Early signs of multi-walled carbon nanotbues degradation in macrophages, via an intracellular pH-dependent biological mechanism; importance of length and functionalization
Landry et al. Particle and Fibre Toxicology
Early signs of multi-walled carbon nanotbues degradation in macrophages, via an intracellular pH-dependent biological mechanism; importance of length and functionalization
Marion Landry 1
Stéphane Tchankouo 1
Audrey Ridoux 1
Jorge Boczkowski 0 1
Sophie Lanone 0 1
0 Université Paris Est-Créteil, Faculté de Médecine de Créteil , 8 rue du Général Sarrail, Créteil F-94000 , France
1 INSERM, U955 , Equipe 4, Créteil F-94000 , France
Background: Carbon nanotubes (CNT) can interact with the biological environment, which could participate in their associated toxicity. We recently demonstrated that pH is an important player of CNT fate inside macrophages. We wanted to further characterize such process, and therefore designed a study dedicated to decipher CNT biodegradation by macrophages, as a function of two major physico-chemical properties in regard with nanotoxicology; length and degree of functionalization. To achieve our aim, we synthesized, following a single initial production process, four MWCNT differing in length and/or surface chemistry: S-CNT (short), SF-CNT (short functionalized), L-CNT (long) and LF-CNT (long functionalized). Results: Raman spectroscopy analysis performed on CNT recovered after exposure of RAW 264.7 macrophages for 6, 24, or 48 h demonstrate that CNT show early signs of biodegradation over time inside macrophages. The modulation of CNT length and functionalization, resulting in the modification of iron accessibility, both represent critical determinants of the biodegradation process; short pristine CNT were more prone to biodegradation than long CNT (pristine or functionalized), while short functionalized CNT were protected. Incubation of cells with Concanamycin completely prevents CNT from being modified, demonstrating that this biodegradation process is dependent on an intracellular pH-dependent mechanism. Interestingly, and despite evidence of degradation via Raman spectroscopy, the CNT length and diameter were not altered during the course of the study. Conclusions: In conclusion, our results identify a new mechanism of CNT biodegradation inside macrophages. This could give new insights for the understanding of CNT-associated toxicity, and represent important tools to develop safe(r)-by-design nanomaterials.
Carbon nanotubes; Biodegradation; pH; Functionalization; Length
Carbon nanotubes (CNT) are a family of nanomaterials
featuring unique properties and presenting a large range
of length, diameter, and number of walls (single-walled
-SWCNT-, or multi-walled -MWCNT-). The current
applications for SWCNT range from chemical sensors, to
conductive heating films, conductive nanoink, nanodevice
or display. These applications need high quality but small
amount of CNT, whereas those utilizing MWCNT as
conducting paints, composite materials (heat exchanger,
reinforced materials, …) or developed for energy storage
require large amounts of CNT [1–4]. Due to this
expansion of MWCNT usage and the resulting likely increase of
human exposure, the potential adverse effects of CNT,
and particularly those of MWCNT on human health are
of great concern. A large body of literature indicates that
CNT can be toxic, depending on numerous
physicochemical characteristics including length, diameter, structural
defects, surface area, tendency to agglomerate,
dispersibility in solution, presence and nature of catalyst residues, as
well as surface chemistry (see  for review).
It is now well accepted that nanomaterials, including
MWCNT, can interact with the biological environment,
which could participate in their associated toxicity . This
is demonstrated by the rapid formation of a bio-corona
(proteins, lipids, other biomolecules) around the
nanomaterials, which potentially confers them new or different
(surface) identity. More recently, data from the literature
also evidenced that SWCNT can undergo enzymatic
biodegradation. So far, the majority of the studies dedicated to
evaluate the degradation of CNT have been conducted with
SWCNT in a-cellular systems supplemented with
recombinant enzymes. Indeed, recent reports have demonstrated
that the plant horseradish peroxidase (HRP), and the
animal peroxidases myeloperoxidase (MPO) and eosinophil
peroxidase (EPO) are able to catalyze the degradation of
CNT [7–12]. However, because of their experimental
setup far from real-life exposure (a-cellular systems, use of
recombinant enzymes, high doses of H2O2 to catalyze the
reaction, …), these studies, although informative, remain of
limited impact in the context of human health. More
recently, a few studies have been conducted in cell cultures,
demonstrating that SWCNT can be biodegraded inside
most inflammatory cell types (neutrophils, eosinophils or
monocytic cells), via a MPO- or EPO-dependent
mechanism [8, 13, 14]. Surprisingly, despite the unique role of
macrophages in the in vivo elaboration of CNT-induced
inflammation , almost no study so far have been
conducted to evaluate CNT biodegradation in this cell type
[14, 16, 17]. Such evaluation could be particularly relevant
since it is long known that CNT-loaded macrophages are
present at the site of exposure and/or distributed
throughout the body, up to 24 months after the initial exposure to
CNT . Moreover, these studies were most exclusively
conducted with SWCNT although they do not represent
the majority of the CNT produced, and therefore might
not represent the major risk for human exposure. Finally,
another aspect that is currently absent in the literature is
the importance of CNT physico-chemical characteristics in
their biodegradation, although, as major determinants of
CNT toxicity, one can imagine that these characteristics
might largely influence CNT biodegradation as well .
