons in a rat stroke
model.19Nevertheless, carbon nanotubes have the
ability to cross the blood?brain barrier and can
accumulate in brain tissue, making therefore the
neurotoxicity of CNTs an extremely important issue.50
This need was recently highlighted by a recent study
on the biodistribution of PEGylated SWCNTs that
clearly indicated their ability to reach brain tissue.
The surface modification of SWCNTs by PEGylation
allows a highly individual dispersion of the nano-
tubes, but also these nanostructures were found to
reside for longer periods of time in the blood circula-
tion, making them more prone to reach various
organs including the brain.51
PC12 cells, derived from pheochromocytoma of the
rat adrenal medulla, represent one of the best biologi-
cal models to study the cytotoxicity and neurotoxicity
of any chemicals or nanomaterials. The PC12 cell culture
system can synthesize, take up, and release one impor-
used to screen the potential cytotoxicity and neurotoxi-
city of the central nervous system to addictive drugs,
successfully used to evaluate the neurotoxin potential
and investigate the toxic mechanism of a number of
nanomaterials including silica nanoparticles, carbon
nanotubes, graphene, quantum dots, iron oxide, silver,
and titanium dioxide nanoparticles.11,47,52?57Therefore,
PC12 cells were selected as a model forthis mechanistic
toxicity study of the impact of nanotube surface chem-
istry at cellular and molecular levels.
Recently, we reported that the shape of carbon
nanomaterials plays a pivotal role in adversely affect-
ing biological systems.11The surface coating of
SWCNTs is another important parameter, both for
the biological properties of these nanomaterials and
their toxic potential. Given the high potential of
SWCNT structures to be used as delivery carriers for
neural reconstruction, regeneration, and photother-
mal therapy and imaging in the nervous system, it is
crucial to understand and study the interaction be-
tween such nanomaterials and neuronal cells. Due to
cells and possibly cause toxicity. In particular, the
mechanism of cellular uptake is not clear since tradi-
tional imaging techniques, such as transmission elec-
tron microscopy (TEM), generally fail to detect and
especially quantify SWCNTs in cells and tissues due to
the similar contrast of the SWCNTsand tissues. Due to
the strong Raman signature of SWCNTs, Raman spec-
troscopy was used successfully to detect the uptake
and the biodistribution of SWCNTs in tissue culture
and in vivo.58Therefore, the surface functionalization
of carbon nanomaterials by PEGylation is proposed to
affect the biological behavior in PC12 cells. This study
presents an evaluation of the neurotoxicity and
ZHANG ET AL.VOL. 5
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and SWCNT-PEGs and their underlying molecular me-
chanisms in neuronal PC12 cells. We found that PEG
functionalization significantly reduces the side effects
of SWCNTsin vitro in neuronal PC12 cells,as well as the
RESULTS AND DISCUSSION
Characterizations of SWCNT and SWCNT-PEG. It is critical
to perform physical and chemical characterization of
nanomaterials before nanotoxicology studies.59Figure 2
shows the TEM images of SWCNTs and SWCNT-PEGs.
The average diameter of the SWCNTs was in the range
0.7?1.6 nm with a length of 0.2?3 μm and an overall
purity in excess of 97%, based on EDS elemental
analysis. After chemical functionalization with PEG
molecules, the average diameter of SWCNT-PEG sam-
to 0.1?1 μm, clearly indicating the formation of a PEG
coating on the SWCNT surface. High-resolution TEM
analysis of both the SWCNT and SWCNT-PEG samples
indicated the presence of a uniform, thin, noncrystal-
line film associated with the PEG presence around the
both SWCNTs and SWCNT-PEGs appears, as expected,
to be nanofibrous, and it can be observed that the
coating is relatively uniform throughout the entire
that the carbon nanotubes, before and after PEG coat-
ing, were found to be mostly dispersed in the cell
medium individually or in few-nanotube bundles.
The physical or chemical properties of the nanoma-
terials may be altered in biological systems due to the
surface protein binding, and the nanotubes' surface
charge may affect their interactions with cells or other
biological systems. As a result, it is critical to fully
understand their surface chemistry in addition to their
shape and size, especially when introduced into the
medium used for cellular cultures. Therefore, we mea-
sured the surface charge of the carbon nanotubes
(before and after PEGylation) in cellular media. The
zeta potential values of the as-obtained SWCNTs and
?15.53 and ?30.14 mV, respectively, indicating that
SWCNT-PEGs are possibly more stable in medium
suspension compared to the uncoated SWCNTs. Both
species of nanotubes were found to be stable in the
cellular media during the duration of the experiments.
To further prove the PEG coating of the nanotubes,
we performed Raman scattering analysis, which is a
characterize various organic and inorganic materials. It
is commonly used to characterize carbon nanotubes,
since it provides extremely important information
about the structure and purity of such graphitic
nanomaterials.60The D band and G band are charac-
teristic of the graphitic carbon structures of the
SWCNTs. The D band is associated with the defects
present in the graphitic structure, and the G band
corresponds to the E2gmodes (stretching vibrations).
