Toxicology 333 (2015) 2536

Contents lists available at ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Effects of nitrogen-doped multi-walled carbon nanotubes compared to

pristine multi-walled carbon nanotubes on human small airway
epithelial cells
Amy L. Mihalchik a,c , Weiqiang Ding b , Dale W. Porter c , Colleen McLoughlin c,
Diane Schwegler-Berry c, Jennifer D. Sisler c , Aleksandr B. Stefaniak d ,
Brandi N. Snyder-Talkington c, Rodolfo Cruz-Silva e, Mauricio Terrones e,f ,
Shuji Tsuruoka e, Morinobu Endo e, Vincent Castranova a , Yong Qian c, *
a

Pharmaceutical and Pharmacological Sciences, West Virginia University School of Pharmacy, Morgantown, WV 26505, United States
Shared Research Facilities, West Virginia University, Morgantown, WV 26505, United States
Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV
26505, United States
d
Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, Morgantown, WV 26505, United States
e
Research Center for Exotic Nanocarbons, Shinshu University, Nagano, Japan
f
Departments of Physics, Chemistry, Materials Science & Engineering, and Center for 2-Dimensional and Layered Materials, The Pennsylvania State University,
University Park, PA 16802, United States
b
c

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 30 January 2015
Received in revised form 13 March 2015
Accepted 18 March 2015
Available online 20 March 2015

Nitrogen-doped multi-walled carbon nanotubes (ND-MWCNTs) are modied multi-walled carbon

nanotubes (MWCNTs) with enhanced electrical properties that are used in a variety of applications,
including fuel cells and sensors; however, the mode of toxic action of ND-MWCNT has yet to be fully
elucidated. In the present study, we compared the interaction of ND-MWCNT or pristine MWCNT-7 with
human small airway epithelial cells (SAEC) and evaluated their subsequent bioactive effects.
Transmission electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and X-ray
diffraction suggested the presence of N-containing defects in the lattice of the nanotube. The NDMWCNTs were determined to be 93.3% carbon, 3.8% oxygen, and 2.9% nitrogen. A doseresponse cell
proliferation assay showed that low doses of ND-MWCNT (1.2 mg/ml) or MWCNT-7 (0.12 mg/ml)
increased cellular proliferation, while the highest dose of 120 mg/ml of either material decreased
proliferation. ND-MWCNT and MWCNT-7 appeared to interact with SAEC at 6 h and were internalized by
24 h. ROS were elevated at 6 and 24 h in ND-MWCNT exposed cells, but only at 6 h in MWCNT-7 exposed
cells. Signicant alterations to the cell cycle were observed in SAEC exposed to either 1.2 mg/ml of NDMWCNT or MWCNT-7 in a time and material-dependent manner, possibly suggesting potential damage
or alterations to cell cycle machinery. Our results indicate that ND-MWCNT induce effects in SAEC over a
time and dose-related manner which differ from MWCNT-7. Therefore, the physicochemical characteristics of the materials appear to alter their biological effects.
Published by Elsevier Ireland Ltd.

Keywords:
Multi-walled carbon nanotubes
Functionalized multi-walled carbon
nanotubes
Reactive oxygen species

1. Introduction
As the eld of nanotoxicology rapidly expands, researchers are
working towards the identication of key toxicity parameters that
can be applied across materials. Since their introduction, multi-

* Corresponding author at: NIOSH, HELD, PPRB, 1095 Willowdale Dr., Morgantown, WV 26505, United States. Tel.: +1 304 285 6286.
E-mail address: [email protected] (Y. Qian).
http://dx.doi.org/10.1016/j.tox.2015.03.008
0300-483X/ Published by Elsevier Ireland Ltd.

walled carbon nanotubes (MWCNT) have been used in a wide

variety of industrial applications due to their unique physicochemical properties; however, these unique characteristics may
pose a threat to workers and the public during production, use, and
disposal (NIOSH, 2013; Oberlin et al., 1976). The high aspect ratio,
surface characteristics, durability, and redox potential of MWCNT
contribute to their bioactivity, therefore suggesting potential
routes for improving their safety prole. (NIOSH, 2013). Pristine
MWCNT (including MWCNT-7) have been shown in numerous
studies to induce acute inammatory and chronic brogenic

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A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