We therefore developed a study dedicated to characterize
the biodegradation of MWCNT by macrophages, as a
function of three major physico-chemical properties in regard
with nanotoxicology; their length, degree of
functionalization and their iron-based catalyst residual content . To
achieve our aim, we synthesized, following a dedicated
initial process, four MWCNT specifically devoted to our study
and differing in length, and that were functionalized to
concomitantly modify their surface chemistry and/or iron
content (Additional file 1: Figure S1): S-CNT (short), SF-CNT
(short and functionalized), L-CNT (long) and LF-CNT
(long and functionalized), functionalized CNT (SF- and
LFCNT) having a different iron content from their
nonfunctionalized counterpart (S- and L-CNT) (Table 1). The
biodegradation of these CNT was addressed in RAW 264.7
murine macrophages in vitro, by the mean of Raman
spectroscopy performed on CNT recovered in cells, after 3
different exposure time points; 6, 24 or 48 h. Moreover, as we
recently demonstrated that pH could be an important
player in the fate of CNT inside cells , we also
investigated the role of pH in CNT biodegradation process. Our
results demonstrate that MWCNT can be biodegraded
inside macrophages, in a time-dependent manner, via an
intracellular pH-dependent biological mechanism. The
modulation of CNT length and functionalization,
concomitantly resulting in the modification of iron accessibility,
both represent critical determinants of the biodegradation
The main physicochemical characteristics of CNT,
deduced from the comprehensive analysis presented below,
are summarized in Table 1. Transmission Electron
Microscopy (TEM) and Optical Microscopy (OM) images of
the initial CNT carpets are presented in Fig. 1a and b.
TEM observations showed that all CNT batches
contained iron-based particles either attached at their external
basis and encapsulated in carbon sheets, or mainly
entrapped inside their hollow core (Fig. 1a). The four CNT
batches showed similar mean external diameter (Table 1)
and distribution (Fig. 1c); neither the shortening nor the
acidification treatment modified the external diameter of
the samples. Mean length was measured at 1.7 and 1.6 μm
for S- and SF-CNT respectively, and at 6.4 and 9.2 μm for
L- and LF-CNT respectively (Table 1). Length distribution
Table 1 Characteristics of the CNT
Iron content (TGA), mean (weight %)
Intrinsic ROS production, mean ± SD
SD standard deviation, ND not detectable
for S- and SF-CNT on one hand, and L- and LF-CNT on
the other hand, was similar (Fig. 1d). Thermogravimetric
analysis (TGA) showed that the average iron content was
similar for S- and L-CNT (4.4 and 4.5 wt% respectively),
and for SF- and LF-CNT (1.3 and 1.0 wt% respectively,
Table 1). X-ray-induced photoelectron spectroscopy (XPS)
analysis revealed a higher content of oxygen atoms (8.1
at% and 8.3 at% respectively) for SF- and LF-CNT as
compared to S- and L-CNT (1.4 at% and 1.8 at% respectively),
consistent with the functionalization by acid treatment
and the presence of various functions (mainly: C-OH, C =
O and O-C-O in different possible groups e.g. carboxylic,
anhydride, ester) at the surface of CNT (Table 1 and
Fig. 1e-h). Moreover, SF- and LF-CNT showed traces of
sulfur, most likely due to the use of sulfuric acid during
the acid treatment. No detectable endotoxin content could
be detected regardless of the CNT. Finally, measurement
of the intrinsic ROS production showed higher content
for S-CNT as compared to L- and LF-CNT, and no/very
low ROS production could be detected for SF-CNT.