After the SWCNT surface was functionalized by PEG
groups, it was found that the Raman D band intensity
increased. In this case, the relative intensity value of ID/
IGincreased from 0.075 to 0.71, as shown in Figure 2,
indicating the presence of more noncrystalline struc-
tures on the surface of the SWCNTs. At the same time,
the D band shifted to 1585 cm?1, which can be
attributed to the PEG polymer coating.61This finding
showedthat the magnitudeofthe upshiftiscorrelated
with the bundles or individual SWCNTs coating with
polymer. The low magnitude of upshift would suggest
that the SWCNT-PEG sample was composed largely of
individually dispersed SWCNTs.
Toxicity Evaluation. Previous studies have shown that
CNT nanomaterials elicit toxicity in both cell cultures
and rodent models.32?43However, SWCNT-PEG, a pro-
mising functionalized carbon nanomaterial given its
structure and properties, is expected to have potential
applications in the nanomedicine field.22,20Therefore,
the cytotoxicity of SWCNT and SWCNT-PEG was deter-
mined using multiple end point evaluation, and the
potential toxic mechanism was investigated.
It hasbeen noted thatmultiple approaches should
be used in nanotoxicology evaluation studies.62
The MTT assay, a commonly employed method to
evaluate the adverse effect of nanomaterials in cell
Figure 1. Diagram of the experimental procedure. Single-walled carbon nanotubes (SWCNTs) and PEG-coated SWCNTs
(SWCNT-PEGs) were incubated with PC12 cells, and their toxicity was assessed by a combination of biochemical and gene
ZHANG ET AL. VOL. 5
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culture, was used to measure the mitochondrial func-
tion of the cells. However, it has been reported that
MWCNTs may affect the MTT assay and sometimes
provide false positive results.63Coccini et al. evalu-
ated the cytotoxicity of MWCNTs by different meth-
ods in vitro and indicate that MWCNTs appear to
interact with tetrazolium salts in the medium, thus
reflecting a false positive cytotoxicity.64Another
study has shown differential cytotoxicity results of
a modified procedure was used in our studies to
attempt to avoid the interference. Two additional
steps were involved in the MTT testing procedure.
CNTs in the medium were extensively washed before
adding the MTT solution, and the supernatant was
transferred to another plate for reading after centri-
been reported that the XTT assay is a good approach
for MWCNT toxicity evaluation,63and thus we verified
the mitochondria cytotoxicity by using the XTT assay.
mitochondria toxicity was also observed in our com-
parative studies involving MTT and XTT assays. It has
Figure 2. Physical and chemical characterization of SWCNTs and SWCNT-PEGs. The sizes, size distribution, shapes, surface
coating, surface charges, and purities were characterized using differential techniques described in the Methods.
ZHANG ET AL. VOL. 5
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the most sensitive methods for cytotoxicity evalua-
could not exclude the possibility that the MTT salts
may have interactions with the SWCNTs like MWCNT
and produce false positive results. Further investiga-
tions need to be conducted, and an evaluation of
SWCNT using the MTT assay should be additionally
validated by other methods. For the MTT analysis, the
in a concentration-dependent manner after 24 h of
effects in the PC12 cells were observed for both the
SWCNTs and SWCNT-PEGs at high concentrations
(100 μg/mL) using MTT and XTT assays (Figure 3A,B).
SWCNT-PEG was found to be significantly less toxic
than uncoated SWCNT in high concentration, sug-
gesting that PEG-functionalized SWCNTs possess a
higher level of biocompatibility compared to the
LDH release is a method of measuring the mem-
brane damage, a hallmark of necrosis. In our studies,
significant LDH release was noted after 24 h of expo-
after functionalization, indicating the less cytotoxic
effect of the SWCNT-PEG nanotubes compared to the
Figure 3. Effect of surface modification of SWCNTs on
mitochondrial toxicity and membrane damage evaluated by
(A) MTT assay, (B) XTT assay, and (C) LDH release (cell
concentrations of nanomaterials for 24 h. At the end of the
incubation period, MTT reduction, XTT reduction, or LDH
assays were performed to evaluate the cytotoxicity as de-
from three independent experiments. (*) indicates statisti-
cally significant from control; (#) indicates statistically signif-
icant within the same concentration group (p < 0.05).
Figure 4. Morphological changes induced by SWCNTs and
SWCNT-PEGs in PC12 cells after incubation of the carbon
nanotubes for 24 h. (A) Normal morphology of the PC12
cells. (B) SWCNTs appear to induce PC12 cells to form the
dendrite growth (arrows).