responses both in vitro and in vivo (Kasai et al., 2014; Mercer et al.,
2011; Pacurari et al., 2011; Porter et al., 2010; Snyder-Talkington
et al., 2013a). In order to improve MWCNT safety and biocompatibility, material scientists have begun implementing safety by
design practices in the synthesis of carbon nanotubes (CNT) with
alternative physicochemical properties. With this in mind, nextgeneration MWCNT may be more or less toxic compared to their
rst generation counterparts and may cause toxicity through
alternative mechanisms.
MWCNT-7 have been shown to induce inammation and
brosis in vivo at occupationally relevant doses in mice and rats
(Mercer et al., 2010, 2013; Porter et al., 2010, 2013). At an inhalation
exposure of 5 mg/m3, MWCNT-7 were capable of bypassing the
defenses of the mouse lung due to their small size, with
approximately 84% of the MWCNT-7 found in the alveolar region
one day post-exposure (Mercer et al., 2011). In a 2010 study, Porter
et al. showed that mice exposed to 10, 20, 40 or 80 mg of pristine
MWCNT-7 exhibited short-term polymorphonuclear leukocyte
inltration and lactate dehydrogenase release, and pulmonary
brosis at 7 days post-exposure. A recent inhalation study in mice
suggests that MWCNT-7 are biopersistent, can remain in the lung,
and may travel throughout the periphery approximately one year
post-exposure (Mercer et al., 2013).
A review of MWCNT in vitro studies indicated that exposures up
to 200 mg/ml of MWCNT are commonly used which are irrelavent
to doses achieved within the mouse and human lung (SnyderTalkington et al., 2012). However, some in vitro studies utilize
lower exposures ranging from 0.1 mg/mL to 6 mg/mL that further
support the in vivo results, suggesting that pristine MWCNT,
including MWCNT-7, are capable of inducing molecular signals
potentially responsible for the toxic effects observed in vivo (Ding
et al., 2005; Pacurari et al., 2012; Mishra et al., 2012; SnyderTalkington et al., 2013a,b,c; Wang et al., 2014). In this study, the
dosage of 1.2 mg/ml of MWCNT-7 or ND-MWCNT was selected to
be reective of previously conducted in vivo studies, roughly
correlating with a 60 mg dose of MWCNT in mice based upon the
alveolar surface area of the mouse lung and surface area of a cell
culture dish (Porter et al., 2010). These in vivo doses were roughly
reective of lung burdens due to concentrations of airborne
MWCNT measured in actual workplaces and account for MWCNT
mass median aerodynamic diameter, minute ventilation, and
human alveolar epithelium surface area (Han et al., 2008; SnyderTalkington et al., 2012).
The addition of defects or heteroatoms, such as nitrogen, to
MWCNT can change their crystallinity and reactivity for industrial
use, and may also impact their bioactivity. ND-MWCNT have been
synthesized using a variety of starting materials, catalysts, and
methods, resulting in CNT with varying percentages of nitrogen and
levels of disruption to the graphene lattice. ND-MWCNT are
suspected to grow by a base-growth model limited by diffusion of
carbon atoms toward the catalyst molecules, coverage of the catalyst
sites by amorphous carbon, or constraints brought on by pyridinic Nincorporation (Shari et al., 2012; Terrones et al., 2002). Nincorporation can be pyridinic (sp2 coordinated, N atom part of
hexagon ring connecting two C atoms) or pyrrolic (sp3 coordinated, N
atom part of pentagon ring connecting two C atoms) depending on
the percentage of nitrogen in the CNT and the doping conditions.
Therefore, since ND-MWCNT fabrication is not standardized,
physicochemical characterization is of great importance for
understanding their biological effects and potential toxicities.
Traditional pristine MWCNT have limited electrical conductance compared to doped variants; however, the addition of
nitrogen to the carbon lattice confers MWCNT n-type semiconductor properties (Ayala et al., 2010). ND-MWCNT typically lack a
hollow core common to pristine MWCNT and have bamboo-like
sections walled off by layers of graphene. Nitrogen creates defects

in the MWCNT by forcing rearrangement of the carbons around the

heteroatoms, thereby introducing disorder into the graphene
layers and curvature of the CNT (Ayala et al., 2010). Since nitrogen
has one additional electron as compared to carbon, it is believed
that this extra electron can carry current and also potentially
interact with ROS, which are highly unstable radicals containing at
least one oxygen atom. Tsuruoka et al. (2013a) suggested that
unpaired electrons on the surface of MWCNT may participate in
reactions with ROS. They assessed a variety of MWCNT with
different modications for ROS quenching through electron spin
resonance studies, and showed that doping and surface chemistry
altered the ability of CNT to quench ROS. The ability to produce ROS
is a key characteristic of MWCNT that can be controlled by surface
modication and composition and directly alters MWCNT-induced
potential toxicity in biological systems (Tsuruoka et al., 2013b).
Addition of functional groups to the surface of MWCNT, such as
nitrogen groups, carboxyl groups, polyethelene glycol, or others,
can also alter the uptake and biocompatibility of MWCNT as
recently shown by Li et al. (2013).
ND-MWCNT are used in a variety of applications including gas
sensors, matrix llers in composite materials, eld emission devices,
and improved lithium storage in lithium batteries; however,
information on ND-MWCNT bioactivity is limited (Ayala et al.,
2010). Carrero-Sanchez et al. (2006) showed that ND-MWCNT (24%
nitrogen) are associated with lesser toxicities and pathological
conditions compared to pristine CNT in an in vivo rat model after
nasal, oral, intratracheal, and intraperitoneal exposures up to 5 mg/
kg. Only the highest dose (5 mg/kg) led to hyperplasia, inammatory cell inltration, broblastic proliferation, and some granulomas in ND-MWCNT treated rats, but was lethal in pristine CNTexposed animals. Limited in vitro results suggest their biocompatibility with blood cells with minimal toxicity at higher doses (20 mg/
ml), which could be useful for potential intravenous drug delivery
(Zhao et al., 2011). Elas et al. (2007) have also demonstrated that
ND-MWCNT are the only type of CNT tested that do not reduce cell
viability in amoeba (even at higher doses), thus indicating that these
N-doped CNTare less toxic when compared to other types. However,
others have shown that tube length may play a key role in toxicity,
with longer ND-MWCNT being more toxic than other functionalized
CNT (Boncel et al., 2011).
The present study assessed the bioactivity of ND-MWCNT and
MWCNT-7 at low exposures in h-TERT immortalized human small
airway epithelial cells (SAEC) derived from the lower bronchioles.
Through assessment of physical characteristics, uptake into SAEC,
cell viability, ROS production, and cell cycle analysis, we provide an
in vitro benchmark for ND-MWCNT toxicity as compared to
MWCNT-7 in a model of lung epithelium.
2. Materials and methods
2.1. ND-MWCNT and MWCNT-7
The ND-MWCNT used in this study were a gift from Mauricio
Terrones (Pennsylvania State University, University Park, PA), and
Morinobu Endo, and Shuji Tsuruoka (Shinshu University, Nagano,
Japan). ND-MWCNT were characterized at the Morgantown
National Institute for Occupational Safety and Health and West
Virginia University Shared Research Facilities. The MWCNT-7 used
in this study were originally obtained through the Mitsui & Co., Ltd.
(MWCNT-7, lot #05072001K28) and previously characterized
(Porter et al., 2010).
2.2. ND-MWCNT and MWCNT-7 preparation
For cell culture studies, ND-MWCNT were prepared in
dispersion media (DM) consisting of PBS (pH 7.4, Ca/Mg-free)