Typical Raman spectra, that reflect the degree of
defects in CNT, are given in Fig. 2a. The calculated ID/IG
ratio (Fig. 2b) appears higher for the functionalized (0.55
and 0.42 for SF- and LF-CNT respectively) as compared
to the non-functionalized CNT (0.35 and 0.31 for S- and
L-CNT respectively, p < 0.05), which indicates that the
grafting of chemical species involves also the formation
of defects in the graphene carbonaceous structure as
often reported in the literature .
CNT are modified inside macrophages
Figure 3a shows representative transmission electronic
microscopy (TEM) images of macrophages exposed to
CNT for 6 h. All CNT could be internalized by
macrophages, mainly inside vacuoles although to a lesser extent
for SF-CNT (Fig. 3b). Whatever the CNT, images showing
CNT penetrating through the vacuole wall could also
been observed. To address the issue of a potential
modification of CNT while incorporated inside the cells, the
cellular (Cell) and supernatant (SN) fractions of macrophage
cultures were recovered and separately analyzed after
exposure of RAW 267.4 macrophages during 6, 24 or 48 h.
As shown in Fig. 4a, ID/IG ratio obtained 6 h after the
initial exposure were similar to those obtained for CNT
powders (Fig. 2), and no difference was observed between
the different fractions considered (Cell or SN). However,
starting from 24 h for S- and 48 h for L- and LF-CNT, we
could observe a significant increase of the ID/IG ratio in
the cellular fraction only as compared to the supernatant
one (0.58 ± 0.05 and 0.29 ± 0.01 for SCNT-Cell and
SCNT-SN respectively after 24 h -Fig. 4b, and, after 48 h,
0.58 ± 0.06 and 0.3 ± 0.01 for SCNT-Cell and SCNT-SN
respectively, 0.42 ± 0.02 and 0.3 ± 0.01 for LCNT-Cell and
LCNT-SN respectively and 0.56 ± 0.02 and 0.43 ± 0.01 for
LFCNT-Cell and LFCNT-SN respectively - Fig. 4c, and
Additional file 2: Figure S2, Cell versus SN, p < 0.05).
Whatever the time point and CNT studied, the ID/IG ratio
obtained in SN fractions were similar to those of the
original powders of CNT (Fig. 2). In addition, no significant
modification of CNT length could be detected in Cell or
SN fractions of RAW macrophages exposed up to 48 h,
regardless of the CNT used (Fig. 5).
CNT modifications are driven by an intracellular
As previous studies from our laboratory demonstrate the
role of pH in the detachment of iron catalyst nanoparticles
from SWCNT and subsequent toxicity , we next
examined intracellular pH modifications in RAW 264.7
macrophages exposed to the various CNT. As shown in
Fig. 1 Microscopy images and XPS analysis of CNT powders. Panel a Transmission electron microscope (TEM) images of S-, SF-, L- and LF-CNT. Scale bar:
100 nm. Panel b Optical microscope images of S-, SF-, L- and LF-CNT. Scale bar: 2 μm. Panel c CNT external diameter distribution. Panel d CNT length
distribution. Typical spectra of the C1s (Panel e and f) and O1s core level (Panel g and h) obtained for L- (Panel e and g) or LF-CNT (Panel f and h). S-CNT:
short-carbon nanotubes; SF-CNT: short functionalized carbon nanotubes; L-CNT: long carbon nanotubes; LF-CNT: long functionalized carbon nanotubes
Fig. 6, all but SF-CNT induced the acidification of
lysosomal compartment after 6 h exposure. Such acidification
remains detectable, although faintly, only in S-CNT
exposed macrophages after 24 h, and was no more observed
after 48 h treatment (Additional file 3: Figure S3).
In order to assess the role of intracellular pH in the
observed biodegradation of CNT, Raman spectroscopy
was performed in the cellular fraction (Cell) of
macrophages exposed to CNT in presence of the H+-ATPase
inhibitor Concanamycin A. As shown in Fig. 7,
incubation of cells with Concanamycin completely prevents
CNT from being modified, suggesting that CNT
modifications were the result of an intracellular pH-dependent
mechanism. This is also confirmed by observations
showing no modification of Raman spectra in the
supernatant fraction (SN) of macrophages exposed to CNT in
presence of Concanamycin, irrespective of the time
point and CNT considered (data not shown).