ZHANG ET AL.VOL. 5
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uncoated SWCNTs. These results are in line with the
already published reports that indicate the role that
surface modification plays in nanoparticle cellular up-
take, bioactivity, and possible toxic effects.67However,
the lesser cytotoxicity of SWCNT-PEGs suggested that
materials. These mitochondrial effects and mem-
brane damages can be explained by the surface func-
tionalization of these nanomaterials and their biological
interaction with the cellular systems.
an elongated shape, while SWCNT-PEGs donot seem to
the cellular shape induced by the SWCNTs is in good
agreement with our previously published studies that
showed thatosteocytic bonecells change their shapein
a similar manner when exposed to SWCNTs.10This
shape/size change was definitely related to the higher
SWCNTs and SWCNT-PEG were found to accumulate in
the cells after 4 h of exposure (Figure 5). As previously
presented, the “snaking” effects of the SWCNTs, given
their tubular shape, promote penetration of various
membranes, uptake by cells, and strong interactions
with various protein systems.11Due to their surface
functionalization, the SWCNT-PEG nanostructures were
expected to have less interaction with the cellular
membranes. It has been reported that different degrees
of agglomeration of SWCNTsinfluencethe cellprolifera-
accumulation of SWCNTs on the cell membranes and
cytoplasm after 4 h of incubation may partially contri-
bute to their toxic properties. During these studies
(optical microscopy and Raman spectroscopy), we did
not observe variations in the amounts of SWCNTs or
SWCNT-PEGs that were internalized by the cells,
although this is a topic for further investigation.
Toxicity Mechanisms. Several studies have shown that
nanomaterials could induce oxidative stress, which
may be considered as a biomarker of toxicity to many
nanomaterials.68,69In biological systems, the oxidation
Figure 5. Intracellular distribution of SWCNT-PEGs in PC12 cells. (A) Optical image of PC12 cells that were incubated at 37 ?C
for 4 h in PEGylated SWCNT dispersion. The colored circles in the optical micrographs represent the specific regions of the
PC12 cell where spectra were acquired. (B) Colored Raman spectra acquired from cytoplasmic (red circle) and nuclear (green
circle) regions of image (A). SWCNT-PEGs were localized in the cytoplasm evaluated using confocal Raman microscopy after
4 h of exposure to PC12 cells.
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and reduction are coupled processes supported by a
group of enzymes known as oxidoreductases. This
process involved charged reactive radicals referred to
as reactive oxygen species (ROS) such as superoxide
(H2O2). ROS, as a short half-life molecule, could be
inactivated by antioxidant molecules and associated
with the enzymatic activities of oxidoreductases.
Although ROS play important roles in maintaining
the physiological functions of the body, the persis-
tence of ROS is generally considered harmful. ROS can
oxidize macromolecules, such as DNA, proteins, and
lipids, resulting in a high degree of cytotoxicity. As a
result, the generation of reactive oxygen species was
investigated in order to determine the toxic mechan-
isms that are responsible for the two types of nano-
materials studied in this work: SWCNTs (uncoated) and
carbon nanotubes on this fluorescence assay, cell-free
medium withnanoparticles only wasused asa control.
quenching the fluorescence by themselves. Figure 6A
shows the measured levels of the generated reactive
oxygen species to be concentration-dependent after
24 h of cellular exposure to the SWCNTs and SWCNT-
PEGs. Surface modification by PEG reduced the capa-
increase in ROS as compared to the control samples,
while the same dose of SWCNT-PEGs induced only a
5-fold increase in ROS as compared with the controls.
On the other hand, 1 μg/mL of SWCNT was found to
induce 3-fold ROS compared with the controls, while
no detectable increase in ROS was noted for the
SWCNT-PEG treatment group in the same concentra-
tion. SWCNTs at 100 μg/mL were found to generate
substantial cell death based on the LDH release assay,
and the intracellular ROS signal decreased compared
the previous findings that have shown that SWCNTs
induce oxidative response in lung epithelial cells at
(GSH), a ubiquitous sulfhydryl containing molecule in
stasis, can scavenge ROS directly and indirectly, playing
an important role in toxicant metabolism.71A significant
depletion of GSH was noted at 10 and 100 μg/mL of
SWCNTs relative to control cells. In contrast, significant
cellular GSH changes were observed at 100 μg/mL of
SWCNT-PEGs. Taken together, GSH depletion and ROS
that the cytotoxicity of SWCNTs is involved in the
oxidative stress mechanism.
