Article

Toxicology and Industrial Health

2019, Vol. 35(1) 79–87
An assessment of the cytotoxic effects © The Author(s) 2018
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of graphene nanoparticles on the DOI: 10.1177/0748233718817180
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epithelial cells of the human lung

Nafiseh Nasirzadeh1, Mansur Rezazadeh Azari2,

Yahya Rasoulzadeh1 and Yousef Mohammadian1,2

Abstract
Nanomaterials are widely used nowadays in a range of technological and biomedical fields. Graphene as a
nanomaterial used in the health-care sector and in workplaces has raised some concerns about its toxicity. This
study aimed to evaluate the cytotoxicity of graphene nanoparticles (GNPs) on the A549 epithelial cells of the
human lung. The GNPs were synthesized from graphite by the modified Hummer method. The physico-
chemical characteristics of GNPs were identified by the transmission electron microscope, the scanning
electron microscope, and the Brunauer–Emmett–Teller method. The hydrodynamic size of GNPs in the
dispersion media was examined using the dynamic light scattering technique. The GNPs were dispersed, after
which the A549 cells were cultured. Finally, the cell viability was assayed by the MTT assay. The statistical
analysis of variance was used to describe the relationship between the concentration/time variables and the
GNP-induced cell deaths. The probit regression model was also used to achieve toxicological indicators. The
results showed that the toxicological effects of GNPs on the A549 epithelial cells of the human lung are dose-
and time-dependent. The GNPs were more cytotoxic after a 72-h exposure period compared to a 24-h and
48-h exposure period. The inhibitory concentration of 50% and “no observed adverse effect concentration”
were estimated to be 40,653.1 and 0.059 mg/mL, respectively. The results of this study can be helpful in
developing the occupational exposure limit for GNPs and in improving occupational health programs in
workplaces. However, more investigation is needed to specify the toxicological mechanisms of GNPs.

Keywords
Nanomaterials, graphene nanoparticles, cytotoxicity, exposures, MTT, TLC, IC50, NOAEC

