Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions
Loret et al. Particle and Fibre Toxicology
Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions
Thomas Loret 1 2
Emmanuel Peyret 2
Marielle Dubreuil 2
Olivier Aguerre-Chariol 0
Christophe Bressot 0
Olivier le Bihan 0
Tanguy Amodeo 0
Bénédicte Trouiller 2
Anne Braun 2
Christophe Egles 1 3
Ghislaine Lacroix 2
0 Institut National de l'Environnement Industriel et des Risques (INERIS), (DRC/CARA/NOVA) , Parc Technologique ALATA-BP 2, Verneuil-en-Halatte F-60550 , France
1 Laboratoire BioMécanique et BioIngénierie (BMBI), Université de Technologie de Compiègne (UTC), UMR CNRS 7338 , Compiègne 60205 , France
2 Institut National de l'Environnement Industriel et des Risques (INERIS), (DRC/ VIVA/TOXI) , Parc Technologique ALATA-BP 2, Verneuil-en-Halatte F-60550 , France
3 Department of Biomedical Engineering, Tufts University , Medford, MA , USA
Background: Recently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO2 and one nano-CeO2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the airliquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed. Results: Deposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used. Conclusions: We showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.
Nanomaterials; In vitro; Alveolar cells; Co-culture; Air-liquid interface; Submerged conditions; Toxicity
The growing utilization of nanomaterials (NMs) in
nanotechnologies leads to an increased risk of human exposure
, raising concerns about public health and safety [2–4].
Metallic and poorly soluble NMs are among the most
widely used  and a major exposure route for these NMs
is inhalation . Nevertheless, occupational and
environmental atmospheres have not been well characterized in
terms of NMs , which partly explains the lack of
epidemiological data on the relationship between exposure
to airborne NMs and potential adverse human health
effects. However, based on epidemiological studies
showing an association between exposure to environmental
ultrafine particles and adverse health effects , the
potential toxicity of NM has been taken into consideration and
been widely studied in cell cultures and animal models
[9, 10]. Results from animal experimentations remain
the most reliable [11, 12], especially because of the
similar level of complexity compared with the human
body. Besides ethical considerations, in vitro studies are
widely used to study mechanisms of toxicity because
they are usually cheaper, faster and easier to implement
than in vivo studies . Nevertheless, the relevance of
in vitro studies to predict in vivo effects needs to be
In vivo, inhaled NMs can deposit in the alveolar region
[14, 15] and interact with components of the alveolar
barrier at the air-liquid interface (ALI) . At the apical
side of the barrier, insoluble NMs first interact with the
thin layer of surfactant secreted by pneumocytes .
This layer covers the entire alveolar surface and
transport of NMs occurs from the air to the aqueous surfactant
phase . NMs can then be taken up by circulating
macrophages to be eliminated or interact directly with
pneumocytes [15, 17]. If NMs cross the alveolar barrier
[18, 19], they can interact with other components of the
barrier such as endothelial cells or immune cells and be
transferred to the blood and other organs [19, 20]. As a
consequence of the particle-cell interactions, mechanisms
of defense can become activated and cell damages can
occur such as cell function impairment, release of
proand anti-inflammatory cytokines, production of
intracellular Reactive Oxygen Species (ROS) and anti-oxidant
species, and genotoxicity [4, 6, 21].
In vitro, monocultures of pulmonary cells are usually
exposed in submerged conditions to suspensions of
NMs to determine mechanisms of toxicity  or high
throughput screening of novel compounds .
However, these experimental conditions do not reflect
cellcell communications and cell-particle interactions
occurring in vivo in the lung, making in vitro results difficult
to interpret [11, 12, 22–24]. Moreover, in submerged
conditions, cell-particle interactions are dependent on
the medium composition [25, 26]. NMs can interact
with components of the culture medium, resulting in
the formation of a medium specific corona [26, 27] and
can agglomerate into larger particles of different sizes.
Furthermore, in suspensions the dose delivered to the
cells depends on the NM properties in suspension and
capacity to settle, which makes in vitro dosimetry
To overcome these difficulties, more complex cellular
models [31, 32] and new in vitro exposure methods
[23, 33] have been and are still being developed to
study NM toxicity. Co-culture models are used to
mimic communications occurring between different
cell types in vivo in the lungs [9, 22]. These models
associate various cells such as epithelial, macrophage,
endothelial or dendritic cells . In vitro systems
exposing cells to aerosols of NMs at the ALI have been
developed to accurately mimic the cell-particle
interactions occurring in the lungs . With these ALI
systems, NM deposition on cells occurs through diffusion
and/or gravitational mechanisms . However, due to
current technical limitations, the maximum doses
achieved in these systems remain generally low
compared to those achievable through suspension
exposure. More recently, in order to improve the deposition
rate, exposure devices using electrostatic deposition of
charged particles [23, 34] or thermal precipitation 
have been introduced. However, it has not yet been
clearly defined whether in vitro simulation of in vivo
exposure conditions to test NM toxicity gives more
As a first step to address this question, the aim of our
study was to assess whether ALI exposures to NM
aerosols give similar results in terms of cellular responses,
compared to submerged exposures to NM suspensions.
For this purpose, we set up an in vitro system allowing
exposure of cell cultures to aerosols of poorly-soluble
metallic NMs. We exposed two alveolar models in
inserts: monocultures of alveolar epithelial cells A549
and co-cultures of A549 and macrophage like cells
THP1 at the ALI to aerosols of three TiO2 and one CeO2
NM. In parallel, we performed submerged exposures in
inserts to suspensions of the NMs, using similar culture
conditions and exposure kinetics. We also performed
classical culture and exposure conditions in plates. We
assessed the biological activity of the cells (release of
pro-inflammatory markers, cell functionality, cell
integrity, intracellular reactive oxygen species (ROS) levels)
after 24 h of exposure and compared the toxicities of the
NMs between aerosol and suspension exposure.
Results and Discussion
The aim of our study was to evaluate if exposing alveolar
cells at the ALI to aerosols of inhalable and poorly
soluble NMs would generate different toxicity patterns
and/or biological activation levels compared to submerged
exposures to suspensions (Fig. 1).
Exposures at the air-liquid interface to aerosols of NM
To simulate inhalation in vitro and to study the toxicity of
inhalable and poorly soluble metallic NMs on cells, a
system using VitroCell® chambers was set up in our
laboratory (Fig. 2). NM aerosols were generated by nebulization
of NM suspensions, using a nebulizer. The deposition of
NMs using similar VitroCell® systems was validated in
several studies [36–39]. Moreover, it was shown that the
deposited doses were sufficient to observe biological
adverse effects, even at low exposure doses [36, 39]. To
validate our system, four different metallic and poorly
soluble NMs: three TiO2 (NM105, NM101, NM100) and
one CeO2 NM (NM212), possessing different
physicochemical characteristics (Table 1) were used. TiO2 and
CeO2 NMs were selected because they are inhalable
and commonly used in toxicity studies at the lung level
[12, 20, 40].
Characterization of NM in aerosols
To evaluate the deposition of the NMs on the cells, we
first characterized the NM aerosols that were generated
in our system. By gravimetric measurements, NM mass
concentrations of around 10, 50 and 100 mg/m3 were
measured in the aerosols, for suspension concentrations
in the nebulizer of 1, 5 and 10 g/L, respectively (Table 2).
The mass size distributions of the aerosols were
evaluated (Additional file 1: Figure S1) and the total aerosol
volume concentrations were determined to assess the
aerosol effective densities (Table 2). Consistently with
the Cosnier et al. study , the aerosol effective
densities were much lower than the NM primary densities.
The number size distributions of the aerosols were
evaluated (Fig. 3a): we observed some isolated
primarysized particles, but most particles were agglomerated, as
indicated by higher Geometric Mean Diameters (GMDs)
than the NM primary particle diameters (Table 2). This
may explain why primary densities were low and why
high Volumetric Mean Diameters (VMDs) were
calculated (Table 2), indicating that most of the aerosol mass
was due to agglomerates. Furthermore, we observed that
the VMDs and GMDs of the NMs in the aerosols were
correlated to the NM mass concentrations measured in
Characterization of NM deposition on cells
After 3 h of exposure of the cells at the ALI, the size
distribution of the particles deposited on the cell surfaces
(Fig. 3) seemed to correlate with the size distribution of
the particles in the aerosols. Thus, the complete range of
particle sizes present in the aerosols was able to deposit
on the cell surfaces during exposure (Fig. 3b).