To further investigate the SWCNT-induced oxida-
tive stress mechanism, the expression of the oxidative
stress-related genes was further analyzed. Reverse
transcription and real-time PCR array were used in
our studies to evaluate 84 genes related to oxida-
tive stress at the same time. Since real-time reverse
transcription polymerase chain reaction (RT-PCR) is
considered one of the most sensitive and reliable
methods for gene expression analysis, this method
was used to detect alterations in gene expression
levels associated with the toxicity caused by SWCNTs
and SWCNT-PEGs in the PC12 cells. ArrayTack, a public
array data management and analysis software, was
used in this study to analyze and interpret the array
data. ArrayTack was developed at the National Center
for Toxicology Research (NCTR) of the Food and Drug
Administration (FDA) and provided an integrated solu-
tion to manage the array data.72This software tool
examine the data quality, perform statistical analysis,
and link significant gene lists to biological databases
for functional pathway analysis.72In this study, 84
oxidative stress-related genes were examined within
one control group and two treatment groups. There
were three biological replicates in each group. Hier-
archical cluster analysis (HCA) and principle compo-
nent analysis (PCA) were used to determine the gene
Figure 6. Effect of surface modification of SWCNTs on
generation of ROS and GSH levels in PC12 cells. Cells were
treated with different concentrations of nanomaterials for
24 h. At theend of theincubation period,ROS or GSH levels
were determined as described in theMethods section. Data
were expressed as mean ( standard error (SE) based on at
least triplicate observations from three independent ex-
(#) indicates statistically significant within concentration (p
ZHANG ET AL.VOL. 5
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expression profiles for control, SWCNTs, and SWCNT-
PEG-treated groups. HCA showed the gene expression
was very homogeneous within the SWCNTs, SWCNT-
PEGs, and the vehicle treatment group. In other words,
there were three clusters according to these treatment
groups (Figure 7A). It was not surprising that PCA
showed a clear separation among these three treat-
PEG nanomaterials induced distinct gene expression
patterns (Figure 7B). Therefore, the results from HCA
and PCA indicate the high-quality and reproducible
array data in the study.
Toxicogenomics is focused on patterns of global
gene expression as a whole rather than on individual
gene expression. When these tools are used to investi-
gate toxicological mechanisms, the resulting outcomes
are generally complex, makingtheir interpretation diffi-
cult. It should be noted that the fold changes in gene
expression may not be proportional to the biological
significance and relevancedue tothe complexity ofthe
signal transduction and amplification process. Slight
changes of the upstream gene expression of the signal
transduction pathways may cause significant gene
changes in the downstream biological processes. In
related oxidative stress, the criteria of a fold change
in comparison to the vehicle group were used. With
p values of less than 0.05 considered significant, only
3 out of 79 genes were found in common between
SWCNT and SWCNT-PEG exposures. In contrast, there
were a number of genes (12 out of 79) that were
different between SWCNTs and the control group,
while 8 out of 79 genes were found to be different
between SWCNT-PEGs and the control group, respec-
tively. It should be noted that five genes in the PCR
PC12 cells. It appears that more reduced gene expres-
to SWCNT-PEGs compared with SWCNTs, indicating
that distinct gene expression profiles are induced by
specifically, the SWCNTs can induce the up-regulation
in the expression of the genes involved in ROS meta-
bolism, such as Txnip, Nos2, Ucp3, Nox4, and oxygen
transporter gene Hbz, and induce down-regulation in
the expression of the ROS metabolism or antioxidant
genes, such as Idh1, Dhcr24, Gpx3, Scd1, Gpx7, and
Ncf2. However, SWCNT-PEGs induce the down-regula-
tion of ROS-associated oxygen transporter, antioxidant,
Figure 7. Effect of surface modification of SWCNT on oxidative stress genes. (A) Hierarchical cluster analysis (HCA) and (B)
principal component analysis (PCA) of nine array data from PC12 cells treated with SWCNTs, SWCNT-PEGs, and vehicle. Blue
dots, yellow dots, and red dots represent the SWCNTs, SWCNT-PEGs, and vehicle-treated samples, respectively. (C) Venn
diagram of the gene significantly changed by SWCNT or SWCNT-PEG treatment.
ZHANG ET AL.VOL. 5
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and ROS metabolism genes, such as Nox4, Hba-a2,
Noxo1, Fancc, Ptgs1, Noxa1, and Dhcr24. These distinct
gene expression profiles may indicate the unique cel-
PEGs (Table 1). It has been reported that intratracheal
instillation of 0.5 mg of SWCNTs in mice induced
pulmonary alveolar macrophage activation, chronic
inflammation responses, and severe pulmonary granu-
SWCNTs by macrophages resulted in the generation of
oxidative stress in mitochondria involved in manga-
dent oxidatase (NOXs).73Macrophages could take up
triggered in cells. In our study, the SWCNTs were found
to accumulate in the cell membrane and then migrate
to the cytoplasm due to their unique fibrous structure
and surface chemistry. The observation of the effect of
the mitochondrial function change and mitochondrion
gene changes provides evidence of the interactions
between the SWCNT and the mitochondrion in PC12
strongly involved in the generation of the reactive oxygen
species, leading to major pathogenicity of many neurode-
generation diseases.74Therefore, the uptake and cellular
translocation of SWCNTs may partially contribute to the
toxicity of the SWCNTs.