Received 18 April 2018; Revised 2 October 2018; Accepted 9 November 2018

Introduction Consequently, concerns have been raised about the

Graphene is a nanomaterial that was developed in
2004 using a simple technique (Sasidharan et al.,
2013). It is an allotrope (form) of carbon consisting
of a single layer of carbon atoms arranged in a hex-
agonal lattice (Geim, 2009). Nowadays, graphene
nanoparticles (GNPs) are used in the health-care sec-
tor as a gene transducer and biosensor for the diag-
nosis of diseases and tissue engineering (Singh,
2018). GNPs have also attracted much attention
because of their great potential for food safety and
packaging (Becaro et al., 2015), agriculture (Hill
et al., 2015), and industrial applications. This means
workers worldwide are potentially exposed to GNPs.
toxicity of GNPs (Goede et al., 2018). Several studies
on the cytotoxicity of GNPs have indicated that the
reactive oxygen species (ROS) increases in cells.
ROS can damage proteins, DNA, and lipids, and may
1
Department of Occupational Health Engineering, Faculty of
Health, Tabriz University of Medical Sciences, Tabriz, Iran
2
Department of Occupational Health Engineering, Shahid
Beheshti University of Medical Sciences, Tehran, Iran
Corresponding author:
Yahya Rasoulzadeh, Faculty of Health, Tabriz University of
Medical Sciences, Tabriz, Iran.
Email: rasoulzadehy@tbzmed.ac.ir
80
lead to many diseases (Seabra et al., 2014). Apoptosis,
the programmed cell death (Sanchez et al., 2011), and
necrosis, a type of cell injury that results in the pre-
mature death of cells in living tissue by autolysis,
are important processes that accelerate cell death
by GNPs (Li et al., 2012). The genotoxicity eva-
luation of GNPs has confirmed its carcinogenic
potential in animal models (Zhang et al., 2015).
Wang et al. (2011) have reported that a dose of
0.4 mg graphene oxide (GO) could cause granu-
loma formation in the kidneys, lungs, liver, and
spleen and could not be filtered by the kidneys.
Recent studies have shown that when respiratory
exposure to nanoparticles occurs, macrophages and
neutrophils in the pulmonary system can be reacti-
vated. In that case, it could produce an inflammatory
reaction (Kennedy et al., 2009).
Since in vitro systems can assess the primary
mechanisms of toxicity, they are known to be useful
methods for the evaluation of toxicity of nanomater-
ials. Some of the advantages of in vitro systems are
efficiency, rapidity, cost-effectiveness, and the fact
that they provide base studies for animal research
(Huang et al., 2010). However, the results of in vitro
methods vary because of the differences in size, sur-
face structure, functionalization, charge, impurities,
aggregations, and the corona effect, and the sample
preparation of nanomaterials. Therefore, cytotoxicity
studies on nanomaterials need to be extended (Jastr-
zebska et al., 2012).
There are still uncertainties about the toxicity of
GNPs. Research on the toxicological effects of GNPs
is still limited, and even occupational exposure lim-
its (OELs) have not yet been defined for GNPs
(Mihalache et al., 2017). A better understanding of
the relationship between the cytotoxicity of nanoma-
terials and the OEL would significantly extend our
knowledge for establishing safe workplaces. A
recent review showed that most of the studies on the
cytotoxicity of GNPs are limited to the evaluation of
the total lethal concentration (TLC) and inhibitory
concentration of 50% (IC 50 ) (Jastrzebska et al.,
2012). The current study evaluated the “no observed
adverse effect concentration” (NOAEC) as the high-
est experimental point without any adverse effects.
Therefore, this study aimed to evaluate the cytotoxi-
city of GNPs on A549 epithelial cells of the human
lung and provide basic data for identifying toxicolo-
gical aspects of graphene clearly. This may be help-
ful in future risk assessment of exposed working
groups in industries.
Toxicology and Industrial Health 35(1)
Method and materials
Synthesis of GNPs
GNPs were purchased with a purity of 99.98% from
the Research Institute of Petroleum Industry (RIPI,
Iran) (Sereshta et al., 2013). GNPs are synthesized
from graphite by the modified Hummer method
through RIPI. In this method, some graphite powder
is added to a mixture of concentrated H2SO4 and fum-
ing HNO3. The mixture is sonicated for 10 min and
stirred for 30 min at 120 C. Then, it is cooled, diluted
with deionized water, and neutralized with Na2CO3.
The solution is filtered and dialyzed in a dialysis bag
for 2 days. After the solution is concentrated on a
rotary evaporator and filtered through 0.22-mm fil-
ters, a black solution is obtained. To obtain the gra-
phene, the oxygen functional groups have to be
eliminated. Hydrazine hydrate is used as the reducer.
Finally, the solution is sonicated using the ultrasound
method, heated in an oil bath, and refluxed under
magnetic stirring at 70 C. The product is filtered
and washed with additional ethanol to remove the
residual hydrazine hydrate. Then, it is dried in an
oven at 100 C.
Characterization of GNPs
The particle size and distribution of GNPs were mea-
sured by a transmission electron microscope (TEM;
CM30-Philips, Japan) and a scanning electron micro-
scope (SEM; S4160-Hitachi, Japan). The specific sur-
face area and pore volume were calculated using the