Furthermore, homogeneous distributions of the particles on the
cell surfaces were observed (Fig. 3b). To assess the mass
of NM deposited, QCM and ICP-MS techniques were
used. Some differences in results were observed, which
could be explained by technical differences between the
two methods. In the QCM technique, NMs get
deposited on an inert quartz surface instead of cells and there
is no discrimination between NMs and potential
contaminants in the deposited mass detected. However, the
ICP-MS methodology reveals the mass of NMs
deposited on the cell surface by direct dosage. For these
reasons, we took the ICP-MS method as reference and the
ICP-MS measurements were used to determine the
deposited mass of the NMs on the cells at the ALI for
the rest of the study. Deposited masses of around 0.1, 1,
3 μg/cm2 were measured for nebulized suspensions at
Fig. 1 In vitro comparisons between ALI and submerged exposure. Alveolar cells in monoculture or in co-culture were cultured in inserts or in
plates and exposed at the ALI to aerosols or in submerged conditions to suspensions of four poorly soluble NMs. Final doses were reached within
3 h in inserts and 24 h in plates. Total deposited doses were measured at the ALI or estimated in submerged conditions and cell biological
activity was assessed after 24 h of exposure to the NMs, performing cell viability, stress oxidative and inflammation assays. Comparisons were
performed between the biological activation levels determined after statistical analysis
Fig. 2 Exposure of cells at the ALI to aerosols of NMs. Cells at the ALI in inserts were exposed simultaneously to aerosols of NMs or to air in two
different VitroCell® exposure chambers. NM aerosols were generated at a 5 L/min flow rate by nebulization of suspensions using a nebulizer.
Aerosols were dried using a dryer to reduce relative humidity to 90 %. The aerosols were sucked using a vacuum pump to allow the NM
deposition on the cells. At the cell level, the flow rate was reduced to 5 mL/min/well using flow controllers to prevent cell damage. Aerosols
were characterized in real time using a SMPS and a COP, to assess the size distribution and by gravimetric measurements, to assess the mass
concentration. The deposition of NMs on the cells was assessed by performing QCM and ICP-MS measurements and TEM analysis, to assess the
mass, shape, size and distribution of the NMs on the cells
concentrations of 1, 5 and 10 g/L, respectively (Table 1).
The deposition efficiency on the cells was calculated
based on the deposited mass, the mass concentration of
the aerosol, the duration of exposure (3 h) and the flow
rate in the VitroCell® chambers (5 mL/min). Depending
on the NM physicochemical characteristics and on the
initial suspension concentrations, mean depositions
ranging from around 4 to 20 % were observed (Table 2).
The maximum deposition efficiency observed in our
study was either higher or lower than those reported in
several studies using similar exposure systems (4, 2,
1.1 % [37, 42, 43] and 70 % ). These discrepancies
could be due to differences in the physicochemical
characteristics of the NMs and aerosols generated or the
methods used to characterize the deposition.
Nevertheless, the maximum doses deposited in our system,
although remaining high when put into perspective with
real exposure scenarios , were low compared to
those reachable through submerged exposure .
Devices allowing electrostatic deposition of charged
particles [23, 34] or thermal precipitation  have been
introduced to improve deposition efficiency. For example,
Panas et al.  increased the deposition percentage of
SiO2 NMs of 50 nm from 0.5 to 11 % using an
Table 1 TiO2 and CeO2 physicochemical properties
electromagnetic field. However, it remains unclear
whether modification of the overall particle charge may
alter the particle-cell interactions. Furthermore, the mean
deposition rates obtained with our system were sufficient
for further toxicological assessments and we decided to
test the NM toxicities without using this approach.
Cellular models used to assess toxicity of NM at the
We assessed the toxicity of TiO2 and CeO2 NMs at the
ALI with our aerosol exposure system. Two cellular
models of the alveolar epithelium were chosen.
Monocultures of A549 cells, which is one of the most studied
alveolar epithelial cell line, were used for the ability of
these cells to form a cell layer (although without
functional tight junctions), grow at the ALI in inserts and
secrete surfactant [44, 45]. This cell line was successfully
exposed at the ALI to NM aerosols in several studies in
which similar exposure systems were used [36, 38]. A
co-culture model of A549 cells and THP-1 cells,
differentiated into macrophages, was also used to mimic
complex cell-cell interactions and communications
occurring in vivo. This model was chosen to represent
the complex alveolar structure comprising macrophages,
Surface area, BET (m2/g)
Primary density (g/cm3)
Fig. 3 Number size distribution of the aerosols and respective deposition in inserts. Aerosols were generated by nebulization of suspensions of
TiO2 (NMs 105, 101, 100) and CeO2 (NM212) at concentrations of 1 g/L (light grey), 5 g/L (dark grey), 10 g/L (black). The size distributions of the
NMs in the aerosols were measured using a SMPS and an OPC, and particles ranged from 10 to 1095 nm and 300 to 34 000 nm, respectively (a).
The deposition of the NMs on the TEM grids was assessed after exposure (b). TEM grids were placed on the apical side of inserts and exposed
3 h to aerosols generated with suspensions of 10 g/L in the nebulizer. After exposure, the grids were analyzed by TEM to assess the sizes, shapes
and distributions of the deposited NMs
in close contact with alveolar cells, which are involved in
the mechanisms of defense against particles [46, 47].
Circulating in the lumen of the alveolar space,
macrophages have the ability to produce pro-inflammatory
markers and internalize particles, underlining their
relevance in the study of host responses to NMs [46, 47]. To
mimic physiology, a ratio of ten A549 cells to one THP-1
cell was used, which is among the highest pneumocyte to
macrophage ratio observed in normal human lungs .
To enable ALI exposure to NM aerosols, the cellular
models were grown in 0.4 μm pore inserts. In this
configuration, after reaching confluence it was possible
to keep the A549 monocultures at the ALI, in the
presence (for co-cultures) or not of differentiated THP-1 cells,
without observing non-physiological medium
translocation from the basolateral to the apical compartment. To
maintain physiological conditions and prevent damage
from exposure to air, the cultures were exposed to
aerosols with 90 % humidity and exposure times of 3 h with
5 mL/min flow rates were used. In these conditions, no
decrease in mono or co-culture viability was observed,
compared to control cells kept at the ALI in the incubator
(Additional file 1: Figure S2).
NM toxicity at the air-liquid interface
To assess the potential adverse effects generated by TiO2
and CeO2 NMs, mono and co-cultures were exposed to
one (about 3 μg/cm2) and three doses (about 0.1 μg/cm2,
1 μg/cm2 and 3 μg/cm2) of NMs, respectively (Table 2).
These final doses were achieved by exposing the cells at
the ALI to the aerosols of NMs continuously for 3 h. After
apical NM deposition, the cells were kept at the ALI in
the incubator during 21 h with fresh medium
containing 10 % Fetal Bovine Serum (FBS) in the basolateral
compartment. Twenty-four hours after exposure, we
generally observed significant biological adverse effects
in the co-culture models and none in the monocultures.
Across the different biological assays performed,
adverse effects were detected at medium and high doses
of NMs (1 and 3 μg/cm2) (Figs. 4 and 5) and none were
observed at doses of 0.1 μg/cm2.