the differential cellular responses of the cells when
exposed to SWCNTs and PEGylated SWCNTs. The up-
regulated and down-regulated gene functions are
shown in Table 2. Genes were up-regulated in most of
the categories after exposure to SWCNTs. Many of the
genes involve nucleic acid (RNA and DNA) biosynthesis,
metabolism,catabolic processes, mitochondria, and oxi-
doreductases and antioxidant activity. The only down-
regulated category was that for the cellular component
organization and biogenesis. Collectively, the data sug-
gest an increase in nucleic acid metabolism and bio-
synthesis, mitochondrial function, and antioxidant
activity after the cell responses to oxidative stress, thus
resulting in protective adaptation to stress. In contrast,
PEGylated SWCNTs result in the up-regulation of lipid
metabolic and biosynthetic processes. The PC12 re-
sponse to the polymer PEG coating is responsible for
the lipid biosynthesis, therefore reducing the cytotoxi-
city. This molecular observation is consistent with the
biochemical and cellular responses. The influence of
SWCNTs on DNA biosynthesis in our studies is an adap-
previous studyin whichmixed neuro-glial cultures were
exposed to 30 μg/mL SWCNTs, which significantly de-
the Hoechst staining assay.32Previous findings have
reported that 4 or 100 nm PEG-coated gold nanoparti-
cles can induce significant gene expression changes
categorized as apoptosis, cell cycle, inflammation, and
metabolic processes in mouse liver tissues.75Through
this analysis, we found that the oxidative damage
induced by SWCNTs primarily results from changes in
well as alteration of mitochondrion functions, while the
results of this analysis are consistent with the other
findings of this study. The SWCNTs appear to be more
TABLE 1. Differentially Expressed Genes Induced by SWCNT or SWCNT-PEG Treatmenta
similar to serine (or cysteine) proteinase inhibitor, clade B, member 1b
thioredoxin interacting protein
nitric oxide synthase 2, inducible
uncoupling protein 3 (mitochondrial, proton carrier)
NADPH oxidase 4
hemoglobin alpha, adult chain 2
NADPH oxidase organizer 1
Fanconi anemia, complementation group C
prostaglandin-endoperoxide synthase 1
NADPH oxidase activator 1
isocitrate dehydrogenase 1 (NADPþ), soluble
glutathione peroxidase 3
stearoyl-coenzyme A desaturase 1
glutathione peroxidase 7
neutrophil cytosolic factor 2
ap value and fold change were considered as treatment versus control group. The genes with p value <0.05 and the absolute value of fold change >1.5 were considered
as significantly changed.
ZHANG ET AL. VOL. 5
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toxic to PC12 cells than SWCNT-PEGs, as demonstrated
by the effect of mitochondrial functions and membrane
damage. This phenotypic biological response is dose-
dependent as well as the capacity to generate oxidative
species at the cellular and molecular levels.
On the basis of our studies, a possible mechanism of
of oxidoreductases, which are involved in 24 oxidative
genes. ROS was generated via a Fenton reaction with
NADP as a cofactor. The lowest level of oxidative stressis
associated with the induction of antioxidant and detox-
ification enzymes such asglutathione. At higher levelsof
depletes the GSH levels, leading to membrane damage
and mitochondria cytotoxicity. In contrast, SWCNT-PEGs
migrated into PC12 cells and appeared to interact with
cells and up-regulate the six genes involved in the lipid
metabolic process as demonstrated in our studies. Fewer
ROS were also generated, which may be attributed to the
mitochondria function was noted in comparison with un-
by using flow cytometry analysis to understand the long-
term, chronic toxicity effects that are responsible for indu-
cing cellular apoptosis or necrosis. The combination of
assays described in this research work proved to have a
high sensitivity and clearly indicate the possible undesir-
chemical compositions, but different surface chemistry
morphologies. On the basis of our results, it can be clearly
concluded that the surface properties of nanomaterials
play important roles in their corresponding biological
responses. Such studies could open the field for the
engineering to cancer targeting and destruction.
city in PC12 cells, and cytotoxicity is involved in the
oxidative stress mechanism. The surface function-
alization of carbon nanomaterials plays an important role
in their interaction within biological systems. The differ-
ential oxidative stress-mediated toxicity mechanisms of
the SWCNTs/SWCNT-PEGs have been shown in this re-
port. In this toxicity evaluation study, the maximum
exposure level of nanomaterials may be higher than the
practical use of carbon-based nanomaterials in various
biomedical applications. However, a lower level of expo-
sure could open the possibility of using nanomaterials
with various coatings in advanced biomedical applica-
tions. This study performed in vitro is the foundation of
further in vivo studies aimed to elucidate the complex
interactions between carbon nanotubes and biological
chemistry ofthese nanomaterials.Further,itisinteresting
to study the possible defense mechanisms developed by
such biological systems to prevent or minimize the toxic
effects of the engineered nanomaterials. Future work will
focus on in vivo mechanistic toxicity studies at low dose
to be a promising strategy for delivering drugs and
growth factors to neuronal cells with potential in treating
major neurodegenerative diseases.