Brunauer–Emmett–Teller method (Romero et al.,
2012). Because nanoparticle size and size distribution
are important factors that affect in vitro toxicity, it
was needed to characterize these parameters via
dynamic light scattering (DLS). DLS is used as a
simple method for analyzing suspension stability and
measurement of particle size in solution to evaluate
any nanoparticles such as metal and metal oxide and
carbon-based nanomaterials (Murdock et al., 2007).
The hydrodynamic sizes of GNPs in the dispersion
media were examined using the DLS technique
(Malvern Instruments Ltd, Zetasizer version 6.01) in
a laboratory service company (Daypetronic, Iran).
Preparation of stock solution
The GNPs were dispersed using the generic nano-
genotoxic dispersion protocol standard operation pro-
cedure (Jensen et al., 2011). In total, 15.36 g GNP was
used to prepare a 2.56 mg/mL stock dispersion in
Nasirzadeh et al.
6 mL of ethanol and bovine serum albumin water
(BSA-water). Then, 0.5% velum ethanol (96% or
higher) and 99.5% velum sterile-filtered BSA-water
(0.05% w/v) were used to disperse the GNPs.
Cell culture and exposure of GNPs
Human epithelial cell line, A549, was bought from the
Cell Bank (Pasteur Institute of Iran, Tehran, Iran).
The cells were cultured in Dulbecco Modified Eagle
Medium (DMEM; BIO-IDEA, Iran) supplemented
with 10% fetal bovine serum (BIO-IDEA, Iran), 100
U/mL penicillin, and 100 l mg/mL streptomycin (pen
stripe). Then, the cells were incubated at 37 C in a
humidified atmosphere (5% CO2 and 95% air).
After 24 h of incubation, the cells were seeded in a
96-well culture plate (1  103 cells/mL) and allowed
to be attached to the plate for 24 h. The suspensions of
the GNPs were dispersed by sonication (160 W, 20
kHz, 5 min) and diluted in various concentrations.
Then, these were suspended in DMEM of 10 different
concentrations (0.1, 1, 10, 50, 100, 200, 300, 500, 600,
1000 mg/mL) for 24, 48, and 72 h. The cells main-
tained in DMEM without GNPs were used as the
control group. Two control groups, negative control
and blank, were used in this study. In the negative
control group, the wells contained only culture
medium and cells. While, the wells containing culture
medium and GNPs was specified as the blank control.
Cell morphology. The A549 epithelial cells were plated
in the 96-well plates (103 cells per well) and incubated
for 24 h. The GNPs were introduced to the cells in a
predetermined concentration in the culture medium.
The cells cultured in the medium without the addition
of the GNPs were the control. The cell morphology
was observed under an optical microscope (Olympus
1x71, equipped with Olympus DP72 Camera 12.8
megapixel, Japan) after 24 h.
Cell viability
The effect of GNPs on cell viability was determined
by the MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphe-
nyltetrazolium bromide) assay. The MTT assay is a
colorimetric assay based on the ability of viable cells
to reduce a soluble yellow tetrazolium salt to a purple
Formosan crystal by the mitochondrial succinate
dehydrogenase activity of viable cells.
According to this assay, the A549 epithelial cells were
washed twice with phosphate-buffered saline (PBS).
Ten microliters of MTT (5 mg/mL) supplemented
81
with 150 mL complete culture medium were added to
the plates and incubated for approximately 4 h. The
surface medium was vacated and 150 mL dimethyl
sulfoxide was added to dissolve the insoluble purple
Formosan product into a colored solution. The cell
plates were put into a shaker for 20 min. Finally, the
optical density was read using a microplate reader
(ELX800, BioTek model, Winooski, USA) at 570 nm.
In MTT method, some nanomaterials such as car-
bon nanotubes may interact with or adsorb the dye/
dye products leading to invalid results. Therefore, the
cells were washed twice with PBS before reading the
adsorption of GNPs at 570 nm.
Statistical analysis and determination
of toxicological indicators
The data were evaluated using SPSS-V16 software.
To determine the relationship between concentration/
time variables and GNP-induced cell death, the sta-
tistical analysis of variance (ANOVA) was used. The
critical value for statistical significance was p < 0.05
with 95% confidence.
The collected data were also entered into the
Minitab 18.1 software and the probit regression model
was used to achieve the toxicological indicators. In
addition, the descriptive data from three independent
experiments were reported as the mean + standard
deviation.
Results
The obtained results were classified into three parts:
characterization of GNPs, cell morphology, and
toxicological indicators.
Characterization of GNPs
Figure 1 shows the SEM and a high-resolution TEM
image of the GNPs. They have six sheets. The GNP
sheets have micro and macro wrinkles and they fold at
the edge, while the plates are larger. The main struc-
tural parameters of the GNPs are shown in Table 1. As
it is shown, the pore size of the GNPs and the average
pore diameter are approximately 2–50 nm and 13.28
nm, respectively.
The average hydrodynamic diameter and the width
of GNPs in the aqueous suspension were 323.3 nm
and 80.05 nm, respectively. Figure 2 illustrates the
size and number distribution of the GNPs in the
DMEM culture medium.
82 Toxicology and Industrial Health 35(1)