The protection against CNT biodegradation brought by
Concanamycin treatment could result, beside from
pHdependent mechanism driven by an intracellular event,
from 3 different events 1/ a direct effect of Concanamycin
on CNT structure, 2/ an effect of Concanamycin on cell
secretome which could, in turn, modify CNT structure, or,
finally, 3/ a direct effect of pH on CNT. The experiments
performed to test these 3 last hypotheses demonstrate that
none of them were correct. Indeed, CNT Raman spectra
are not modified when CNT were incubated for 48 h 1/ in
culture medium supplemented with Concanamycin (Fig. 8a),
2/ in supernatant from cells treated with Concanamycin
(Fig. 8b), or 3/ in an artificial solution representative of
lysosomal compartments (Fig. 8c). Finally, incubation of CNT
with H2O2 for 48 h didn’t induce any modification in
Raman spectra (Fig. 8d). Overall, these results are the first
evidence of the degradation of CNT inside the cells via an
intracellular pH-dependent mechanism.
Fig. 2 Raman spectroscopy analysis of CNT powders. Panel a Typical
Raman spectra obtained on S-, SF-, L-, and LF-CNT. Panel b ID/IG ratio
calculated from Raman spectra of S-, SF-, L-, and LF-CNT powders. Data
are given as mean ± SEM of minimum 5 values. *: p < 0.05 vs
nonfunctionalized counterpart. Abbreviations as in Fig. 1
Overall, our data demonstrate that CNT can be
biodegraded over time inside macrophages, with a combined
influence of both CNT length and acidic
functionalization; short pristine CNT were more prone to
biodegradation than long CNT (pristine or functionalized), while
short functionalized CNT were protected. Moreover, we
showed that this biodegradation process is dependent on
an intracellular pH-dependent mechanism, and not
associated to a modification of CNT length or diameter that
could be detected during the time course of the study.
All but SF-CNT show early signs of degradation in
macrophages, starting at 24 h for S-CNT, and 48 h for L- and
LF-CNT. Pretreatment of cells with the V-ATPase
inhibitor Concanamycin protects all CNT from their
biodegradation by macrophages, which strongly suggests that this
biodegradation process is pH-dependent. The exclusive
intracellular localization of this pH-dependent mechanism
is also strongly suggested by the absence of Concanamycin
effect on CNT structure (directly or by the mean of
cellular secretome) or any direct effect of pH on CNT. To the
best of our knowledge, this is the first evidence of such a
specifically intracellular-driven mechanism as the few
studies studying CNT degradation in cellular conditions
have been performed on samples containing both cells
and supernatant together, which could lead to a confusion
on the origin of the biodegradation process [8, 13, 14].
Indeed, thanks to our experimental protocol, we were able
to isolate CNT present inside the cells from those present
in the supernatant of the exposed cells. The
pHdependence of CNT biodegradation process is also
confirmed by the fact that the only CNT batch that was not
biodegraded (SF-CNT) was also the only one that did not
induce an intracellular acidification after 6 h. This could
be related to the lesser internalization of SF-CNT inside
phagocytic vacuoles as compared to the other batches of
CNT. Indeed, it has been described that, in macrophages,
phagocytosis is rapidly accompanied by the recruitment of
V-ATPases to the phagosome membrane, leading to a
rapid decrease of pH . We recently showed that
intracellular acidification of macrophages leads to the
detachment of iron-based catalyst nanoparticles initially attached
to CNT . This accessible iron could react, via the
Fenton reaction, and, in turn, lead to the generation of ROS,
which are proposed to be important mediators of CNT
degradation . Interestingly, the overall biodegradation
of CNT in our experimental set-up strictly followed the
intrinsic ROS production by CNT per se, with high levels
for S-CNT, lower (and similar) levels for L- and LF-CNT,
and absence of ROS production by SF-CNT. Recent
studies have proposed peroxidases, and most frequently
myeloperoxidase (MPO), as potential candidate for the
biodegradation of SWCNT [7, 8, 13, 22, 23], together with
reactive intermediates that are considered to be key
factors for SWCNT degradation by cells. However,
macrophages are relatively poor in MPO or NADPH oxidases
(NOX), and both MPO and NOX are inactive at acidic
pH, which rules out their potential role in the observed
biodegradation process at least in the time-course of our
study [24–26]. Finally, and beside a direct reactivity of
CNT surface because of the acidic environment, this
pHdependent intracellular mechanism could rely on protein
neosynthesis (or a cascade of protein synthesis) or other
biological actions triggered by acidic pH (activation of
acidic hydrolases, …). Indeed, given the delay between
cellular acidification (present at 6 h but no more or only
faintly for S-CNT after 24 h) and the first observable signs
of CNT biodegradation, the intracellular acidification that
occurs in response to CNT might be the initial event
driving their subsequent surface modifications. Such
Fig. 3 TEM images of RAW 264.7 macrophages exposed to CNT for 6 h. Panel a Typical TEM images of RAW 264.7 cells exposed to CNT for 6 h.