Among all the markers tested, the pro-inflammatory
mediators were the most sensitive. In the co-cultures, all
the cytokines tested were high above the quantification
limit. In the monocultures, IL-1β levels were between
detection and quantification limits, IL-6 and TNF-α
levels were just above the quantification limit and IL-8
levels were largely above the quantification limit. We
observed significant differences in cytokine levels in the
co-cultures with all NMs tested, compared to control
(Fig. 4). TiO2 NMs105 and 101 triggered pro-inflammatory
responses at lower doses than TiO2 NM100 and CeO2
NM212. After exposure to aerosols of TiO2 NM105, we
observed increased levels of IL-1β, IL-6, IL-8 and TNF-α at
doses of 1 and 3 μg/cm . With TiO2 NM101, we observed
significant increases in IL-1β, IL-6, TNF-α levels at doses
of 1 and 3 μg/cm2 and in IL-8 levels at 3 μg/cm2,
compared to control. Intriguingly, we observed lower levels of
response to TiO2 NM101 at high doses than at medium
doses. Although cytokines were dosed in the basolateral
compartment to avoid bias due to potential interactions
with the NMs, we cannot rule out the possibility that apical
cytokines coated with NMs at the apical side may not have
translocated to the basal compartment inducing variability
in the results at high doses. With TiO2 NM100, we
observed a significant increase in IL-8 levels at doses of
1 μg/cm2 and a significant increase of IL-6 levels at doses
of 3 μg/cm2, compared to control. With regards to CeO2
NM212, we observed a significant increase in IL-1β and
IL-6 levels and a significant decrease in IL-8 levels at doses
of 3 μg/cm2, compared to control.
Cell functionality was not affected across the
conditions. Regarding cell integrity, we observed a slight but
statistically significant decrease in cell integrity (around
5 %) in the co-cultures exposed to medium doses of
NM105, but not with the other NMs (Fig. 5). However,
probably due to high variability, we didn’t observe a
significant loss of integrity in the co-cultures exposed to
high doses of NM105. The integrity of the cells was not
impaired with the other NMs (Fig. 5). Finally, a
significant increase in intracellular ROS levels was observed in
co-cultures exposed to high doses of NM101 but not
with the other NMs (Fig. 5).
The potential adverse effects of poorly soluble NMs
were assessed at the ALI in a few other studies. For
example, in two linked studies [49, 50], the cytotoxicity
Fig. 4 Levels of pro-inflammatory mediators IL-1β, IL-6, IL-8 and
TNF-α in culture medium of cells exposed at the ALI to aerosols.
Mono (A549) and co-cultures (A549 + THP-1) were exposed for 3 h
at the ALI to aerosols of TiO2 (NM105, NM101, NM100) and CeO2
(NM212) or air and kept in the incubator at the ALI for 21 h, with
NMs deposited on their surface. Deposited doses were around 0.1, 1,
3 μg/cm2. At 24 h, IL-1β, IL-6, IL-8 and TNF-α levels in the culture
medium were measured by ELISA multiplex at the basal side. For
exposures at the ALI, specific air and positive controls (LPS 20 μg/
mL) (not shown on the graph) were used for each NM used and for
each concentration tested. Data represent the mean ± Standard
Deviation (SD) of three independent experiments. A Kruskal-Wallis
test followed by Dunn’s post-hoc test were performed to compare
treated groups to controls (*p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 5 Functionality, integrity and intracellular ROS levels of cells
exposed at the ALI to aerosols. Mono (A549) and co-cultures (A549
+ THP-1) were exposed for 3 h at the ALI to aerosols of TiO2
(NM105, NM101, NM100) and CeO2 (NM212) or air and kept at the
ALI for 21 h in the incubator, with NMs deposited on their surface.
Deposited doses were around 0.1, 1, 3 μg/cm2. At 24 h, Alamar blue®
and LDH assays were performed to assess functionality and integrity
of the cells, respectively. A DCF assay was performed to measure
intracellular ROS levels and H2O2 (1 mM) was used as positive
control for the DCF assay (not shown on the graph). Specific air
and incubator controls (cells kept in the incubator) (not shown on
the graph) were used for each NM used and for each
concentration tested. Data represent the mean ± SD of three
independent experiments. A Kruskal-Wallis test followed by
Dunn’s post-hoc test were performed to compare treated
groups to control (*p < 0.05; **p < 0.01; ***p < 0.001)
of TiO2 NM105 was evaluated in A549 cells using a
CULTEX® Radial flow system module. With a deposited
mass of 25 μg/cm2 per 15 min on the cells during 15, 30
and 60 min, they showed a strong decrease in cell
viability 24 h after exposure, of about 50, 60 and 70 %,
respectively. Using an electrode device to enhance
deposition, Panas et al.  exposed A549 cells to
aerosols of Aerosil200 (SiO2) and SiO2-50 with deposited
doses of 52 μg/cm2 and 117 μg/cm2, respectively. They
showed increased lactate deshydrogenase (LDH) and
IL8 release after exposure to Aerosil200 and increased
IL8 release after exposure to SiO2-50.
To conclude, it was shown in several studies that ALI
systems can be used to assess the toxicity of soluble
NMs in monocultures and co-cultures [36, 39, 51, 52]
with deposited doses of about 1-3 μg/cm . Assessment
of poorly soluble NM toxicity was also proven to be
feasible with alveolar epithelial cells in monocultures, by
increasing the deposited doses to around 25–100 μg/
cm2 [38, 49]. Our results indicate that it is also possible
to evaluate the toxicity of metallic and poorly soluble
NMs at the ALI at lower deposited doses, by using more
sensitive cell models like the co-cultures. The enhanced
sensitivity of co-culture models including macrophages
compared to monocultures of alveolar epithelial cells
was also observed in several studies in submerged
conditions [53–55]. More physiological culture models such
as human primary alveolar epithelial cells could also be
used. However, these are not commercially available and
their use requires to have access to human biopsies .
Exposure in submerged conditions to suspensions of NM
Since we could only observe biological adverse effects
with the co-cultures after exposure to aerosols of
NMs at the ALI, we focused on this model for the
following experiments. The co-cultures were exposed
in submerged conditions to suspensions of TiO2 NMs
105, 100, 101 and CeO2 NM212 in inserts and plates
with culture medium containing 10 % FBS. In inserts,
the final deposited dose was achieved within 3 h,
after which the suspensions were replaced with fresh
medium and the cells were kept in the incubator
during the remaining 21 h with the deposited NMs
on their surface. In plates, the final deposited dose
was achieved within 24 h.
Characterization of suspensions and deposited doses on cells
For accuracy, we characterized the NMs in suspension
and estimated the real mass deposited on the cells after
3 h of exposure in inserts and 24 h of exposure in
plates, using the in vitro sedimentation diffusion and
dosimetry (ISDD) model  (Table 3). First, Dynamic
Light Scattering (DLS) measurements were performed
in 2.56 mg/mL sonicated stock suspensions in Milli-Q
water and in 0.4 mg/mL suspensions in culture medium
(Additional file 1: Figure S3), to assess the size
distribution and hydrodynamic diameter of the NMs (Table 3).
We observed that the NMs were all polydispersed and
that most of the particles were agglomerated in
suspension, with sizes ranging approximately from 100 to around
1000 nm (Additional file 1: Figure S3). Furthermore, we
observed similar distributions in the Milli-Q water and
the culture medium. The effective densities of the NMs in
culture medium were measured following the Volumetric
Table 3 Characterization of suspensions and deposition of NM on cells
Hydrodynamic diametera, Z-average (nm) (n = 6)
Effective densityb (g/cm3) (n = 3)
Centrifugation Method (VCM) developed by Deloid and
coworkers  (Table 3). Once the NM suspensions were
characterized, we used these values to estimate the mean
deposited fractions of the NMs on the cells after 3 h
and 24 h of exposure, using the ISDD model (Table 3).
It is important to note that for each NM we assumed
that all the particles were agglomerated, had the same
size in suspension and had the same density. To
estimate more precisely the deposition, which depends
highly on the size and effective density, it would have
been necessary to measure the sizes and effective
densities of all the agglomerates separately, as
mentioned by Deloid et al. .
Initial concentrations in suspensions were adjusted
according to the estimated deposited fractions to
determine the real dose deposited on the cells (Table 4). As
shown by Deloid et al., we observed that the particles
were able to settle faster when the hydrodynamic diameter
and the effective density were higher. Furthermore, as it
was shown that NMs could interfere in assays [58–60]
leading to misinterpretation of results, we assessed the
potential interactions between the NMs and the cytokine
and LDH assays (Additional file 1: Figure S4).
after 24 h in platesc
after 3 h in insertsc
NM toxicity in submerged conditions
Co-cultures were exposed to suspensions of NMs in
inserts using similar culture conditions and exposure
kinetics to the air-liquid interface, to assess whether the
cells were more sensitive to NMs when exposed to
aerosols at the ALI. Cells were exposed for 3 h to NM
suspensions to achieve deposited doses of around 1, 3,
and 10 μg/cm2 (Table 4). Cells were then kept in the
incubator with fresh medium during the remaining 21 h
with the deposited NMs on their surface, and biological
adverse effects were assessed at 24 h.