Figure 1. (a) SEM and (b) high-resolution TEM image of GNPs. SEM: scanning electron microscopy; TEM: transmission
electron microscopy: GNP: graphene nanoparticle.

Table 1. TEM, BET parametric structures of GNPs. Almost similar results were obtained from 24- and
48-h exposures at the lowest concentration (less than
Parametric Parametric
300 mg/mL), but the results were different for 72-h
structures Dimension structures Dimension
exposure. For example, for 100 mg/mL of GNP, the
Main pore size 2–50 Pore diameter 13.28 cell viability was 54.71% at 24 h and 54.48% at 48 h,
belongs to (nm) but it was 47.56% after 72 h of exposure (Figure 4).
nanopores (nm) Mean of cell death was 45.28 + 20.14, 56.2 + 27.14,
Number of layer 6 Crystal thickness 2
and 60.9 + 23.61 in 24, 48 and 72 h, respectively.
(nm)
Layer distance (Å) 3.4 specific surface 195.97 According to the ANOVA test results, a significant
(m2/g) relationship was observed between cell death with
Pore volume 0.0651 time of exposure to GNPs (p ¼ 0.03) and exposed
(cm3c/g) concentration (p ¼ 0.00).
Nutrient depletion by nanomaterials is known to be
TEM: transmission electron microscopy; BET: Brunauer–
Emmett–Teller; GNP: graphene nanoparticle. a reason for nanotoxicity. Hence, the toxicity of GNPs
on A549 cell culture was evaluated.
The DMEM medium was pretreated with separate
Morphological changes of A549 cells induced GNP samples for 24 h, and then the supernatant liquid
by GNPs was collected for A549 cell culture. Had the cells died
The cell morphology, as the main indicator, expresses in the GNP-pretreated culture medium, it would be
the status of the cells. The morphological changes concluded that the adsorption of nutrients on to the
were not observed in the GNP-treated and control nanomaterials influenced the toxicity of the culture
cells following the GNP exposure. Neither the GNP- medium. Nevertheless, the cells grew and so did the
treated nor the control cells showed any differences in control cells. The cell viability did not decrease dur-
their adhesion to the culture medium dish. The major- ing the exposure.
ity of the cells had a spindle shape and adhered to the
substrate normally (Figure 3). Toxicological indicators
The toxicological indicators explain the relationship
Cell viability between the dose and the response. The TLC is the
The cell viability was assayed by MTT to demonstrate concentration of an inhibitor that reduces the response
the cytotoxicity of GNPs in the A549 cells. The to zero. The IC50 is defined as the concentration of an
adsorption of GNP itself (0.012) was measured using inhibitor, which reduces the response by half.
similar methods and conditions and considered in cal- NOAEC is defined as the highest experimental point
culations. Cell death was observed at higher concen- without any adverse effect. The NOAEC indicator is
trations of GNPs. For example, for 200 mg/mL GNP, defined as the concentration of an inhibitor by which
the cell viability was 48% after 24 h of exposure. the response is reduced to 10% for new materials and
Nasirzadeh et al.
Figure 2. Size distribution by number in DMEM culture medium. DMEM: Dulbecco Modified Eagle Medium.
Figure 3. Optical microscopy images of GNPs-treated
A549 cells: (a) GNPs at 24 h, (b) the control at 24 h,
(c) GNPs at 48 h, (d) the control at 48 h. GNP: graphene
nanoparticle.
unknown toxic materials such as nanomaterials (ISO,
2016). The toxicological indicators such as TLC, ICs,
and NAOEC were attained by the probit regression
model (Figure 5). The probit analysis is commonly
used in toxicology to determine the relative toxicity
of chemicals in living organisms/cells (Finney and
Stevens, 1948). Toxicological data obtained were also
displayed in Table 2.
The probit regression was also used to illustrate the
dose–response relationship based on the concentra-
tion regardless of the exposure time (Figure 6). In this
83
respect, the TLC, IC50, and NOAEC were estimated
to be 40,653.1, 41.15, and 0.059, respectively.
Discussion
The results of this study show that the GNPs reduced
cell viability at high concentrations/doses. In other
words, GNPs can be characterized as a toxin for the
A549 epithelial cells of the human lung. There was a
relatively good correlation between cytotoxicity and
the exposure time. In addition, toxicological indica-
tors illustrated the relationship between dose/time and
response for GNPs. Several studies have confirmed
the adverse effects of GNPs on organs and cells, too
(Shang et al., 2014; Su et al., 2015). A study indicated
that GNPs with 100–110 nm diameters had an
increasing cytotoxic potential for pheochromocytoma
and PC12 cells in rats. In addition, lactate dehydro-
genase (LDH), ROS, and apoptosis were also released
at 10–100 mg/mL for 48 h (Zhang et al., 2010).
Another study reported that GNPs with a size of
500–1000 nm increased the ROS levels and triggered
apoptosis in murine RAW 264.7 macrophages in
5–100 mg/mL concentrations after a 48-h exposure
(Li et al., 2012). Ma et al. found that the cytotoxicity
of GO depends on the size of nanoparticles. Small
nanosheets of graphene enter the cells mainly by
endocytosis, whereas large graphene sheets are
adsorbed onto the plasma membrane. Then, they enter
the cells by phagocytosis, which may lead to the
enhanced production of inflammatory cytokines and
recruitment of immune cells (Ma et al., 2015). There-
fore, the cytotoxicity of GNPs depends on the size of
84 Toxicology and Industrial Health 35(1)