A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

27

supplemented with 0.6 mg/ml mouse serum albumin and

0.01 mg/ml 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
as previously described (Porter et al., 2008). DPPC was prepared
fresh as a 1 mg/ml stock solution in 100% ethanol. Transmission
electron microscopy (TEM) micrographs of MWCNT dispersed in
DM demonstrated that DM promotes signicant dispersion of
MWCNT (Porter et al., 2008). ND-MWCNT and MWCNT-7 were
prepared in DM by indirect sonication at 4
C for 5 min (Hielscher
ultrasonic processor, UIS259L; Ringwood, NJ) at amplitude 100%
and cycle 1. Following indirect sonication, the suspension was
directly sonicated for 5 min at 5 W output and 30% duty cycle in
1 min increments (Branson Sonier 450; Danbury, CT). The stock
solution (0.5 mg/ml) of ND-MWCNT or MWCNT-7 was kept at 4
C
and used within 23 weeks. The ND-MWCNT and MWCNT-7 stock
solutions were directly sonicated for 1 min at 5 W output and 30%
duty cycle prior to cell culture experiments. Cells were treated with
DM, ND-MWCNT, or MWCNT-7 at 1.2 mg/ml, a concentration which
was extrapolated from previous murine in vivo exposure experiments based upon alveolar surface area (Porter et al., 2010).

for approximately 2 h before being transferred to an ultra-high

vacuum chamber for analysis. The sample was stimulated by a
focused aluminum K-a X-ray source at 1486 eV energy and 25 W
power with an X-ray spot size of 100 mm. A survey scan was carried
out using an analyzer pass energy of 117.40 eV, and high-resolution
scans for carbon, oxygen, and nitrogen elements were carried out
at an analyzer pass energy of 23.50 eV. The spectra were referenced
to the C1s peak at a binding energy of 284.8 eV.

2.3. Cell culture

2.8. X-Ray diffraction (XRD)

SAEC were a gift from Tom K. Hei (Columbia University, New

York, NY) (Piao et al., 2005). SAEC were cultured in serum-free
SABM medium supplemented with a SAGM SingleQuot kit of
growth factors, cytokines, and supplements (Lonza Walkersville,
Inc., Walkersville, MD). SAEC were maintained in an incubator at
37
C with 5% CO2. For cell culture experiments, SAEC were serum
starved 24 h prior to ND-MWCNT or MWCNT-7 exposure in serumfree SABM medium.

XRD patterns of the MWCNT were collected with a PANanalytical XPert Pro powder X-ray diffractometer. ND-MWCNT or
MWCNT-7 powders were lled into stainless steel powder sample
holders and radiated with a Cu K-a X-ray source at 1.8 kW power in
a theta/theta scan mode. The XRD spectra of the samples were
collected over a 2-theta range of 1590
at a step size of 0.05
with
a solid state X-ray detector.

2.4. Transmission electron microscopy (TEM)

2.9. Zeta potentials

Samples of ND-MWCNT were diluted in double distilled H2O

(ddH2O) followed by vortexing, and a drop of solution was placed
on a formvar-coated copper grid and allowed to air dry. Images
were photographed under a JEOL 1220 transmission electron
microscope (Peabody, MA). Additionally, SAEC interaction with and
engulfment of ND-MWCNT and MWCNT-7 were then analyzed by
TEM. SAEC were grown to conuence and exposed to 1.2 mg/ml
DM, ND-MWCNT, or MWCNT-7 for 6 or 24 h. Cells were trypsinized
with ReagentPack Subculturing reagents (Lonza Walkersville, Inc.,
Walkersville, MD) per manufacturer guidelines and harvested by
centrifugation at 400  g for 5 min. Cells were xed in Karnovksys
xative (2.5% glutaraldehyde and 3% paraformaldehyde in 0.1 M
sodium cacodylate, pH 7.4), washed three times in 0.1 M sodium
cacodylate, and postxed in 1% osmium tetraoxide, followed by
washing with 0.1 M sodium cacodylate and distilled water. The
cells were dehydrated by sequential washings in 25, 50, and 100%
ethanol and embedded in LX-112 (Ladd, Williston, VT). Ultrathin
sections were stained with uranyl acetate and lead citrate and
examined with a JEOL 1220 transmission electron microscope.

The electrophoretic mobility (motion of particles relative to a

uid under the inuence of an electric eld) of particles was
determined using light scattering in an applied electric eld and
converted to values of zeta potential (the potential difference
between a dispersion medium and stationary layer of uid
attached to a particle in the medium) using the Henry equation.
For each material, an independent sample was drawn for
determination of electrophoretic mobility. The instrument operability was previously veried using U.S. National Institute of
Standards and Technology Standard Reference Material 1980:
Positive Electrophoretic Mobility, having a certied value for
mobility of 2.53  0.12 mm cm/(V s), corresponding zeta potential
of 32.5 mV at 25
C, and prepared using the protocol described in
the SRM certicate. The pH of the experimental samples was
determined after measurement of electrophoretic mobility using a
calibrated electrode attached to a volt meter. The pH probe tip was
rinsed thoroughly with 18 MV cm ddH2O and blotted dry before
measurements. The PBS had a pH of 7.2, and SAEC serum-free
media had a pH of 7.6. The parameters for the dispersants were
based on PBS (refractive index = 1.334, viscosity = 0.9110 cP, dielectric constant = 79.0, and Smoluchowski approximation, f(ka)
value = 1.5). Zeta potential is used as an indicator of the stability
characteristics of a dispersion. Riddick (1968) developed categories
to describe the stability of dispersions based on zeta potential:
threshold of agglomeration (10 to 15 mV), threshold of delicate
dispersion (16 to 30 mV), moderate stability (31 to 41 mV),
fairly good stability (41 to 60 mV), and very good stability
(61 to 80 mV). All measurements were performed at 25
C using
a Malvern Zetasizer Nano ZS90 (Worcestershire, UK) equipped
with a 633 nm laser at a 90
scattering angle. Samples were
equilibrated inside the instrument for 2 min, and ve measurements (10 s delay between measurements), each consisting of ve
runs (2 s delay between runs), were recorded.