Panel b quantification of the percentage of cells presenting CNT inside phagocytic vacuoles. Abbreviations as in Fig. 1
mechanisms however deserve further studies to be fully
S-CNT were the more susceptible to biodegradation as
compared to L- and LF-CNT, both in terms of kinetics
(modifications visible as soon as after 24 h) and extend
(magnitude of the modification in ID/IG ratio between the
cellular and supernatant compartments). This could be
linked to their better iron accessibility. Indeed, TEM
observations showed that both S and L-CNT samples presented
almost no carbon-based by-products such as amorphous
carbon, but did contain Fe-based particles (i.e. catalyst
particles) mainly entrapped inside their hollow core. Assuming
that the direct consequence of length reduction from L- to
S-CNT is a higher amount of CNT extremities for the
SCNT as compared to L-CNT, and even though the total Fe
content was similar in S-CNT and L-CNT, the accessibility
of Fe nanoparticles encapsulated in S-CNT cores should be
higher than in L-CNT. Therefore, during cell exposure, the
Fe nanoparticles present in the S-CNT may have been
more rapidly, and thus, for a longer time, in contact with
the biological medium as compared to those present in the
L-CNT. This preferential accountability of iron
nanoparticles on the biodegradation of S-CNT is however not visible
at a global morphological and structural scale at the
timepoints studied. Indeed, no noticeable CNT length/diameter
reduction or holes occurrence was observed up to 48 h
from optical and TEM analyses, as opposed to what
Elgrabli et al. recently described . It cannot be excluded,
Fig. 4 Raman spectroscopy analysis of CNT recovered from cell cultures. ID/IG ratio calculated from Raman spectra of S-, SF-, L-, or LF-CNT
recovered from cellular (Cell) or supernatant (SN) fractions of RAW 264.7-exposed macrophages for 6 (Panel a), 24 (Panel b) or 48 h (Panel c). Data
are given as mean ± SEM of minimum 5 values. Abbreviations as in Fig. 1. *: p < 0.05
however, the occurrence of such modifications at longer
time-points. Our results are difficult to compare with those
of the literature, as, as of now, the majority of the studies
available in the literature describe modifications occurring
in only one batch of CNT [8, 13, 14]. So far, only two
studies compared the degradation of different batches of CNT,
but none of them utilized pristine CNT, and thus could not
be compared to the physico-chemical differences present in
the MWCNT of the present study [16, 27].
We are fully aware that our study was performed on a
static in vitro system, which represents a far less complex
environment than what should happen in vivo in the lung.
However, macrophages represent the first line of defense
after inhalation of exogenous material, and should,
therefore, be the first to recognize and take CNT in charge
while inhaled. As such, a large amount of studies
combining both in vitro approach using RAW 264.7
macrophages, and in vivo experiments in mice, demonstrates
the relevance of the findings obtained in RAW 264.7 cells,
and their potential translation to what may occur in vivo
[28–31]. We therefore strongly believe that our results are
relevant to what occurs in vivo, although this particular
issue should be specifically addressed by dedicated studies
that were beyond the scope of the present study.
Our results identify new determinants of CNT
biodegradation inside macrophages. This could give new insights for
understanding CNT-associated toxicity, and represent
important tools to develop safe(r)-by-design nanomaterials.
The initial aligned multi-walled CNT powder (at the gram
scale) was produced by aerosol-assisted catalytic chemical
vapor deposition (CCVD) . This method is based on
the catalytic decomposition of liquid hydrocarbons by
injecting mixed aerosols containing both the hydrocarbon
and the metallic sources which simultaneously and
continuously fill the reactor. A solution composed of ferrocene
dissolved in toluene (1.25 wt. %) was used to synthesize the
nanotube samples at 800 °C under Ar/H2 atmosphere
(70%/30%). The presence of dihydrogen in the vector of the
aerosol allowed to obtain a small external diameter
(compared to the 40 nm mean external diameter obtained for a
synthesis performed under Ar only), as previously described
by Celia Castro et al. . Following this procedure, the
sample was formed of aligned CNT carpets covering the
reactor walls. The duration of the growth was set at 45 min.