Figure 4. Dose–response graph for GNPs-treated A549 cells at different times. GNP: graphene nanoparticle.

Figure 5. Dose–response relationship for GNPs and A459 cell death, based on time, prepared by probit regression
model. GNP: graphene nanoparticle.

Table 2. TLC, IC50, and NOAEC indicators for GNPs.

the nanoparticles. In the current study, GNPs at con-
Toxicology indicators (mg/mL) centrations lower than 0.19 mg/mL exhibited remark-
able cytotoxicity in the lung cells during the 24-h
Time exposure (h) NAOEC IC50 TLC
exposure period, while the diameters of GNPs were
24 0.19 134.771 100,081 2–50 nm. The results of the current study are in agree-
48 0.057 41.19 30,585 ment with previously mentioned studies.
72 0.029 21.51 15,978 The mode of dispersion of the nanomaterials in the
TLC: total lethal concentration; IC50: inhibitory concentration of exposure tests is decisive for the outcome of in vitro
50%; GNP: graphene nanoparticle. studies. The agglomeration of GNPs, due to their
Nasirzadeh et al.
Figure 6. Dose–response relationship for GNPs and A459 cell death, based on total versus concentration, by probit
regression model. GNP: graphene nanoparticle.
differential dispersion ability, critically affects their
ability to interact with the cells and, in turn, affects
their internalization within the cells (Stone et al.,
2009). In this study, DMEM containing 5% serum
was chosen for dispersion, whereas the agglomeration
of GNPs was moderate, and the size of the GNPs was
323.3 nm. In another study, the graphene agglomera-
tion occurred following the dispersion in double-
distilled water cells and on the cellular membrane,
while the mean size of the nanoparticles was 349 +
24 nm (Majeeda et al., 2016). It can be concluded that
the agglomeration of GNPs might be related to their
size.
The functionalized surface of GNPs may also
affect cytotoxicity. Majeeda et al. found that solubi-
lity and toxicity could be influenced by changes in the
surface properties. With respect to graphene surface
chemistry, the pristine graphene exhibited the highest
toxicological behavior (Majeeda et al., 2016). GNP-
COOH and GNP-NH2 caused DNA damage, geno-
toxicity, and hypomethylation in human bronchial
epithelial cells (BEAS-2B cells) at 10 and 50 mg/L
after 24 h of exposure. Chatterjee et al. reported GNP
toxicity higher than GNP-COOH and GNP-NH2 in a
severe form of DNA damage. So, the concentration of
50 mg/mL was reported as the lowest effective con-
centration (Chatterjee et al., 2016). In this study,
GNPs were pure and without a functionalized surface,
but the TLC (or 99% death) of GNPs was obtained at
40.65 mg/mL. The obtained TLC results confirm that
85
pristine GNPs are more toxic than functionalized sur-
face GNPs.
According to the study by Chang et al., when GNPs
were oxidized to GOs, cell viability decreased at a
high concentration. They found that the GO did not
enter the A549 cell and thus had no obvious cytotoxi-
city. Although the GO created ROS, IC 20 was
reported for 200 mg/mL in 24 h (Chang et al.,
2011). In another study, cytotoxicity was observed
at the highest GO concentration of 100 mg/mL (Schin-
wald et al., 2013; Zhang et al., 2012). When even
graphene is changed to reduced graphene oxide
(rGO), toxicity occurred in the highest concentration
(100 mg/mL) (Akhavan et al., 2012; Jaworski et al.,
2015). Chong synthesized the graphene quantum dots
(GQD) from graphite and GO and evaluated the cyto-
toxicity. This compound of GO, which could improve
the aqueous stability of GO, resulted in approximately