2.5. Field emission scanning electron microscopy (FESEM)

The particles were dispersed in ddH2O and ltered with a
0.2 mm nucleopore lter. The lter was attached with double-stick
carbon tape on an aluminum mount and sputter coated with gold/
palladium. Images were collected on a Hitachi (Tokyo, Japan)
S-4800 eld emission scanning electron microscope.
2.6. X-ray photoelectron spectroscopy (XPS)
XPS analysis was carried out with a Physical Electronics
VersaProbe 5000 XPS (Chanhassen, MN). ND-MWCNT powder
was pressed into a small pellet and evacuated in an entry chamber

2.7. Raman spectroscopy

Raman spectra of the MWCNT were collected using a Renshaw
Invia Raman spectrometer with a CCD detector (Hoffman Estates,
IL). ND-MWCNT or MWCNT-7 powders were directly placed on a
microscope glass slide, which was then mounted under the 50
objective lens of the Raman microscope. Samples were excited
with a 532 nm green laser at around 0.23 mW laser power for data
collection.

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A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

2.10. ROS production

ROS measurements by confocal microscopy were performed
according to methods previously described (Qian et al., 2010). SAEC
were cultured on glass cover slips, serum starved 24 h, and exposed
to 1.2 mg/ml DM, ND-MWCNT, or MWCNT-7 for 6 or 24 h.
Dihydroethidium (DHE) (SigmaAldrich, St. Louis, MO) was added
at a nal concentration of 5 mM for the last 30 min of exposure in
the dark. After incubation, cells were washed twice with PBS
(pH 7.4), xed with 4% paraformaldehyde, washed three times over
15 min with PBS, and mounted on slides with ProLong Gold antifade (Invitrogen; Carlsbad, CA). A Zeiss LSM 510 microscope was
used to obtain images with a 20 objective. DHE staining was
determined by uorescence at 546 nm. Five independent experiments were carried out with representative images taken from
each slide. Images from a single experiment overall reective of
these experiments are found in Fig. 7.
2.11. Cell proliferation assay
A doseresponse assessment of cellular proliferation was
carried out by seeding SAEC (1 104 cells) in 100 ml complete
media in 96-well plates, followed by 24 h serum starvation prior to
exposure to DM, ND-MWCNT, or MWCNT-7 for 24 h at 0, 0.12, 1.2,
12, or 120 mg/ml. Twenty microliters of CellTiter 961 Aqueous One
Solution (Promega; Madison, WI) was added to each well during
the last 4 h of exposure. Absorbance was read at 490 nm using a
BioTek Synergy H1 plate reader (Winooski, VT), and statistical
analyses were carried out using the Data Analysis pack in Microsoft
Excel (Redmond, WA). Three experiments were performed in
triplicate; treatment groups were averaged by triplicate and
compared using two sample t-tests assuming unequal variances.
Error bars are reective of standard error.
2.12. Cell cycle assessment
SAEC were grown to subconuency, serum starved for 24 h, and
treated with 1.2 mg/ml DM, MWCNT-7, or ND-MWCNT. Cells were
collected by trypsinization, followed by centrifugation at 1100  g
for 7 min. Cell pellets were washed in PBS and subsequently
resuspended in 100 mL PBS. Cells were xed in 70% ethanol and
stored at 4
C until assessed by ow cytometry. Fixed SAEC were
centrifuged and washed twice with PBS, treated with 50 mL of
100 mg/ml RNase (SigmaAldrich, St. Louis, MO), and stained with
a nal concentration of 10 mg/ml propidium iodide (Sigma
Aldrich, St. Louis, MO). Samples were run on an LSRII cell analyzer
(BD Biosciences, San Jose, CA). Ten thousand events were collected
per sample, and analysis was carried out using FlowJo software
(Ashland, OR). Gating was set to exclude debris and only include
cells by forward and side scatter. Gating for singlet discrimination
was through pulse width versus area for phycoerythrin, forward
scatter, and side scatter. The Watson model was used to determine
the percentage of cells in each phase. Cells in each phase were
analyzed from two independent trials in biological triplicate (n = 6)
from which 10,000 events were collected from each individual
sample. Statistical analysis was done using the Data Analysis pack
in Microsoft Excel (Redmond, WA). Treatment groups were
compared using two sample t-tests assuming unequal variances
and error is presented as standard error in Table 2.
2.13. Western blotting
Whole cell protein extraction was carried out using RIPA buffer
(150 mM NaCl, 10 mM Tris pH 7.4, 2 mM EDTA, 1% IGEPAL, 1%
sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented
with a 10 mg/ml protease inhibitor cocktail and 10 mg/ml

phosphatase inhibitor (Thermo Fisher Scientic, Waltham, MA).