The levels of the pro-inflammatory mediators IL-1β,
IL6, IL-8 and TNF-α were assessed after submerged
exposure in inserts, and similarly to at the ALI we
generally observed significant effects at lower doses with TiO2
NMs 105 and 101 than with TiO2 NM100 and CeO2
NM212 (Fig. 6). With NM105, we observed significant
increases in IL-1β, IL-8 and TNF-α levels at doses of 3 and
10 μg/cm2 and 10 μg/cm2 for IL-6. Significant effects were
observed with NM101 at 3 and 10 μg/cm2 for IL-6, IL-8
and TNF-α and at 10 μg/cm2 for IL-1β. Significant
inductions were observed for IL-6 and IL-8 with NM100, at
doses of 3 and 10 μg/cm2 and 10 μg/cm2, respectively.
24 h deposition in plates
3 h deposition in inserts
Table 4 Dose deposited in submerged conditions in function of nominal concentration in suspensions
Fig. 6 Levels of pro-inflammatory mediators IL-1β, IL-6, IL-8 and TNF-α in culture medium of cells exposed in submerged conditions in inserts.
Co-cultures (A549 + THP-1) were exposed in inserts for 3 h to suspensions of TiO2 (NM105, NM101, NM100) and CeO2 (NM212), to achieve
deposited doses of around 1, 3 and 10 μg/cm2. Suspensions were then replaced by fresh medium and cells were kept for 21 h at the incubator
with NMs deposited on their surface. At 24 h, IL-1β, IL-6, IL-8 and TNF-α levels were measured by ELISA multiplex in cell culture medium (apical
and basal sides). A specific control (cells exposed to culture medium) and positive control (LPS 20 μg/mL) (not shown on the graph) were used
for each NM used. Data represent the mean ± Standard Deviation (SD) of three independent experiments. A Kruskal-Wallis test followed by Dunn’s
post-hoc test were performed to compare treated groups to controls (*p < 0.05; **p < 0.01; ***p < 0.001)
Finally, we observed significant effects only with IL-8 and
TNF-α, at doses of 10 μg/cm2 with NM212.
Cell functionality and integrity were measured to
evaluate cell viability (Fig. 7). We observed slight (below
5 %) but significant decreases in cell functionality at the
dose of 10 μg/cm2 with the NMs 105 and 212 only,
compared to control. However, we observed significant
decreases in cell integrity with all the NMs tested: at
doses of 3 and 10 μg/cm2 for the NMs 105 and 101 and
at doses of 10 μg/cm2 for the NMs 100 and 212.
Intracellular ROS levels were also measured and we observed
significant increases only after exposure to doses of
10 μg/cm2 with NMs 105 and 100 (Fig. 7).
Co-cultures were also exposed in plates for 24 h to
deposited doses of about 1, 3, 10 and 20 μg/cm2 (Table 4),
to compare the ALI results with those obtained by
Fig. 7 Functionality, integrity and intracellular ROS levels of cells
exposed in submerged conditions in inserts. Co-cultures (A549 +
THP-1) were exposed in inserts for 3 h to suspensions of TiO2
(NM105, NM101, NM100) and CeO2 (NM212), to achieve deposited
doses of around 1, 3 and 10 μg/cm2. Suspensions were then
replaced by fresh medium and cells were kept for 21 h at the
incubator with NMs deposited on their surface. At 24 h, Alamar
blue® and LDH assays were performed to assess functionality and
integrity of the cells, respectively. A DCF assay was performed to
measure intracellular ROS levels and H2O2 (1 mM) was used as
positive control for the assay (not shown on the graph). Data
represent the mean ± SD of three independent experiments. A
Kruskal-Wallis test followed by Dunn’s post-hoc test were
performed to compare treated groups to controls
(*p < 0.05; **p < 0.01; ***p < 0.001)
classic submerged protocols (Figs. 8 and 9). In plates, we
only observed significant biological effects with the TiO2
NMs 105 and 101, compared to control. We observed
significant increases in the pro-inflammatory mediators
IL-1β and IL-6 at doses of 10 and 20 μg/cm2 for NMs 105
and 101 (Fig. 8). Significant increases in TNF-α levels at
doses of 10 and 20 μg/cm2 for NM101 and 20 μg/cm2 for
the NM105 were also observed, compared to control.
Nevertheless, we didn’t observe any significant effects on
IL-8 levels, probably due to NM-cytokine interactions
Fig. 8 Levels of pro-inflammatory mediators IL-1β, IL-6, IL-8 and TNF-α
in the culture medium of cells exposed in submerged conditions in
plates. Co-cultures (A549 + THP-1) were exposed in plates for 24 h to
suspensions of TiO2 (NM105, NM101, NM100) and CeO2 (NM212), to
achieve deposited doses of around 1, 3 and 10 and 20 μg/cm2. IL-1β,
IL-6, IL-8 and TNF-α levels were measured by ELISA multiplex in the
culture medium. A specific control (cells exposed to culture medium)
and positive control (LPS 20 μg/mL) (not shown on the graph) were
used for each NM used Data represent the mean ± SD of three
independent experiments. A Kruskal-Wallis test followed by Dunn’s
post-hoc test were performed to compare treated groups to controls
(*p < 0.05; **p < 0.01; ***p < 0.001)
(Additional file 1: Figure S4). Regarding cell functionality,
decreases were observed in cells exposed to suspensions
of NM105 (at doses of 3, 10 and 20 μg/cm2) and NM101
Fig. 9 Functionality, integrity and intracellular ROS levels of
cells exposed in submerged conditions in plates. Co-cultures
(A549 + THP-1) were exposed in plates for 24 h to suspensions
of TiO2 (NM105, NM101, NM100) and CeO2 (NM212), to achieve
deposited doses of around 1, 3 and 10 and 20 μg/cm2. Alamar
blue® and LDH assays were performed to assess functionality
and integrity of the cells, respectively. A DCF assay was
performed to measure intracellular ROS levels and H2O2
(1 mM) was used as positive control for the assay (not
shown on the graph). Data represent the mean ± SD of
three independent experiments. A Kruskal-Wallis test followed
by Dunn’s post-hoc test were performed to compare treated
groups to controls (*p < 0.05; **p < 0.01; ***p < 0.001)
(at doses of 20 μg/cm2) (Fig. 9). We observed a loss in cell
integrity at doses of 10 and 20 μg/cm2 (Fig. 9).
Finally, we observed significant increases in
intracellular ROS levels at doses of 10 and 20 μg/cm2 with
NM105 only (Fig. 9).
As reported in several studies, we observed that TiO2
NM [20, 40] and CeO2 NM [61, 62] were slightly toxic
but could induce oxidative stress, inflammation, and
cytotoxicity after exposure to high concentrations in
Comparison between air-liquid interface and submerged
We observed significant pro-inflammatory effects in
the co-cultures both at the ALI and in submerged
conditions (Table 5). However, we observed less
significant impacts in cytotoxicity and oxidative stress
(Table 6). To provide accurate comparisons between
ALI and submerged exposures, the potential
interactions between the NMs in suspension and the LDH
and cytokine assays were assessed in cell free
conditions, as described in the materials and methods
section. No interactions between the LDH assay and
the NMs were observed (Additional file 1: Figure S4a).
Although interactions were detected between the
proinflammatory markers and the NMs (Additional file 1:
Figure S4b), these interactions did not prevent appropriate
data interpretation. Release of pro-inflammatory
mediators by the cells appeared to be the most sensitive
indicator of biological adverse effects to the NMs at 24 h. For
this reason, we focused on the pro-inflammatory
responses to the NMs to perform the following comparisons
between ALI and submerged exposures.
Comparisons according to deposited doses
We observed significant pro-inflammatory responses in
cultures exposed to lower deposited doses at the ALI
compared to submerged exposures (Table 5). Moreover,
in submerged conditions, we observed significant effects
at lower doses in inserts compared with plates (Table 5).
Nevertheless, in vitro effects were observed at extremely
high doses (at least 100 fold higher) compared to
realistic human exposure scenarios .