85% A459 cell viability at a concentration of
640 mg/mL (Chong, 2014). In the present study, IC50
was achieved at around 134.77 mg/mL in 24 h. The
results of the current study justified the high toxicity
of pristine GNPs, which is consistent with the results
of similar studies.
This study indicated that cytotoxicity depends on
the exposure period. GNPs were more cytotoxic after
72 h of exposure compared to a 24-h and 48-h expo-
sure period. Chang et al. (2011) have reported similar
results after 24, 48, and 72 h of exposure to GO and
confirmed that the cytotoxicity of GNPs depends on
86
the exposure period. Hence, cytotoxicity was dose-
and time-dependent.
The obtained NOAEC results showed that GNPs
are toxic for A459 cells in a 0.19 mg/mL concentration
after an exposure for 24 h. In other words, IC10/24 for
pristine GNPs was 0.19 mg/mL. Chang et al. (2011)
reported IC20/24 of 200 mg/mL for GO. Since pristine
GNPs are more toxic than other forms of GNPs (such
as GO, rGO, and GQD), it can be proposed that the
structure of nanomaterials can influence the NOAEC.
Zhang et al. (2010) observed that a low dose of GNPs
(0.01 mg/mL) could decrease PC12 cell viability,
while it could not affect metabolic activity, LDH
release, and ROS. It can be concluded that 0.01 mg/
mL was the NOAEC indicator for GNPs. In other
studies, Li et al. (2012) have reported that less than
20% of the RAW 264 cells remained in a concentra-
tion of 20 mg/mL. This indicates that the type of cell
may influence the cytotoxicity.
Although the MTT measurement is a reliable
toxicity marker, the assay has some inherent lim-
itations. This measurement, which is a colorimetric
assay, was used to determine the effect of cytotoxi-
city; it can evaluate cell viability without consid-
ering the toxicity mechanisms. It is recommended
that toxicity mechanisms, such as oxidative stress,
apoptosis, LDH, superoxide dismutase, and autop-
hagy, which can influence cytotoxicity, be consid-
ered to evaluate the cytotoxicity of GNPs. A better
understanding of toxicity mechanisms will justify
the cytotoxicity and the toxicological indicators
of GNPs.
Conclusion
The findings of the current study show that the viabi-
lity of the A549 cells decrease with an increase in the
concentration/dose of GNPs and the exposure period.
In other words, the toxicological effects of GNPs on
the A549 epithelial cells of the human lung are dose-
and time-dependent. The NOAEC toxicological indi-
cator for GNPs was 0.059 mg/mL. In addition to that,
NOAEC may be related to the physicochemical prop-
erties of GNPs, the sample preparation, and the type
of cell, which needs to be studied.
The results of this study can be helpful for devel-
opment of the OEL for GNPs and risk evaluation of
exposed population. However, more investigation is
needed to clarify and specify the toxicological
mechanisms of GNPs.
Toxicology and Industrial Health 35(1)
Acknowledgements
The authors thank the Research Institute of Neuroscience
Research Center at the Shahid Beheshti University of
Medical Sciences for laboratory services.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest
with respect to the research, authorship, and/or publication
of this article.
Funding
The author(s) disclosed receipt of the following financial
support for the research, authorship, and/or publication of
this article: This work was financially supported by the
research deputy of the Tabriz University of Medical
Sciences (grant number 57288).
ORCID iD
Yahya Rasoulzadeh https://orcid.org/0000-0003-4499-
5153
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