Protein concentration was measured using a BCA protein assay kit
(Thermo Fisher Scientic, Waltham, MA). Twenty micrograms of
protein per sample was resolved using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
polyvinylidene uoride membranes (PVDF, Pall Corporation;
Pensacola, FL). Membranes were blocked for 1 h in 5% bovine
serum albumin (Thermo Fisher Scientic, Waltham, MA) in Tris
buffered saline with Tween-20 (TBST) (62.5 mM Tris pH 7.4,
150 mM NaCl, 0.05% Tween 20) (Thermo Fisher Scientic,
Waltham, MA) and incubated with CDK4, phospho-tyrosine, or
phospho-threonine (Cell Signaling Technology, Boston, MA)
primary antibodies overnight at 4
C. Membranes were washed
three times with TBST for 10 min each and incubated with speciesspecic secondary antibodies for 1 h at room temperature. Analysis
was carried out using ECL Western Blotting Substrate (Thermo
Fisher Scientic, Waltham, MA). Representative western blots are
shown in Figs. 8 and 9 from a minimum of three independent
experiments per antibody. Densitometry was carried out using
ImageJ software (NIH, Bethesda, MD) on three representative
western blots normalized to respective b-actin blots per antibody.
Error bars in the bar graphs show standard error.
3. Results
3.1. Characteristics of ND-MWCNT and MWCNT-7 in suspension
Low magnication TEM (Fig. 1A, B) and FESEM (Fig. 1C, D)
indicated that the ND-MWCNT were 5.28  2.07 mm long and
79.7  19.4 nm wide, after assessment for size in Adobe Photoshop
CS5 (San Jose, CA). The ruler tool was used to measure single
nanotubes from the TEM and SEM images. As previously
determined, the MWCNT-7 were determined to be 3.86 (GSD
1.94) mm long and 49  13.4 nm wide (Porter et al., 2010) (Table 1).
Zeta potentials were measured for ND-MWCNT and MWCNT-7 in
DM diluted in PBS and serum free SAEC media. In PBS (pH 7.2),
values were determined to be 13.9  0.485 mV and 15.8  0.450
mV, respectively. In serum free media (pH 7.6), values were
determined to be 12.9  0.835 mV and 12.2  0.283 mV, respectively (Table 1), and there was no appreciable difference in zeta
potentials between particles in PBS and those in serum free SAEC
media.
3.2. Physical characteristics of ND-MWCNT and MWCNT-7
Several analytic methods were used to assess the physical
characteristics of the ND-MWCNT and MWCNT-7, including XPS,
Raman spectroscopy, and XRD. The MWCNT-7 were previously
characterized by Porter et al. (2010) as pristine MWCNT with
2050 walls per nanotube and with minimal trace metal
contamination, including sodium (0.41%) and iron (0.32%). XPS
determined the elemental composition and surface chemistry of
the ND-MWCNT. The XPS survey scan revealed the existence of
carbon, oxygen, and nitrogen elements within ND-MWCNT. The
corresponding atomic concentration was determined from high
resolution spectra of carbon, oxygen, and nitrogen elements,
showing that the ND-MWCNT were composed of approximately
93.3% carbon, 3.8% oxygen and 2.9% nitrogen (Fig. 2A). The spectra
of these elements were analyzed, and different chemical states
were identied. The carbon atoms were found to be in three
functional groups: sp2-hybridized graphitic carbon (284.8 eV),
C
N (285.9 eV), and CO (287.3 eV) (Fig. 2B). The nitrogen atoms
were found to be in two chemical states: amino 
NH2 (399.0 eV)
and pyrrolic CN (400.3 eV) types (Seran et al., 2012; Zhang et al.,
2013) (Fig. 2D). The oxygen atoms were found to be in two
chemical states: CO (531.8 eV) and 
OH (533.6 eV) (Fig. 2C).

A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

[(Fig._1)TD$IG]

29

Fig. 1. TEM of ND-MWCNT at 5000 (A) and 15000 (B) and eld emission scanning electron micrographs of ND-MWCNT at 2000 (C) and 5000 (D).

These XPS results further showed the inclusion of nitrogen atoms

into the graphitic carbon lattice of ND-MWCNT (pyrrolic C
N
bonding) and the existence of lattice structural disorder.
To further characterize these materials, Raman spectroscopy
was used to assess the carbon lattice structure condition of NDMWCNT and MWCNT-7 samples. Raman peaks at around
1358 cm1, 1590 cm1, and 2700 cm1 were identied and assigned
to the D-band, G-band, and 2D(G0 )-band of carbon structure
(Ferrari and Basko, 2013). The intensity ratio of the defect-derived
D-band to the G-band (ID/IG), an indicator of the structural
integrity of the graphitic carbon lattice, was calculated based on
Raman peak tting results. A D-band to G-band intensity ratio of
0.132 was obtained for MWCNT-7, while the ND-MWCNT had a
ratio of 1.440, indicating signicant structural disorder induced in
the graphitic carbon lattice of the ND-MWCNT due to nitrogen
doping (Fig. 3).
XRD was performed to study the crystallinity condition of the
MWCNT. The strong diffraction peaks at 26.3
and 42.5
were
assigned to the (0 0 2) and (1 0 0) diffractions of the graphitic lattice
of the MWCNT (Zhao et al., 2014). The full width at half maximum
(FWHM) values of the (0 0 2) peak in MWCNT-7 and ND-MWCNT
spectra were 0.91
and 1.21
, respectively. Compared to MWCNT-7,

the ND-MWCNT had a broader (0 0 2) peak with lower intensity,

indicating a decrease of graphene crystallinity due to nitrogen
doping induced structural damage (Fig. 4).
3.3. ND-MWCNT and MWCNT-7 exposure alters SAEC proliferation
An MTS assay was carried out to determine the effects of NDMWCNT and MWCNT-7 on SAEC proliferation. SAEC were treated
with DM or 0.12, 1.2, 12, and 120 mg/ml of ND-MWCNT or MWCNT7 for 24 h. SAEC treated with 0.12, 1.2, and 12 mg/ml ND-MWCNT
exhibited a trend toward increased proliferation, which decreased
at 120 mg/ml when compared to DM. Treatment of SAEC with
MWCNT-7 at 0.12 mg/ml signicantly increased proliferation
compared to DM and 0.12 mg/mL ND-MWCNT, while treatment
with 1.2 mg/ml and 12 mg/ml exhibited a lessened proliferative
effect compared to DM. At 120 mg/ml, MWCNT-7 signicantly
decreased proliferation below that of DM (Fig. 5). In summary,
MWCNT-7 signicantly enhanced epithelial cell proliferation at a
lower dose than ND-MWCNT. Additionally, a high dose of 120 mg/
mL of MWCNT-7 was cytotoxic to the SAEC, while ND-MWCNT was
not. Therefore, MWCNT-7 appear more potent in inducing
proliferation and cytotoxicity than ND-MWCNT. A dose of

Table 1
Zeta potentials of ND-MWCNT and MWCNT-7 in PBS (pH 7.2) and serum-free media (pH 7.6).
Dimensions
ND-MWCNT
MWCNT-7

Length (mm)
Width (nm)
Length (mm)
Width (nm)

5.28  2.07
79.7  19.4
3.86 (GSD 1.94)
49  13.4 nm

Zeta potential (mV)

(Mean  standard error)

PBS (pH 7.2)

Serum-free media (pH 7.6)
PBS (pH 7.2)
Serum-free media (pH 7.6)

13.9
12.9
15.8
12.2






0.483
0.835
0.450
0.283

30

[(Fig._2)TD$IG]

A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

Fig. 2. X-ray photoelectron spectroscopy survey scan spectrum of ND-MWCNT sample (A), high resolution X-ray photoelectron spectroscopy spectra for carbon (B), nitrogen
(C), and oxygen elements (D).