Once detached from the reactor walls by scrapping, the
precursor sample was treated in de-ionized water
(Millipore, 18.2 MΩ.cm), with a dispersing agent (1% biliary salts,
composed of 50% sodium deoxycholate (≥98%) and 50%
sodium cholate (99%, Acros Organics). An ultrasonic probe
Bioblock Vibracell 75043 working at 20 kHz and 375 W in
pulse mode (1 s/1 s amplitude, 50% power) was used in
order to control the CNT shortening and reach a desired
length distribution . Two different durations of
ultrasonic treatment (7 h or 5 min) were applied to obtain two
distinct groups of CNT: a short and a long group
respectively. Both CNT sample powders were then purified at
Fig. 5 Microscopy images of CNT after RAW 264.7 macrophages exposure for 48 h. Panel a TEM images of S-, SF-, L- and LF-CNT recovered in
RAW 264.7 cells exposed for 48 h to the different CNT. Panel b Optical microscopy images of S-, SF-, L- and LF-CNT recovered in RAW 264.7 cells
exposed for 48 h to the different CNT. Panel c Length of S-, SF-, L-, and LF-CNT recovered from the cellular (Cell) or supernatant (SN) fractions of
48 h-exposed RAW 264.7. Abbreviations as in Fig. 1. Data are given as mean ± SEM
1000 °C under Ar atmosphere after filtration in order to
remove/burn traces of dispersing reagent. Each dry samples
was then separated in two sub-groups, treated or not by an
acidic solution (75% H2SO4 and 25% HNO3) at 60 °C for
2 h in order to functionalize the nanotubes by grafting
oxidized groups on their surface. Finally the different
subgroups were extensively washed with de-ionized water, and
final dry samples of CNT were obtained by evaporating
water in a fume hood. Ultimately, four distinct groups were
obtained (see Additional file 1: Figure S1), with controlled
variations in length and surface chemistry: short group
(SCNT), short functionalized group (SF-CNT), long group
(L-CNT) and long functionalized group (LF-CNT).
Optical, scanning electron and transmission electron
To assess the morphology, structure, and presence of
synthesis by-products as well as the diameter and length
distributions of the different CNT, samples were
observed using optical (Olympus BX60 optical microscope
coupled to a color view digital camera, Olympus
Corporation, Japan), scanning electron (SEM; Carl Zeiss
Ultra 55, field emission gun, Carl Zeiss, Germany) and
transmission electron (TEM; Philips CM12 TEM
microscope, Philips Research, The Netherlands) microscopes.
The morphology and thickness of the CNT precursor
Fig. 7 Raman spectroscopy analysis of CNT recovered from cells
cultured in presence or absence of Concanamycin. ID/IG ratio
calculated from Raman spectra of S-, SF-, L-, or LF-CNT recovered
from RAW 264.7 macrophages concomitantly exposed to CNT and
Concanamycin A for 24 h (Panel a) or 48 h (Panel b). Abbreviations
as in Fig. 1. Conca: Concanamycin A. Data are given as mean ± SEM
of minimum 5 values
carpets were investigated by SEM on sections of aligned
CNT carpets fixed on a SEM sample holder with a
carbon adhesive tape. To perform TEM analysis, CNT
powders were dispersed in ethanol and placed in ultrasonic
bath for 1 min (this duration has been chosen to prevent
from the introduction of any supplementary
morphological/structural modifications). One droplet of this
suspension was then deposited on a Cu grid covered with
thin carbon film, and grids were observed at 120 kV.
To determine the sample initial iron content, the
measurement of the remaining iron oxide weight was
performed by thermogravimetric analysis (TGA) with a
TGA 92–16, 18 Setaram apparatus (Setaram
Instrumentation, France) under flowing air at a temperature up to
1000 °C (10 °C/min heating ramp).
Raman spectroscopy was used to evaluate the organization
of carbons at the structural (atomic) scale, and especially
the degree of order. Carbon organization in CNT samples
was analyzed by Raman spectroscopy (Renishaw Invia
spectrometer, Renishaw, UK) at an excitation wavelength of
514 nm in the range of 800–3500 cm−1. Quantitative
Raman parameters were obtained by conventional fitting with
a linear baseline and Voigt functions (a combination of
Gaussian and Lorentzian functions, the proportion of
which is adjusted to each spectrum) using Renishaw Wire
3.2 software. The intensity of the D band and G band were
measured from the peak fitting using 1570–1590 cm−1 and
1340–1360 cm−1 limits for the D and G bands respectively
in order to calculate ID/IG ratio. Indeed, the D-band is
associated with the defect concentration or measure of
disorders in the C–C bonds within graphitic materials, while the
G-band is associated with in-plane vibrations of C–C bonds
and is a measure of graphitization or degree of metallicity
of graphitic materials . The characteristic Raman peak
intensity ratio ID/IG is a commonly used and useful
qualitative and quantitative way of evaluating the structural
defects to graphitization or crystallinity ratio in MWCNTs;
the degree of order increase when the ID/IG ratio decrease
(ID/IG ratio of graphite = 0) .