We determined the Lowest Observed Adverse Effect
Levels (LOAELs) for significant pro-inflammatory levels.
For TiO2 NMs 105 and 101 we observed LOAELs at
doses of 1 μg/cm2 at the ALI, at 3 or 10 μg/cm2 in
submerged conditions in inserts and at 10 or even
20 μg/cm2 in plates. After exposure to TiO2 NM100 or
CeO2 NM212, we observed significant adverse effects at
doses of 1 or 3 μg/cm2 at the ALI, at doses of 10 μg/cm2
in submerged conditions in inserts but no effects after
exposure in plates, even with the maximum
concentration tested. Based on these results, we provided a
ranking of the four NMs used in our study according to the
LOAELs observed. Generally, the rankings were similar
whatever the exposure method used. We observed
significant adverse effects at lower doses with TiO2 NMs
105 and 101, compared to TiO2 NM100 and CeO2
NM212. This was in agreement with both in vitro and in
vivo literature observations. The mass based toxic effects
observed depend on the NM primary particle size
(surface area) . Indeed, despite differences regarding
their crystalline phases, TiO2 NM105 (20 % anatase,
80 % rutile) and NM101 (100 % anatase), with primary
Table 5 Lowest observed adverse effect levels (LOAELs in μg/cm2) determined with the pro-inflammatory effects for each exposure
ALIa Subm insertb Subm platec ALIa Subm insertb Subm platec ALIa Subm insertb Subm platec ALIa Subm insertb Subm plateb
aExposure at the air-liquid interface in inserts (ALI)
bExposure in submerged conditions in inserts
cExposure in submerged conditions in plates
Ø No effects measured at tested doses
particle sizes of 21 and 8 nm respectively, were more
toxic than NM100, which has a primary particle size of
100 nm. The observed toxic effects also depend on the
NM chemical composition and surface properties .
We observed that CeO2 NM212, with a primary particle
size of 29 nm, appeared as toxic as TiO2 NM100, which
has a primary particle size of 100 nm and less toxic than
NM105, which has a similar primary particle size.
To understand why significant effects were observed
at lower deposited doses at the ALI than in submerged
conditions, we attempted to evaluate whether the cells
cultivated at the ALI were more sensitive to xenobiotics
in general than cells cultivated in submerged conditions.
We compared the release of pro-inflammatory mediators
in the co-culture model in insert at the ALI and in
submerged conditions. We stimulated cells cultured at the
ALI and in submerged conditions with a dose of 20 μg/
mL of Lipopolysaccharide (LPS) in the basolateral
compartment (Fig. 10). The stimulation was effective and
significant differences in cytokine levels were observed
for all the cytokines tested, compared to non-stimulated
controls. In ALI conditions we observed cytokine
inductions only at the basolateral side, except for TNF-α
(Fig. 10a) and in submerged exposures we observed
inductions both at the apical and basal sides (Fig. 10a).
Moreover, we observed significantly higher levels of
proinflammatory mediators at the basolateral side but not
at the apical side after stimulation at the ALI compared
to submerged conditions (Fig. 10a). This can be partly
aExposure at the air-liquid interface in inserts (ALI)
bExposure in submerged conditions in inserts
cExposure in submerged conditions in plates
Ø No effects measured at tested doses
attributable to higher basal secretion at the basolateral
side at the ALI. Indeed, when the data was normalized
to non-stimulated cellular secretion, the increase of
cytokine secretion at the ALI was less clear, compared to
submerged exposure (Fig. 10b). At the ALI, we dosed
significantly more cytokines at the basolateral side for
IL-1β and TNF-α, but significantly less for IL-6,
compared to submerged conditions. On the contrary, in
submerged conditions we dosed significantly more
cytokines at the apical side compared to at the ALI. For
IL8, stimulation with LPS induced an unexpected
significant drop in secretion compared to control (Fig. 10a)
with significantly less cytokines at the basolateral side
and more at the apical side in ALI compared to
submerged conditions (Fig. 10b). In summary, there seemed
to be opposite trends in the polarity of cytokine
secretion between the exposure conditions, with more
basolateral secretion at the ALI and more apical secretion in
submerged conditions. Nevertheless, after taking into
account the different cellular responses observed in the
ALI and submerged conditions after exposure to LPS,
we observed that the levels of induction were similar.
We concluded, therefore, that there was no difference in
sensitivity to xenobiotics that could explain the higher
adverse effects observed in the cells at the ALI after
exposure to NMs (Table 5).
We also assessed whether the differences observed
between the ALI and submerged conditions could be
explained by differences in cell densities. In their study,
Fig. 10 levels of pro-inflammatory mediators after stimulation with LPS (20 μg/mL). Co-cultures were stimulated at the ALI or in submerged
conditions in inserts with 20 μg/ml of LPS at the basal side for 21 h. Levels of IL-1β, IL-6, IL-8 and TNF-α were assessed in culture medium at basal
side and in culture medium or washing liquid at the apical side for submerged and ALI exposures, respectively. The control for ALI exposures was
cells exposed at the ALI to air for 3 h in the exposure system and kept at the ALI for 21 h with fresh medium in the incubator. The control for
submerged exposure was cells kept in submerged condition with fresh medium for 21 h. IL-1β, IL-6, IL-8 and TNF-α secretions were measured by
ELISA multiplex in the cell culture medium. Results were first expressed in concentrations (pg/mL), to assess whether cells secreted similar
amounts of cytokines at the ALI and in submerged conditions in inserts, after stimulation (a). Because we observed more basal secretion at the
ALI (secretion by non stimulated cells), the data was also expressed in cytokine levels compared to control (b), to compare ALI and submerged
results more accurately. Data represent the mean ± SD of three independent experiments. A two-way Anova followed by a Bonferroni post-hoc
test were performed to compare treated groups to their respective controls (*p < 0.05; **p < 0.01; ***p < 0.001) or to compare ALI and submerged
exposures (#p < 0.05; ##p < 0.01; ###p < 0.001)
Lenz et al.  observed a strong correlation between
exposures at the ALI in inserts and submerged
conditions in inserts and in plates, when expressing results in
dose per cell units. We measured the number of cells in
the inserts and in the plates during exposure to the
NMs. We counted similar numbers of cells at the ALI
(599 000 cells/cm ) and in submerged conditions (608
000 cells/cm2) in inserts, and fewer cells in submerged
conditions in plates (226 000 cells/cm2). Thus, by
expressing results in doses per cell, the differences
observed in LOAELs remained similar between conditions
in inserts. Moreover, lower cell densities in submerged
conditions in plates conferred higher LOAELs when
expressing the results in doses per cell instead of doses
per surface thus increasing the differences observed
between the ALI and classical submerged conditions. In
our study, cells were exposed to NMs and not to soluble
chemicals (Bortezomib), as was the case in the Lenz et
al. study, which may explain why we did not observe a
correlation of the results between the ALI and
submerged conditions by normalizing the LOAELs to the
number of cells exposed. Indeed, poorly soluble NMs
are toxic through surface reactivity . Moreover, in
contrast to chemicals which are highly soluble, the doses
of poorly soluble NMs are heterogeneously distributed
on cells, especially in the complex co-culture model
presenting an uneven surface. For this reason we thought
that expressing the doses in μg/cm2 rather than in μg/
cells may be better to describe our results. Nevertheless,
further investigations are still needed to assess if μg/cm2
is a better dose metric for poorly soluble NMs.
To conclude, the lower LOAELs observed in the ALI
conditions were not due to higher basolateral cytokine
secretion by the cells or because of different cell
numbers. The differences were unlikely caused by
NMcytokine interactions in the submerged conditions, as
the cytokines were dosed separately in the apical and
basolateral sides to limit this bias (for exposure in inserts
only). For these reasons, we hypothesized that the
differences may have been due to a higher sensitivity of the
cells to the NMs or at least to particles at the ALI.
However, it was not possible to conclude this with certainty.
Indeed, we could not totally exclude that uncertainties
regarding the measurements of the deposited doses on
cells, especially in submerged conditions where no direct
dosage was performed, could explain the differences in
LOAELs, of at least a 3 fold order of magnitude,
observed between ALI and submerged exposure in inserts.