1.2 mg/ml was selected for further studies as it was previously

established as an occupationally relevant dose capable of inducing
cellular effects in SAEC upon treatment with the particles studied
here (Snyder-Talkington et al., 2013b,c,c). Also, no signicant
difference was noted between proliferation of SAEC treated with
1.2 mg/ml of ND-MWCNT or MWCNT-7.

[(Fig._3)TD$IG]

3.4. ND-MWCNT and MWCNT-7 uptake by SAEC

SAEC were treated with DM (Fig. 6A), ND-MWCNT (Fig. 6BD),
or MWCNT-7 (Fig. 6EG) and imaged for particle interaction and
uptake through TEM. ND-MWCNT and MWCNT-7 varieties were
found at the cell surface at 6 h (Fig. 6B, C or E, F, respectively),
followed by internalization at 24 h (Fig. 6D or G, respectively).

[(Fig._4)TD$IG]

Fig. 3. Raman spectra of ND-MWCNT and MWCNT-7 samples.

Fig. 4. X-ray diffraction spectra of ND-MWCNT and MWCNT-7 samples.

A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

[(Fig._5)TD$IG]

31

ND-MWCNT and MWCNT-7 were examined to determine their

ability to induce ROS production in SAEC. SAEC exposed to DM
(Fig. 7A), ND-MWCNT (Fig. 7B, C), or MWCNT-7 (Fig. 7D, E) were
stained with DHE to assess ROS production at 6 and 24 h. ROS
production peaked at 6 h and remained higher than control at 24 h
in ND-MWCNT treated cells (Fig. 7B, C). MWCNT-7 appeared to
induce ROS production at 6 h that resolved to baseline levels at 24 h
(Fig. 7D, E). The more rapid response to MWCNT-7 is consistent
with greater potency compared to ND-MWCNT.
3.6. ND-MWCNT and MWCNT-7 exposure affects SAEC cell cycle and
CDK4 expression

Fig. 5. SAEC were treated with varying concentrations of DM, ND-MWCNT, or

MWCNT-7 for a 24 h period. CellTiter 961 Aqueous One Solution was added 4 h prior
to end of exposure and absorbance was measured at 490 nm. Results indicate that
MWCNT-7 induces a signicant increase in cell proliferation at 0.12 mg/ml
compared to DM and ND-MWCNT at 1.2 mg/ml and decrease at 120 mg/mL
compared to DM. * p  0.05.

3.5. ND-MWCNT and MWCNT-7 exposure induces ROS production in

SAEC
Some bers, including MWCNT-7, are capable of inducing the
production of ROS upon interaction with various cell types (Apopa
et al., 2009; Huerta-Garcia et al., 2014; Pichardo et al., 2012; Shi
et al., 2014; Snyder-Talkington et al., 2013b). In this experiment,

The cell cycle, which regulates cell duplication and division, can
be altered by MWCNT (Ding et al., 2005; Siegrist et al., 2014; Zhang
and Yan, 2012). Here, the effects of ND-MWCNT and MWCNT-7 on
the cell cycle were compared. SAEC were treated with DM, NDMWCNT, and MWCNT-7 for 6 or 24 h prior to xation and cell cycle
analysis. Propidium iodide staining was used to examine potential
ND-MWCNT and MWCNT-7-related changes to the cell cycle.
Results are presented as percentage of cells in each phase in
Table 2. Results indicate that MWCNT-7 induce a signicant change
in the percentage of cells in G2 compared to DM at 6 h, suggestive
of cell cycle dysfunction. At 24 h, a signicant difference in
percentage of cells in G1 and G2 was noted in SAEC exposed to NDMWCNT compared to MWCNT-7. A signicant increase was noted
between 6 and 24 h in G2 in ND-MWCNT exposed cells. Signicant
changes were noted between 6 and 24 h in G1 and S phase in
MWCNT-7 exposed cells (Table 2). To gain a better understanding
of a molecular basis for the cell cycle dysfunction, whole cell
lysates were subjected to SDS-PAGE and probed for CDK4, a serine/
threonine cyclin dependent kinase important to the G1 transition
prior to synthesis of new DNA in S phase (Baker and Reddy, 2012).

[(Fig._6)TD$IG]

Fig. 6. TEM images of SAEC treated with (A) 1.2 mg/ml DM, (BD) ND-MWCNT, or (EG) MWCNT-7 for 6 h (column 2, 3) or 24 h (column 4).

32

[(Fig._7)TD$IG]

A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

Fig. 7. Confocal images of SAEC treated with 1.2 mg/ml DM (A), ND-MWCNT (B, C), or MWCNT-7 (D, E) over 6 (B, D) or 24 h (C, E) followed by staining with DHE for presence of
ROS.

We observed an increase in CDK4 expression in SAEC exposed to

ND-MWCNT over 6 h, followed by a decrease at 24 h, which is
reective of the propidium iodide cell cycle analysis results.
CDK4 does not appear to play a role in the molecular mechanism
employed by MWCNT-7 to induce cell cycle arrest, which was
expected because CDK4 should not be active during G2 (Fig. 8).
3.7. ND-MWCNT and MWCNT-7 exposure induces phospho-tyrosine
and phospho-threonine
The majority of cell signaling processes involve protein
phosphorylation, especially signal transduction pathways and
those essential to cell cycle regulation, cell differentiation, and cell
maintenance (Hunter, 1995; Marshall, 1995). To better understand
how ND-MWCNT and MWCNT-7 may affect these processes,
general cell signaling pathways were probed using pan-phosphotyrosine and phospho-threonine antibodies. SAEC were exposed to
DM, ND-MWCNT, or MWCNT-7 over 6 or 24 h, and whole cell
lysates were collected using RIPA buffer with protease and
phosphatase inhibitors. Lysates were subjected to SDS-PAGE,
transferred to PVDF membranes, and exposed to phosphoantibodies. Phospho-tyrosine expression increased at 6 h for
MWCNT-7 exposed SAEC, suggesting a time-dependent activation

of signaling pathways (Fig. 9A). Changes in phospho-threonine

expression following MWCNT exposure were less obvious (Fig. 9B).
4. Discussion
Although MWCNT have been in production for over twenty
years, functionalization offers an avenue to increase their
dispersibility and industrial applications across a variety of elds.
Surface functionalizations and substitution reactions (with