SWCNT Synthesis. Carbon nanotubes were synthesized over
the Fe?Co/MgO catalyst using the RF-CCVD method and
methane as the carbon source.76The rf inductive heating was
performed using an rf generator operated at a radio frequency
of 350 kHz. A 200 mg amount of catalyst was placed in a
TABLE 2. Gene Functions (gene ontology) Dysregulated
by SWCNTs or SWCNTs Coated with PEG (p < 0.01)a
number of genes
response to chemical stimulus
cellular catabolic process
RNA metabolic process
transcription DNA dependent
RNA biosynthetic process
regulation of RNA metabolic process
biopolymer catabolic process
DNA catabolic process
macromolecule catabolic process
oxidoreductase activity acting on peroxide
regulation of transcription from RNA
polymerase II promoter
Regulation of transcription DNA dependent
transcription from RNA polymerase II promoter
regulation of cellular component organization
lipid metabolic process
lipid biosynthetic process
ap values were considered as treatment versus control group, and p < 0.01 was
considered as a significant change of the gene function.
ZHANG ET AL.VOL. 5
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graphite boat, and the boat was inserted into a quartz reactor.
The catalysts were heated in nitrogen (flow rate of 600 mL/min)
and were annealed at 700 ?C for 1 h. All of the synthesis
reactions were performed at 850 ?C with a methane flow rate
of 80 mL/min, while keeping the nitrogen flow constant. The
temperature of the graphite boat was continuously measured
byan optical pyrometer andwasfound to bevery stable during
a nitrogen flow. The raw SWCNTs were purified by HCl for 24 h
under gentle stirring and washed repeatedly with deionized
water. The suspended nanotubes were separated by vacuum-
filtration using a 0.22 μm Millipore polycarbonate membrane
filter and further washed with water until neutral pH. The
groups through H2SO4/HNO3(3:1) treatment. After consecutive
washing and drying, SOCl2was added and mixed with the CNT-
70 ?C. This process generated COCl-functionalized SWCNTs,
which were further mixed with PEG (Bioworld, biotechnology
grade, MW = 8000); the mixture was then stirred at 140 ?C for
24 h. The resulting solid SWCNT-PEG sample was filtered again
further washed with water. The resulting sample was dried
overnight in a vacuum, generating SWCNT-PEG. The SWNT-PEG
was dispersed into DI water and then put into a dialysis
membrane tubing (cutoff molecular weight is about 12000)
for dialysis to remove the impurities for 2 days before being
cell medium were found to be extremely stable over time and
definitely during theduration ofthe experiments. To exemplify,
the SWCNT-PEG solution was found to remain stable at room
temperature for over six months without any indication of the
agglomeration of the nanotubes. As a result, all of the experi-
ments were performed with nanotubes that were dispersed
either individually or in small bundles, as indicated by micro-
SWCNT and SWCNT-PEG Characterization. Transmission electron
microscopy images were collected on a field emission JEM-
2100F TEM (JEOL Inc.) equipped with a CCD camera. The
acceleration voltage was 100 kV for the SWCNT and SWCNT-
PEG analysis. The nanotube samples were homogeneously
dispersed in 2-propanol under ultrasonication for 30 min. Next,
a few drops of the suspension were deposited on the TEM grid,
dried, and evacuated before analysis. EDX elemental analysis
was collected on a JEOL-7000 (JEOL Inc.) with an accelerating
voltage of 15 kV. The SWCNT sample was mounted on alumi-
num pins, and the corresponding EDX elemental spectra and
data were obtained.
Zetasizer Nano ZS (Malvern Instruments) using dynamic
light scattering (DLS) techniques was an ideal tool for charac-
terization of the hydrodynamic size and zeta potential of the
nanomaterial in solution. SWCNT or SWCNT-PEG samples were
measured after dilution of nanomaterial stock solutions to 100
μg/mL suspensions in RPMI 1640 media. The nanomaterial
suspension was vortexed to a homogeneous solution, and
1 mL of suspension was transferred to a plastic cuvette or clear
zeta potential cell for measurement following the manufac-
Cell Culture. The PC12 cell line obtained from the American
Type Culture Collection (Manassas, VA) was maintained at 37?C
(5% CO2, 95% air) in RPMI-1640 media (Sigma 8758) supple-
mented with 5% fetal bovine serum (Atlanta Biologicals), 10%
horse serum (ATCC), and 1% penicillin/streptomycin (Sigma).
Confluent cells were harvested with 0.25% trypsin/EDTA solu-
tion (Invitrogen). PC12 cells were allowed to attach on 96-well
plates for 48 h before treatment with carbon nanotubes in
serum-free, phenol red-free RPMI media.
MTT Assay. The colorimetric MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide, Sigma) test assesses cell
metabolic activity based on the ability of the mitochondrial
succinate/tetrazolium reductase system to convert the yellow
dye (MTT) to a purple formazan in living cells. The metabolic
activity of the cell is proportional to the color density formed.