Thus, more accurate dosimetry should be considered in
the future to provide better comparisons.
After exposure to NMs, direct comparisons between
ALI and submerged results have been performed in
other studies, leading to different conclusions. As was
observed in our study, Lenz et al. observed effects at
lower doses when cells were exposed to ZnO NPs at the
ALI . Using ZnO NM, Xie et al. observed toxic
effects at “doses that are in the same order of
magnitude” . Using SiO2-50 and Ag NM respectively,
Panas et al.  and Herzog et al.  concluded that
the nanoparticles were less toxic when deposited at the
ALI. In these studies, different NMs were used, which
could explain the discrepancy between the conclusions.
For example, some NMs have the ability to release ions
in suspension  and it was shown that both ions and
particles are able to cause toxic effects on cells .
Thus in some cases, the differences in toxic effects
observed between ALI and submerged exposures can be
attributed to an increase in the proportion of ions
released in the submerged conditions . Differences in
cellular models, methodologies used to expose the cells
to aerosols or suspensions and in methods employed
to characterize the NM deposition on the cells are
additional factors that could also explain the different
Xie et al.  used the same method to prepare the
NM suspensions to generate aerosol and submerged
exposures. In this study, FBS was added to the
suspension, which could induce the formation of a medium
specific corona at the ALI. This might explain why they
observed similar effects between the ALI and
submerged exposures. To improve the deposited dose in
their system, Panas et al.  raised the aerosol flow
rate to 100 mL/min at the cellular level. As an increase
of the flow rate can lead to a decrease in cell viability,
they added 100 μL of PBS on the cell surface, which
can lead to a decrease in the cell surface directly
exposed at the ALI. Moreover in some studies, the real
mass deposited on the cells after exposure to
suspensions was reached after 24 h of exposure, which can
create uncertainties towards the conclusions.
In conclusion, while there is a general trend towards
higher sensitivity of ALI as compared to submerged
exposure conditions, relatively large uncertainties in the
dose estimates for submerged conditions (due to
uncertainties in volume median diameter and effective
density) render this result uncertain. More advanced
dosimetry methods as currently available are required to
resolve this issue with certainty.
Importance of the dose rate
In our study, the final deposited doses were reached
either within 3 h in the inserts, in ALI and submerged
conditions or within 24 h in the plates. We observed
significant effects at lower doses in the inserts than in
the plates, in the submerged conditions (Table 5). Thus,
as shown recently in vivo , it also seems important
to consider the dose delivery rate when assessing NM
toxicity in vitro.
The deposition of NMs on cells via aerosol exposure
was validated in our system. We showed that the
maximum deposited doses achieved were of about 3 μg/cm2
for a 3 h-exposure at the ALI. These doses were low
compared to those reached in submerged exposures, but
were sufficient to observe biological adverse effects
(inflammation, cytotoxicity and oxidative stress) 24 h after
exposure. Thus, we hypothesize that our model can be
used to assess the toxicity of other metallic and insoluble
NMs. Furthermore, biological adverse effects were
observed in A549 + THP-1 co-cultures but not in A549
monocultures, indicating a higher sensitivity of the
coculture model. This underlines the importance of the
cellular model used and offers the possibility to deal with
low deposition doses by using more sensitive and
physiologic cellular models.
To compare the ALI results to those obtained in
classic submerged experiments, we exposed the co-culture
models to suspensions of NMs and assessed for
biological adverse effects (inflammation, cytotoxicity and
oxidative stress). Based on the quantified or estimated
deposited doses on the cells, we performed direct
comparisons of the results between the different exposure
methods used. We observed adverse effects at lower
deposited doses after exposure at the ALI to aerosols of
NMs than in submerged conditions to suspensions of
NMs. Furthermore, comparing submerged exposures in
inserts and plates at the same dose of NMs, we showed
that the biological effects observed were dependent on
the timing of the dose delivery. We provided a ranking
of the NMs according to the biological adverse effects
observed and these were ranked similarly whatever the
exposure method used. Thus, despite the differences in
levels of biological adverse effects observed, we showed
that the two in vitro methods provided reliable results in
the assessment of potential biological adverse effects and
the ranking of poorly soluble and metallic NMs. Future
studies should examine more precisely why biological
effects were observed at lower deposited dose at the ALI
in our study. Studies with accurate dosimetry are still
necessary to confirm if differences in sensitivity exist
when cells are exposed at the ALI to poorly soluble
NMs. It would be also interesting to determine the
influence of surfactant and to assess the importance of the
corona surrounding NMs, both at the ALI and in
The nano-TiO2 NM105 (AEROXIDE® TiO2 P25) was
obtained from Evonik Industries. The nanos TiO2
NM100 and NM101 and nano-CeO2 NM212 were
obtained from the Joint research council (JRC). The TiO2
and CeO2 physicochemical properties (Table 1) were
well characterized by the Joint research council (JRC)
[68, 69]. The endotoxin levels of the NMs were tested by
partners of the European project NANoREG. They were
below the limit of detection (data not shown).
The human type II alveolar epithelial cell line A549 and
the human alveolar monocyte cell line THP-1 were
obtained from our partners of the NANoREG project
(from BAUA and GAIKER, respectively). Both cell lines
were cultivated in RPMI 1640 medium (Gibco, 61870),
supplemented with 10 % Fetal Bovine Serum (FBS)
(Gibco, 15070) and 1 % penicillin-streptomycin (Gibco,
15070) (culture medium) at 37 °C in a humidified
atmosphere containing 5 % CO2 (Sanyo-18AIC). A549
and THP-1 cells were seeded in 75 cm2 tissue culture
flasks (Falcon, 353136), with 700 000 A549 cells/flask
and 3 000 000 THP-1 cells/flask. At 90 % confluency,
A549 cells were trypsinized (Gibco, 25300), and seeded
into 24-well plates (Falcon, 353047) with 50 000 cells/
well, (0.5 mL of culture medium/well) for submerged
exposures in plates or seeded in 6 well plates inserts
(4.67 cm2 of diameter, 0.4 μm pore size, Costar, 3450)
with 80 000 cells/insert (1 mL of culture medium at the
apical side and 2 mL at the basal side/insert) for aerosol
exposures and submerged exposures in inserts. For the
co-cultures, THP-1 cells were differentiated into mature
macrophage-like cells in culture flasks with 300 ng/mL
of Phorbol Myristate Acetate (PMA) (Sigma-Aldrich,
P1585) for 24 h and seeded on the A549 cells 24 h
before exposure, at a ratio of one THP-1 cell to ten A549
cells. To calculate the number of A549 cells at the
exposure time, cells were grown for 5 days in dedicated
inserts (at the ALI and in submerged conditions) or for
72 h in plates, were trypsinized and then counted. To
trypsinize cells in inserts, trypsin was added at the apical
and basal side to promote cell detachment.
Exposure at the ALI to aerosols
For monoculture exposures, A549 cells were grown for
96 h until confluence. The culture medium at the apical
side of the cells was then retrieved to adapt the cells to
the ALI for 20 h before exposure. For co-culture
exposures, A549 cells were grown for 96 h until confluence.
In the meantime, THP-1 cells were differentiated into
mature macrophage-like cells. One day prior exposure,
the differentiated THP-1 cells were washed, trypsinized
(Gibco, 25200), centrifuged and seeded on the A549 cells
with a ratio of one THP-1 cell for ten A549 cells. Four
hours after seeding, the culture medium at the apical
side of the cells was removed to adapt the co-cultures to
the ALI for 20 h before exposure. Five days after A549
seeding, both cell models were exposed for 3 h at the
ALI. Just after exposure cells were placed in new plates
(Costar, 3516) with fresh culture medium containing
10 % FBS at the basal side and kept at the ALI for 21 h
in the incubator. At every step, the condition of the cells
was checked carefully by optical microscopy.