[(Fig._8)TD$IG]

Table 2
SAEC were grown to subconuence and treated with 1.2 mg/ml DM, ND-MWCNT, or
MWCNT-7 over 6 or 24 h prior to ethanol xation, staining with propidium iodide,
and assessment using a FACS SR instrument. Results presented indicate an
increased percentage of cells in G2 compared to DM and signicant changes
between 6 and 24 h in G1 and S phase in MWCNT-7 exposed SAEC, while NDMWCNT induced signicant changes between 6 and 24 h in G2. Signicant changes
are also noted at 24 h between MWCNT-7 and ND-MWCNT exposed SAEC at G1 and
G2.
Treatment

%G1  std error

%S  std error

%G2  std error

MWCNT-7 DM
MWCNT-7 6 h
MWCNT-7 24 h
ND-MWCNT DM
ND-MWCNT 6 h
ND-MWCNT 24 h

79.3  2.03
77.8  0.492*
79.6  0.587*
76.1  2.13
79.1  2.59
76.2  0.848*

14.4  1.70
15.1  0.325*
14.2  0.152*
16.1  1.9
14.4  2.06
16.6  0.848

3.37  0.232
4.25  0.307*
3.80  0.344*
4.16  0.091
3.45  0.350*
4.67  0.222*

p  0.05.

Fig. 8. SAEC were grown to subconuence and treated with 1.2 mg/ml DM, NDMWCNT, or MWCNT-7 over 6 or 24 h. Whole cell lysates were resolved by SDS-PAGE
on ten percent gels and probed for CDK4, which appeared to be increased at 6 h in
ND-MWCNT-treated SAEC. b-actin was used as a loading control. Densitometry
results (n = 3) are presented below the blot.

[(Fig._9)TD$IG]

A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

33

Fig. 9. SAEC were treated with 1.2 mg/ml DM, ND-MWCNT, or MWCNT-7 over 6 or 24 h. Whole cell lysates were resolved by SDS-PAGE on ten percent gels and probed for
phospho-tyrosine (A) or phospho-threonine (B) indicating that cell signaling response may be time and particle dependent. b-actin was used as a loading control.
Densitometry results (n = 3) are presented to the right of each blot.

nitrogen or boron, as examples) to the carbon lattice may increase

nanotube reactivity at sites of curvature and breaks in the lattice
(Kuzmany et al., 2004; Meier et al., 2008). The increase in porosity
greatly increases the surface area (and therefore, reactive surface)
of ND-MWCNT, which could have interesting implications for
biocompatibility (Meier et al., 2008). Nitrogen functionalization
(or doping) of CNT has been shown to signicantly increase the
brittleness, chemical reactivity, and n-type semiconductor activity
of this material (Maldonado et al., 2006). Varying amounts of
nitrogen may be incorporated into the carbon lattice, disrupting
sp2 bonding and creating potential active sites for further
functionalization (e.g., drug molecules) or participation in oxygen
reduction reactions (Kundu et al., 2009; Maldonado et al., 2006).
Nitrogen-containing functional groups, such as amines, can also be
attached to the outermost layer of graphene in CNT for use in
biosensors and epoxies (Arrigo et al., 2008; Deng et al., 2009;
Sharma and Shukla, 2014). However, limited information is
available on how functionalization of CNT with nitrogen alters
their bioactivity.
Both pyridinic and pyrrolic nitrogen atoms are located at either
the structural defect sites within the graphene network or at the
sheet edges of the MWCNT walls. Specically, our ND-MWCNT
appear to have pyrrolic N-incorporation. The nitrogen atoms in
MWCNT, especially at the outermost layer, become attraction sites
for oxygen atoms and other functional groups and signicantly
modify MWCNT surface chemistry. The lengths of ND-MWCNT are
often shorter than pristine MWCNT due to different growth
kinetics, which was observed in this study. Fiber length can play a
role in toxicity and longer bers have often been shown to be more
toxic than their shorter counterparts (Donaldson and Poland, 2013;
Poland et al., 2008). Data from the present study suggesting that
ND-MWCNT are less bioactive than pristine MWCNT are as follows:
(1) in the MTS assay, MWCNT-7 enhanced epithelial cell
proliferation at a 10-fold lower dose than ND-MWCNT; (2)
MWCNT-7 caused signicant cytotoxicity in the MTS assay at a

dose of 120 mg/ml, while ND-MWCNT did not; and (3) MWCNT7 induced an increase in phospho-tyrosine signaling at 6 h which
may be linked to signaling cascades such as the MAPK and PI3K
cascades (Ray et al., 2012). Indications that ND-MWCNT may be
less damaging than pristine MWCNT in vivo and in vitro have also
been reported by other labs (Nel et al., 2006). Due to the inert
character and agglomeration of pristine MWCNT, it is possible that
the ND-MWCNT remain better dispersed in vivo and are therefore
less likely to block the small airways in the lungs (Carrero-Sanchez
et al., 2006). In vitro, the hydrophilic nature and surface charge of
functionalized MWCNT, including ND-MWCNT, can alter their
uptake and subsequent toxicity in pulmonary epithelial and
macrophage cells. Previous studies have shown no signicant
activation of the NRLP3 inammasome in ND-MWCNT exposed
cells when compared to undoped MWCNT, making it a potentially
better choice for use in industry to protect workers and the general
public (Li et al., 2013). Boncel et al. (2011) studied the cytotoxicity
of 3 wt.% ND-MWCNT compared to oxidized MWCNT and pristine
MWCNT and suggested that ND-MWCNT are of intermediate
toxicity, but less toxic than the pristine variant.
In the present study, the bioactivities of ND-MWCNT and
pristine MWCNT in short-term exposure of SAEC were compared.
Our data suggests that ROS could potentially initiate the bioactivity
noted in our study. It has been previously shown that MWCNT7 produce no detectable hydroxyl radical alone or in the presence
of H2O2 from the acellular Fenton reaction and instead scavenged
ROS similar to ND-MWCNT which also scavenged ROS in the
acellular Fenton reaction (Porter et al., 2010; Snyder-Talkington
et al., 2013b, unpublished data). This implies that the SAEC were
generating ROS in response to ND-MWCNT exposure. Within the
past decade, ROS has been increasingly linked to a variety of
cellular functions, including growth factor activation, cytoskeletal
changes, and cell cycle progression, as well as contributing to
inammation, oxidative stress, and general cell signaling (Belousov
et al., 2013; Boonstra and Post, 2004; Ray et al., 2012; Verbon et al.,