X-ray induced photoelectron spectroscopy
The surface chemical composition of CNT samples was
determined by X-ray induced photoelectrons spectroscopy
(XPS) using a Kratos Analytical Axis Ultra DLD
spectrometer (Kratos Analytical Inc., UK) with monochromatic AlKα
X-ray radiation (hν = 1486.6 eV). C1s, O1s and S2p spectra
were recorded at a take-off angle of 90° with a 700 μm by
300 μm slot aperture and 20 eV pass energy. The energy
scale of the instrument was calibrated by setting Au 4f7/2
= 84.0 eV. Data were acquired with Kratos Analytical Vision
2 software, and peak fitting was performed after Shirley
baseline background subtraction using Avantage Thermo
Electron software. A Lorentzian/Gaussian ratio of 30% was
applied to C1s, O1s and S2p peaks. The atomic sensitivity
factors used for semi-quantitative analysis were C1s = 1.0,
O1s = 2.93 and S2p = 1.68, relative to C1s = 1.00.
Endotoxin contamination of CNT
CNT samples were assessed for endotoxin
contamination using the Limulus Amebocyte Lysate assay (Lonza,
Switzerland), performed as recommended by the
Intrinsic ROS production
The intrinsic production of ROS by CNT was measured
in acellular conditions using the properties of φX174 RFI
plasmid DNA (Thermo Fisher Scientific, Waltham, MA)
. This DNA has the ability to decoil when in presence
Fig. 8 Raman spectroscopy analysis of CNT incubated in media presenting various acidity. Panel a ID/IG ratio calculated from Raman spectra of S-,
SF-, L-, or LF-CNT incubated for 48 h in culture medium in presence or absence of Concanamycin A. Panel b ID/IG ratio calculated from Raman
spectra of S-, SF-, L-, or LF-CNT incubated for 48 h in the supernatant of cells cultured in presence or absence of Concanamycin A. Panel c ID/IG
ratio calculated from Raman spectra of S-, SF-, L-, or LF-CNT incubated for 48 h in artificial medium which pH was set-up at 7.2, 6, or 4.5. Panel d ID/IG
ratio calculated from Raman spectra of S-, SF-, L-, or LF-CNT incubated for 48 h in culture medium in presence or absence of H2O2. Abbreviations as in
Fig. 1. Data are given as mean ± SEM of minimum 5 values
of ROS. Two hundred and 90 ng of plasmid DNA were
incubated with 100 μg/mL CNT for 8 h at 37 °C. The PstI
endonuclease (Thermo Fisher Scientific) was used as a
positive control. The different forms of DNA in the
samples (coiled, decoiled and linearized) were then separated
on an agarose gel (0.8%) for 16 h at 30 mV. The intensity
of the different DNA bands was quantified, and the ratio
of the decoiled and linearized DNA intensity to that of
total DNA was calculated.
Cell culture and exposure to CNT
RAW 264.7 murine macrophages, were purchased from
ATCC (Manassas, VA). Cells were cultured in Dulbecco’s
modified Eagle medium (DMEM) supplemented with
10% heat-inactivated fetal calf serum and antibiotics
(streptomycin, 10 mg/mL; penicillin G, 10,000 IU/mL) at
37 °C in a humidified atmosphere of 5% CO2/95% air.
Cells were exposed for 6, 24 or 48 h to 50 μg/mL CNT
prepared by dispersion of the dry material sample in
serum-free cell culture medium. For homogenization
purpose, the CNT suspensions were sonicated and
vortexed just before cell exposure. Culture medium alone
was used as a control. In a subset of experiments, cells
were treated with Concanamycin (Conca, 10 nM;
SigmaAldrich) or Lysosensor DND-189 (10 μM; Thermo
Fisher Scientific) 2 h prior to the end of CNT exposure.
For Raman spectroscopy analysis, at the end of cell
exposure, the cell supernatant was recovered, and cells
were washed three times in PBS. This washing solution
was added to the supernatant, centrifuged at 6000 rpm
for 15 min and washed 3 times with 10 mL of ultrapure
water to obtain a CNT pellet (SN fraction). To obtain
the CNT pellet from the Cell fraction, cells attached to
the cell culture dish were also washed 3 times with
10 mL of ultrapure water and centrifuged at 6000 rpm
for 15 min (Cell fraction). A few drops of each CNT
pellet (obtained from Cell and SN fractions) were deposited
on a glass slide and dried in an oven at 100 °C for
further analysis by Raman spectroscopy.