Aerosol exposure system
A system using VitroCell® devices was set up to expose
mono or co-cultures to aerosols of NMs (Fig. 2). The
system was composed of two chambers (VitroCell®, 6/4
and 6/3 CF Stainless cultivation base modules) in which
cells seeded in inserts were exposed at the ALI to
aerosols of NMs or to filtered air. The NM exposure
chamber contained 4 wells. Three wells were dedicated to
expose cells grown in inserts to aerosols of NMs, and
one to assess the real-time deposition of the NMs on a
Quartz Cristal Microbalance (QCM) (VitroCell®). The
QCM was used to check if the system was running
properly and to quantify the NM deposition on the
inserts. The air exposure chamber contained three wells
in which cells grown in inserts were exposed to filtered
air containing 90 % humidity. Culture medium
supplemented with 25 mM HEPES (Gibco, 156030-056) was
individually supplied to each basolateral compartment of
the inserts to maintain the cells at the ALI during
exposure. The chambers were connected to a water bath to
maintain the cells at 37 °C.
To generate the aerosols, suspensions of 1, 5 and 10 g/
L of NM were prepared in Milli-Q water, and sonicated
for 5 min in an ultrasonic bath (Bioblock, Leo-80). A
nebulizer (Palas, AGK 2000) supplied with filtered air
(Norgren, SPGB/BMR/28262) was used at a flow rate of
5 L/min, to aerosolize the suspension. A dryer composed of silica gel (Roth, P077) was used to prevent condensation into the system, maintaining the relative humidity
above 90 %. To expose the cells, a vacuum pump (KnF,
N840FT-18) and flow calibration valve (VitroCell®) were
used to suck TiO2 and CeO2 aerosols or air into the
VitroCell® chambers at a 5 mL/min flow rate.
Characterization of aerosols and NM deposition on cells
Mass concentrations of the NMs 105, 100, 101 and 212
in the aerosols were measured by a gravimetric method.
Briefly, quartz microfiber filters (Whatman, 1851-037)
were dried for 24 h in a dessicator and weighed before
exposure. Samplings of the aerosols were performed in
triplicates at 4 L/min for at least 10 min using a
sampling pump (Casella, Apex personal air sampler). After
exposure, the filters were dried for 24 h and weighed.
The mass concentration was calculated according to the
mass of NM weighed and the sampling volume. A
Scanning Mobility Particle Sizer SMPS, (Differential Mobility
Analyser (DMA) (Grimm, L-DMA 5400) with
Condensation Particle Counter (CPC) (Grimm, 5.416) and
XRay neutralizer (TSI, 3087), and an optic counter (OPC)
(Grimm, 1.109) were used to measure the size
distribution of the particles in the aerosol. Particles ranging
from 10 nm to 30 μm of diameter were measured.
According to the number size distribution
measurements, the GMDs of the aerosols (equivalent to count
median diameters) were calculated. VMDs were also
calculated assuming perfect spherical geometry of the
NMs in the aerosol for the conversion from number size
distributions to volume size distributions. This
assumption was made based on the observations of spherical
NM agglomerates deposited at the apical side of the
inserts (Fig. 3b). The effective densities of the aerosols
were then calculated by dividing the aerosol
concentration, measured by gravimetry, by the total aerosol
volume concentration, calculated by SMPS and OPC.
The deposition of the NMs on the cells after 3 h of
exposure was also characterized. QCM and ICP-MS
measurements were performed to measure the mass of
NMs deposited on cells. For ICP-MS analysis, A549 cells
at confluence were exposed for 3 h to aerosols of NMs.
At the end of the exposure period, the insert membranes
were directly cut with a scalpel and kept in tubes
(Dutsher, 030402) at -20 °C before analysis. Samples
were mineralized to perform the analysis. According to
the deposited masses measured, the deposition
efficiencies on the cells were calculated. To do this, the
theoretical deposited masses were calculated assuming 100 % of
deposition on the cells, by dividing the mass
concentration of the aerosols by the aerosol volume passing
through the exposure chambers within 3 h. The
deposition efficiencies were calculated for each NM and
concentration used by dividing the mass measured (by
QCM or ICP-MS) by the theoretical deposited mass.
Transmission Electron Microscopy (TEM) grids (Agar
scientific, Quantifoil S143-3) were used to assess the
shapes, sizes, and distributions of the NMs deposited.
TEM grids were deposited on the apical side of the
inserts, exposed for 3 h to aerosols of NMs and then
analyzed by TEM.
Submerged exposure to NM suspensions
To expose co-cultures to suspensions, THP-1 cells were
first differentiated into mature macrophage-like cells with
300 ng/mL of PMA in culture flasks for 24 h.
Differentiated THP-1 cells were trypsinized, washed, centrifuged
and seeded on A549 cells 20 h before exposure, to achieve
a ratio of ten A549 cells to one THP-1 cell. In inserts,
5 days after A549 seeding, the co-cultures were exposed at
the apical side to NM suspensions in culture medium
containing 10 % FBS with concentrations ranging from 23 to
545 μg/mL depending on the NMs used, to achieve
deposited doses of 1, 3 and 10 μg/cm2 after 3 h of exposure
(Table 4). After 3 h of exposure, the apical NM
suspensions and the basal medium were removed carefully, to
ensure that all the particles deposited remained on the cell
surface and were replaced by fresh culture medium
containing 10 % FBS. Cells were kept for the remaining
21 h in the incubator with NMs deposited on their
surface. In plates, 72 h after A549 seeding, cells were
exposed for 24 h to suspensions of NMs in culture
medium containing 10 % FBS, with concentrations
ranging from 4 μg/mL to 200 μg/mL (equivalent to about
1 μg/cm2 to 20 μg/cm2 deposition, depending on the
deposition ratio calculated) (Table 4).
Characterization of suspensions and NM deposition on cells
Stock suspensions of NMs were prepared in Milli-Q
water at 2.56 mg/mL. Suspensions were dispersed with a
sonicator equipped with a cup horn (QSONICA, Q700),
at maximum amplitude, at a frequency of 2 times 1 min
with a pause of 1 min between. The cup horn indicated
the total energy delivered to the volume of water in the
cup (1 L) and to the sample (40 000 J). We also
estimated the energy delivered to the sample experimentally
(97 J), as described in the Additional file 1. To expose
the cells, sonicated suspensions were first diluted in
culture medium and successive dilutions were performed
to achieve the desired concentrations. For each NM,
DLS measurements were performed (Malvern, Zetasizer
Nano S) on stock and 0.4 mg/mL suspensions to
measure the hydrodynamic diameter and assess the size
distribution of the particles in suspension. The effective
densities of each NM were measured in suspension
according to the VCM method developed by Deloid and
The ISDD model  was used to estimate the
deposited fraction on cells after 3 h of exposure in inserts or
24 h of exposure in plates. The primary particle
diameters, the primary densities of the powders, the
hydrodynamic diameters, the effective densities of the
NMs measured in suspensions, the height (0.214 cm for
inserts, 0.25 cm for plates), the temperature (37 °C), the
density (1.00 g/mL), the viscosity (0.00074 Pa.s) and the
nominal NM concentrations (4 to 547 μg/mL) of the
medium were used as input parameters. For results
interpretation, nominal NM concentrations expressed
in μg/cm2 were adjusted according to the estimated
Alamar blue® assay (cell functionality)
After 24 h of exposure to NMs, an Alamar blue®
assay was performed to measure the metabolic
activity of the cells exposed to aerosols or suspensions.
The culture medium was retrieved and the cells were
washed with HBSS (Gibco, 140025). Some washing
liquids (1 mL/sample) from aerosol exposures were
also kept for analysis. Cells were then incubated at
37 °C, in 5 % CO2 for 1 h submerged with 0.5 mL
(in plates) or 1 mL (in inserts) of Alamar blue®
solution (Invitrogen, prestoblue A13261) diluted to 10 %
in culture medium. After 1 h of incubation, 100 μl of
metabolized Alamar blue® was transferred in a 96 well
plate (Falcon, 353072) and the fluorescence was read
(excitation: 555 nm, emission: 585 nm) using a
spectrophotometer (TECAN, infinite 2000). The values of
each sample were expressed in percentage of cell
functionality compared to control. Cells exposed to
clean air at the ALI served as controls for the aerosol
exposures; cells exposed to culture medium served as
controls for suspension exposures.
LDH assay (cell integrity)
LDH releases in apical or basal sides were measured
in culture media retrieved after 24 h of exposure to
suspensions and aerosols and kept at 4 °C until
analysis. Culture media, retrieved at the apical sides,
were centrifuged for 5 min at 13 000 G and 4 °C to
remove the NMs. A commercially available kit
(Promega, CytoTox-ONE Homogeneous Membrane
Integrity assay) was used according to the supplier manual.