34

A.L. Mihalchik et al. / Toxicology 333 (2015) 2536

2012). It has previously been shown that MWCNT are capable of

inducing ROS in a variety of cell types (He et al., 2011; Pacurari
et al., 2012; Snyder-Talkington et al., 2013b; Ye et al., 2009). Here,
we showed that ND-MWCNT are capable of inducing ROS in SAEC
in a time-dependent manner. ROS are an important part of cellular
immunity, act as cell signaling agents, play a role in cytoskeletal
modications and cell cycle regulation, and can lead to the
development of cancer (Verbon et al., 2012). Our results suggest
that ND-MWCNT induced sustained production of ROS over a 24 h
period, while MWCNT-7 induced transient production of ROS at 6 h
that resolved by 24 h. This difference may be due to the
physicochemical properties of the particles and their ability to
scavenge ROS, which has been shown to vary with nanotube
composition, surface characteristics, and redox potential in
acellular systems (Tsuruoka 2013a,b; Tsuruoka et al., 2015Tsuruoka
2013a,b; Tsuruoka et al., 2015 Tsuruoka et al., 2015). As suggested
by Tsuruoka, surface electrons of MWCNT may be able to scavenge
ROS in vivo through electron donation that could be limited by the
ability of cells in the body to resupply electrons to MWCNT to
scavenge additional ROS. When this theory is applied to our results,
SAEC may be better able to resupply electrons on the surface of
MWCNT-7 or MWCNT-7 may have more surface electrons available
to donate to ROS present in our cellular system over a 24 h period
(Tsuruoka et al., 2015).
Although ROS can cause oxidative damage to DNA, lipids, and
proteins, we believe that the sustained levels of ROS induced by NDMWCNT treatment may be alternatively acting as a signaling
molecule as proposed by Boonstra and Post (2004). ROS can
inuence the cell cycle through the activation of growth factor
receptors and phosphorylation and ubiquitination of cell cycle
regulators (Verbon et al., 2012). The cell cycle is regulated through a
series of checkpoints mediated by cyclins and serine/threonine
cyclin-dependent kinases that tightly block or promote cycling.
Several recent publications have discussed functionalized MWCNTinduced cell cycle alterations. Siegrist et al. (2014) studied
carboxylated MWCNTand showed that exposure of Beas2B bronchial
epithelial cells with 24 mg/cm2 MWCNT induced a signicant
increase in cells in S phase, suggesting a block at the G1/S checkpoint.
Zhang and Yan (2012) recently showed in several cell lines that
carboxylated MWCNT caused cell cycle arrest at the transition
between G1/S and a slight arrest in G2 in mouse mesenchymal stem
cells and human neuroblastoma cells. Our ndings suggest that NDMWCNT and MWCNT-7 may be inducing cell cycle aberrations
between 6 and 24 h at G2 phase and at 6 h in G2 phase, respectively.
Our ndings also show that CDK4 levels distinctly vary between
6 and 24 h in SAEC exposed to ND-MWCNT or MWCNT-7, further
suggesting that they may act through alternative mechanisms to
alter the cell cycle. Total phospho-threonine levels are similar to
CDK4 levels for each time point and exposure, suggesting that the
cellular activation may be related. Phospho-tyrosine kinases are
important for a variety of different cell signaling pathways including
mitogen-activated protein kinase pathways, Jak/STAT pathways, and
phosphotidylinoside 3-kinase (Marshall, 1995). These signaling
pathways are related to cell cycle regulation, cell differentiation, cell
migration, and apoptosis (Rawlings et al., 2004). Since dysregulation
of these pathways has been associated with inammation, cancer,
and diabetes, our results warrant further studies of these particles
specic molecular mechanisms. Our lab has previously shown
signicant increases in phospho-NF-kB p65, phospho-p38, and
phospho-Stat3 in human microvascular endothelial cells indirectly
exposed to MWCNT-7 in an alveolar-capillary co-culture system
(Snyder-Talkington et al., 2013c). Others have also shown activation
of p38 MAPK in Beas2B lung epithelial cells and normal mesothelial
cells in response to MWCNT exposure, which may provide direction
for future work (Hirano et al., 2010; Pacurari et al., 2008).

5. Conclusions
In this study, we present the rst in-depth physicochemical
characterization of these ND-MWCNT, suggesting that during
synthesis, nitrogen atoms were incorporated into the MWCNT,
introducing structural disorder and altering their crystallinity. Our
major nding is that the differences in physicochemical properties,
especially disruption of the carbon crystalline lattice by incorporation of nitrogen atoms, impacts the bioactivity of MWCNT.
Overall, our ndings suggest that the bioactivity of ND-MWCNT in
SAEC is somewhat lower than that exhibited by pristine MWCNT
due to differences in physicochemical properties. The time and
dose-dependent nature of observed proliferation, cytotoxicity, ROS
production, cell cycle alterations, and total phospho-tyrosine and
phospho-threonine-altered proteins suggests that the physicochemical properties of MWCNT affect their subsequent biological
effects.
Disclaimer
The ndings and conclusions in this report are those of the
authors and do not necessarily represent the views of the National
Institute for Occupational Safety and Health. The mention of Mitsui
& Co., Ltd. and MWCNT-7 does not constitute product endorsement.
Conict of interest
The authors declare that there are no conicts of interest.
Transparency document
The Transparency document associated with this article can be
found in the online version.
Acknowledgements
A.L. Mihalchik acknowledges support from the National Science
Foundation through the IGERT program under grant DGE-1144676.
We would also like to acknowledge use of the WVU Shared
Research Facilities.
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