Transmission electron microscopy
Samples were analyzed using TEM (JEOL microscope,
Japan) to observe cells and cell components exposed to
50 μg/mL CNT for 6 h. Ultra-thin sections (90 nm) of
the cell samples were prepared as previously described
 and deposited on a Cu grid covered with thin
carbon film. Grids were observed at 80 kV.
RAW 264.7 cells were exposed to 10 μg/mL CNT for 6,
24 or 48 h, and Lysosensr DND-189 (10 μM,
Lifetechnologies) was added for the last 2 h. After exposure,
the cells were fixed and the fluorescence images were
digitally acquired on a Zeiss Axio Imager M2 (Carl
Zeiss). Fluorescence intensity was quantified (arbitrary
units) in at least 20 different cells per condition.
Otherwise mentioned, each value is given as the mean ±
standard error of the mean (SEM) of at least 3
experiments performed in triplicate. Data were analyzed with
the GraphPad Prism 5.01 software (La Jolla, CA).
Comparisons between multiple groups were performed using
Kruskall–Wallis’ non-parametric analysis of variance test
followed, when a difference was detected, by two-by-two
comparisons with Dunn’s multiple comparisons test.
Pvalues <0.05 were considered significant.
Additional file 1: Figure S1. Experimental set-up for CNT synthesis.
Experimental set-up for CNT synthesis leading to the obtainment of 4 final
batches of CNT: S-, SF-, L-, and LF-CNT. Abbreviations as in Fig. 1. (PDF 49 kb)
Additional file 2: Figure S2. Raman spectra. Raw Raman spectra
obtained from CNT recovered in macrophages exposed to the different
CNT for 48 h (Cell fraction). Abbreviations as in Fig. 1. (PDF 32 kb)
Additional file 3: Figure S3. Lysosomal activity assessment. Lysosensor
assay in RAW 264.7 macrophages exposed to CNT for 24 h (Panel a) or
48 h (Panel b). Scale bar: 10 μm. Abbreviations as in Fig. 1. Panel c:
quantification (arbitrary units) of fluorescence intensity at 6 h time point.
Panel d: quantification (arbitrary units) of fluorescence intensity at 24 h
time point. b quantification (arbitrary units) of fluorescence intensity at
48 h time point. *: p < 0.05 vs Control condition. (PDF 587 kb)
CCVD: Catalytic chemical vapor deposition; CNT: Carbon nanotubes;
EPO: Eosinophil peroxidase; HRP: Horseradish peroxidase; MPO:
Myeloperoxidase; MWCNT: Multiwalled carbon nanotubes; NOX: NAD(P)H oxidase;
OM: Optical microscopy; SEM: Scanning electron microscopy;
SWCNT: Singlewalled carbon nanotubes; TEM: Transmission electron
microscopy; TGA: Thermogravimetric analysis; XPS: X-ray-induced
This work was supported by funds from INSERM, Agence Nationale de la
Recherche (grant ANR-13-CESA-0010-01), and Université Paris Est-Créteil. Marion
Landry was a fellow from Agence nationale de sécurité sanitaire de l’alimentation,
de l’environnement et du travail (ANSES). Stéphane Tchankouo was a fellow from
the Institut Thématique Multi-Organismes (ITMO) Physiopathologie, métabolisme
et nutrition. This work also received the support of Labex SERENADE
11-LABX0064 and DHU A-TVB (Département Hospitalo-Universitaire
ML performed the experimental work, analyze data and wrote the first draft
of the manuscript. ST, EC and AR helped in the experimental work. ML, MP
and MM performed the particle synthesis and characterization. JB, MM and
SL critically reviewed the manuscript and worked on its final elaboration. All
the authors have read and approved final manuscript.
1INSERM, U955, Equipe 4, Créteil F-94000, France. 2NIMBE, CEA, CNRS,
Université Paris-Saclay, CEA Saclay, 91191 Gif sur Yvette Cedex, France.
3Université Paris Est-Créteil, Faculté de Médecine de Créteil, 8 rue du Général
Sarrail, Créteil F-94000, France. 4DHU A-TVB, Service d’explorations
fonctionnelles respiratoires, Assistance Publique Hôpitaux de Paris, Hôpitaux
Universitaires Henri Mondor, Créteil F-94000, France.
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