Briefly, PC12 cells were incubated with various concentrations
of SWCNT or SWCNT-PEG for 24 h, then gently washed three
times with PBS (pH 7.4) followed by replacement with 90 uL of
serum-free medium. To avoid potential interferences of SWCNT
in this method, SWCNTs in the medium or attached to the cell
membrane were extensively washed. A10μL portion ofan MTT
stock solution (5 mg/mL) was added to each well followed by
incubation for 4 h at 37 ?C. The supernatant was then removed,
and the formazan in cells was dissolved with 100 μL of DMSO.
Then the fresh supernatant was transferred to another micro-
The absorbance was determined using a microplate reader
(Biotek, USA) at 550 nm with a reference wavelength of
680 nm. H2O2(0.5 mM) was used as a positive control. The
subtracted absorbance of cell free blank was calculated, and
data are presented as percent survival of control cells.
XTT Assay. The cell viability of PC12 cells following exposure
to SWCNT or SWCNT-PEG was determined by measuring the
to a soluble, orange-colored formazan derivative. Briefly, the
cells were treated with different concentrations of SWCNT or
SWCNT-PEG for 24 h, then gently washed three times with PBS
(37.5 μM final concentration), and further incubated for 2 h at
ambient temperature. H2O2(0.5 mM) was used as a positive
control in this experiment. The absorbance was determined
using a microplate reader (Biotek, USA) at 450 nm with a
reference wavelength of 680 nm. The absorbance data were
analyzed and corrected for interference by subtracting absor-
reagents. The subtracted absorbance of control cells (nano-
particle free media) was calculated, and data are presented as
percent survival of control cells.
LDH Assay. Cell membrane integrity can be measured by
lactate dehydrogenase (LDH) leakage assay. LDH leakage was
measured using a cytotoxicity detection kit (Roche Applied
Science). Extracelluar LDH was measured with a coupled enzy-
matic assay that results in the conversion of a tetrazolium salt
(INT) into ared formazan product. In thisexperiment, PC12cells
cultured with 1% Triton-X-100 were used as positive control,
and cells cultured in nanoparticle-free media were used as
negative control. Media with each sample only were set as
respective cell-free blank control. PC12 cells (1 ? 105cells/mL)
were treated at 100 μL of different concentrations of SWCNT or
SWCNT-PEG nanomaterials or positive control 1% Triton-x-100
for 24 h. Then 50 μL of supernatant was transferred to another
plate followed by the addition of 50 μL of substrate mix. The
plate was incubated for 30 min at room temperature followed
by the addition of 50 μL of stop solution to each well. Absor-
bance was recorded at 490 nm in a plate reader (Biotek, USA)
using a reference wavelength of 680 nm. LDH leakage was
calculated using the following formulation: LDH leakage (%) =
100[(sample absorbance ? cell free sample blank) ? mean
media control absorbance]/(mean Ttriton-X-100 positive con-
trol absorbance ? mean media control absorbance).
Measurement of Reactive Oxygen Species. The intracellular reac-
Probes Inc., Eugene, OR). DCFH-DA is ROS probe that undergoes
intracellular deacetylation, followed by ROS-mediated oxidation
to fluorescent species. DAFH-DA can be used to measure ROS
generation in the cytoplasm and cellular organelles, such as
mitochondria. In this experiment, PC12 cells cultured with
nanoparticle-free media were used as control. Media with each
sample only were set as respective cell-free blank control.
Briefly, the cells were seeded in a 96-well cell culture plate at
a density of 1 ? 105cells/mL in 100 μL of growth media and
cultured for 48 h. PC12 cells were gently washed three times
medium for 30 min. PC12 cells were again washed three times
various concentrations of SWCNT or SWCNT-PEG.The
ZHANG ET AL.VOL. 5
’ NO. 9
fluorescence intensity (indicating ROS levels) of the cells was
determined 24 h after the treatment using a plate reader with
an excitation/emission wavelength of 485 nm/530 nm (Biotek,
USA). The values were analyzed and corrected for interference
by subtracting the value of the nanomaterials and cells (blank
control). H2O2(10 μM) was used as a positive control in this
experiment. The values were expressed as a percent of relative
fluorescent units to positive control wells. All of the procedures
were performed without exposure to light.