The fluorescence was measured (excitation: 550 nm,
emission: 585 nm) using a spectrophotmeter (TECAN
infinite 2000). The values of each sample were
expressed in function of the maximum LDH release
by cells, in percentage of cell integrity compared to
control. To measure the maximum LDH release, cells
were lysed for 24 h using a 0,1X solution of triton
(Sigma-Aldrich, T8787). For exposure to suspensions
in inserts, results from the apical and basal sides were
pooled to evaluate the cell integrity.
Dichlorofluorescein (DCF) assay (Intracellular ROS)
After performing Alamar blue® assays, the cells were
washed with PBS (Gibco, 10270). Afterwards, the cells
were incubated at 37 °C, in 5 % CO2 with 10 μM of a
diacetate (CM-H2DCFDA) probe (Life technologies,
C6827) in PBS (0.5 mL/well or insert) for 35 min. After
30 min of incubation, the probe was removed in some
wells, 1 mM of H2O2 in PBS was added and the cells
were incubated for 5 min to serve as positive controls.
After incubation, the cells were washed with PBS and
incubated for 5 min in 90 % Dimethyl Sulfoxide (DMSO)
(Sigma-Aldrich, D2438) in PBS (0.5 mL/well or insert).
Then the cells were scraped using a scraper (TPP,
99002), the well or inserts contents were recovered in
tubes (Eppendorf, 3810X) and the tubes were
centrifuged at 10 000 G, at 4 °C, for 5 min to eliminate dead
cells and remove remnants particles. The tube contents
were transferred into black 96 well plates (150 μL/well)
(Greiner Bio-one, 655076) and the fluorescence of the
samples was read (excitation: 488 nm, emission: 530 nm)
using a spectrophotometer (TECAN, infinite 2000). The
values of each sample were expressed in percentage of
intracellular ROS compared to their respective control.
Cytokine and chemokine quantification by ELISA
Pro-inflammatory mediator levels were measured in
culture media or washing liquids retrieved after 24 h of
exposure from aerosol and submerged exposures and
kept at -80 °C until analysis. Before freezing, culture
media and washing liquids were retrieved from the
apical side of submerged and aerosol exposures and
centrifuged for 5 min at 13 000 G at 4 °C to remove the
nanoparticles. Il-1β, IL-6, IL-8 and TNF-α release was
measured using a commercially available ELISA
multiplex kit (Mesoscale discovery, K15025B) and a multiplex
reader (Mesoscale discovery, Sector Imager 24000),
according to the supplier recommendations. The values
of each sample were expressed in percentage of cytokine
levels compared to the respective control. Cells
stimulated for 21 h to a concentration of 20 μg/mL of LPS
(Sigma-Aldrich, L2880) (at the basal side for exposures
in inserts), were used as positive control.
Interactions between the NMs and the LDH assay
Potential interactions between LDH and NMs were
assessed. 96 well plates (Falcon, 353072) were incubated
under cell-free conditions for 24 h with suspensions of
0, 100, 400 μg/mL of TiO2 and CeO2 in presence of
0275 UI/mL of LDH standard (Roche, 10127876001).
After 24 h of incubation, supernatants were retrieved,
centrifuged for 5 min at 13 000 G at 4 °C to remove the
nanoparticles and the LDH assay was performed.
Interactions between the NMs and the cytokines
Potential interactions between NMs in suspension and
the cytokines were studied. For suspension exposures,
96 well plates (Falcon, 353072) were incubated under
cell-free conditions for 24 h with suspensions of 0, 100,
400 μg/mL of TiO2 and CeO2 in presence of 1250 pg/
mL of IL-1β, IL-6, IL-8 and TNF-α. After 24 h of
incubation, the supernatants were retrieved and centrifuged
for 5 min at 13 000 G at 4 °C to remove the
nanoparticles. IL-1β, IL-6, IL-8 and TNF-α were measured by
ELISA multiplex (Mesoscale discovery, K15025B) in the
All the data was expressed as mean ± standard deviation
(SD) from three independent experiments performed in
triplicates. The statistical analysis was performed using
Graphpad Prism 5.0 (GraphPad Software Inc., San Diego,
CA). Shapiro-wilk’s and Bartlett’s tests were used to assess
the data normality and the variance equality, respectively.
Because variances weren’t equal, results were analyzed by
the non-parametric Kruskal-Wallis test followed by
Dunn’s post-hoc test to compare the different treated
groups to the controls. Cells exposed to air at the ALI
served as control for aerosol exposures. Cells exposed to
culture medium served as control for suspension
exposures. For alamar blue and LDH assays, values from cells
exposed to NMs or control and from cells kept in the
incubator (for ALI exposure only) were included in the
statistical analysis. For the DCF assay, values from the cells
exposed to NMs or to control air and from cells exposed
to 1 mmol H2O2 were included in the statistical analysis.
For ELISA assays, values from cells exposed to NMs or
control and from cells stimulated with 20 μg/mL of LPS
were included in statistical analysis. To assess the level of
pro-inflammatory mediators at the ALI and in
submerged conditions after stimulation with LPS, a
twoway Anova followed by Bonferroni post-hoc test was
performed to compare treated groups to controls or to
compare ALI and submerged exposure. In all the
analysis, p-values < 0.05 were considered significant.
Additional file 1: Figure S1. Mass size distribution of TiO2 and CeO2
aerosols. Figure S2. Functionality and integrity of mono and co-cultures
exposed at the ALI. Figure S3 Number size distributions of TiO2 (NM105,
101, 100) and CeO2 (NM212) NM in suspensions used to expose cells.
Figure S4. Interactions between the NMs and LDH or with cytokines in
suspensions. Table S1. Alamar blue results expressed in percentage of
functionality compared to control. Table S2. LDH results expressed in
percentage of integrity compared to control. Table S3. DCF results
expressed in percentage of intracellular ROS compared to control. Table S4.
IL-1β results expressed in percentage compared to control. Table S5. IL-6
results expressed in percentage compared to control. Table S6. IL-8 results
expressed in percentage compared to control. Table S7 TNF-α results
expressed in percentage compared to control. Table S8. IL-1β levels at the
basal and apical sides after stimulation with LPS (20 μg/mL). Table S9. IL-6
levels at the basal and apical sides after stimulation with LPS
(20 μg/mL). Table S10. IL-8 levels at the basal and apical sides after
stimulation with LPS (20 μg/mL). Table S11 TNF-α levels at the basal
and apical sides after stimulation with LPS (20 μg/mL).
(DOCX 670 kb)
ALI: Air-liquid interface; DLS: Dynamic Light Scattering; DMSO: Dimethyl
Sulfoxide; FBS: Fetal Bovine Serum; ICP-MS: Inductively Coupled
Plasma—Mass Spectrometry; ISDD: in vitro sedimentation diffusion and
dosimetry; LDH: Lactate deshydrogenase; LPS: Lipopolysaccharides;
NM(s): Nanomaterial(s); OPC: Optic counter; PMA: Phorbol Myristate Acetate;
QCM: Quartz Cristal Microbalances; ROS: Reactive oxygen species;
SD: Standard deviation; SMPS: Scanning Mobility Particle Sizer;
TEM: Transmission Electron Microscopy; VCM: Volumetric Centrifugation
We thank Dominique Lison (Université catholique de Louvain) and Sophie
Lanone (INSERM) for their advice regarding the manuscript conception. We
thank our NANoREG partners BAuA and GAIKER, for providing the A549 and
THP-1 cells respectively. We thank Camille Rey for the critical revision and
the language corrections.
The manuscript was written through contributions of all authors. All authors
gave approval to the final version of the manuscript. TL was involved in the
conception, design, performance of the experiments, analysis and
interpretation of the data and wrote the manuscript. EP helped to set up the
aerosol exposure system and in deposition characterization experiments. MD
performed several in vitro exposures to suspensions. CB and OAC performed
the TEM analysis. OLB and TA helped in the interpretation of the aerosol
characterization data. BT, AB, CE were involved in the writing and the
revisions. GL was involved in the conception, design, writing and the
The authors report no conflict of interest.
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