Measurement of GSH Content. The content of GSH was deter-
mined using the GSH-Glo Glutathione Assay kit from Preomega
(Madison, WI) according to the manufacturer's protocol. The
assay is based on the conversion of a luciferin derivative into
luciferin in the presence of glutathione, catalyzed by glu-
tathione S-transferase. The signal generated in a coupled reac-
tion with firefly luciferase is proportional to the amount of
glutathione present in the sample. Briefly, PC12 cells were
plated at 10000 cells/well in a 96-well plate or 1.5 ? 105/well
in a six-well plate and allowed to attach for 48 h. Various
amounts of SWCNT or SWCNT-PEG were added, and the cells
were incubated for 24 h. The medium was removed from wells
in 96-well plates, and 100 μL of GSH-Glo Reagent was added. A
GSH standard curve was generated at the same time as
described in the manufacturer's protocol. Media with each
sample only were set as respective cell-free assay controls to
evaluate if the carbon nanomaterials interfere with the ap-
proach. Diethyl maleate (0.5 mM) was used in this study as a
positive control. The reaction was stopped by adding 100 μL of
Luciferin Detection Reagent, and luminescence was measured
after 15 min. The amount of GSH was calculated on the basis of
the standard curve. The cells in six-well plates were harvested
after 24 h exposure followed by the standard Bradford Protein
assay (Bio-Rad, CA). The glutathione content was normalized as
μmol/mg protein. Control cells (9.43 μmol/mg) were used. Data
are presented as percent GSH content of control cells.
SWCNTs or SWCNT-PEGs (10 μg/mL) were incubated with PC12
cells on collagen-coated coverslips lying in the six-well plate for
up to 24 h. The medium was removed, and the PC12 cells were
rinsed three times with sterile PBS. The coverslip containing cells
was sealed on the glass slide using nail polish. Images were
captured using a dark-field microscope (Cytoviva Inc.).
Confocal Raman Microscopy. Raman scattering spectra of the
SWCNTs and SWCNT-PEGs were acquired at room temperature
using a Horiba Jobin Yvon high-resolution LabRam Raman
microscope system equipped with a charge-coupled detector
were set up with a 150 μm entrance slit and a 400 μm pinhole.
The 633 nm laser excitation was provided by a helium?neon
laseroperatingat5mW.Raman shift calibration wasperformed
using the 521 cm?1line of a silicon wafer. In the cellular
distribution study, adherent cells in a coverslip were brought
into focus by transmitted white-light images obtained through
a CCD video camera followed by Raman spectra collection. All
spectra were plotted as the average of three scans.
RNA Isolation. Total RNA was isolated with a mirVana miRNA
isolation kit (Ambion Inc., Austin, TX) from cells treated with
either 10 μg/mL SWCNT, SWCNT-PEG, or vehicle by following
the total RNA isolation procedure suggested by the manufac-
turer. The quality and quantity of RNA were measured by a
Nanodrop ND-1000 spectrophotometer (Nanodrop Technolo-
gies, Rockland, DE). All of the RNA samples have an OD260/280
ratio > 1.9, indicating the high quality of RNA.
Reverse Transcription and Real-Time PCR Array. TotalRNAsamples
were analyzed using an RT2Profiler PCR Array System
(SABioscience, Frederick, MD, #PARN-065C). The first strand
synthesis was carried out by the RT2PCR Array First Strand kit
followed by real-time PCR with Oxidative Stress RT2Profiler PCR
Array on an ABI 7500 PCR machine (Applied Biosystems Inc.,
95 ?C for 10 min, 40 cycles of 95 ?C for 15 s, and 60 ?C for 1 min.
PCR Array Analysis. The PCR array data were analyzed using
PCR array data analysis software provided by SABiosciences.
The raw Ct was extracted by 7500 software V2.0 (Applied
Biosystems) by setting the threshold line as 0.15. The baseline
was automatically determined by the software. The PCR array
system contains built-in positive control elements for the
proper normalization of the data, for the detection of genomic
DNA contamination, for the quality of the RNA samples, and for
general PCR performance. Normalization was conducted by
subtracting the mean Ct value of five house-keeping genes,
Rplp1, Hprt1, Rpl13a, Ldha, and Actb, from the raw Ct value of
each gene present in the PCR array. p-Values and fold changes
were calculated by t test and the ΔΔCt method, respectively.
Gene expression was considered significantly changed when
the fold change was g1.5 and p e 0.05. The normalized Ct
AR) for principle-component analysis and hierarchical cluster
analysis. Functional analysis was carried out by GSEA software.
( standard error based on at least triplicate observations from
three independent experiments. Statistical significance was
determined by two-way analysis of variance (ANOVA) followed
by Dunnett's multiple comparison test. A value of p < 0.05 was
considered statistically significant. The analyses were con-
ducted using the Prism software package (GraphPad Software).
Acknowledgment. This research was supported in part by
an appointment (Y.Z.) to the Research Participation Program at
the National Center for Toxicological Research administered by
the Oak Ridge Institute of Science and Education through an
and the U.S. Food and Drug Administration. Portions of these
(NanoCore) located at the U.S. Food & Drug Administration's
National Center for Toxicological Research (NCTR) and Office of
Regulatory Affairs Arkansas Regional Laboratory (ORA/ARL)
campus. The views presented in this article do not necessarily
reflect those of the U.S. Food and Drug Administration. The
financial support of the U.S. Army Telemedicine and Advanced
Research Center program is highly appreciated. The editorial
assistance of Dr. Marinelle Ringer is also acknowledged.
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