In vitro biological responses to nanofibrillated cellulose by human dermal, lung and immune cells: surface chemistry aspect
Lopes et al. Particle and Fibre Toxicology
In vitro biological responses to nanofibrillated cellulose by human dermal, lung and immune cells: surface chemistry aspect
Viviana R. Lopes 0
Carla Sanchez-Martinez 1
Maria Strømme 0
Natalia Ferraz 0
0 Nanotechnology and Functional Materials, Department of Engineering Sciences, Uppsala University , Box 53475121 Uppsala , Sweden
1 Present affiliation: Ocular Biology and Therapeutics, UCL Institute of Ophthalmology , 11-43 Bath Street, EC1V 9EL London , UK
Background: Nanocellulose, and particularly nanofibrillated cellulose (NFC), has been proposed for a diversity of applications in industry and in the biomedical field. Its unique physicochemical and structural features distinguish nanocellulose from traditional materials and enable its use as an advance nanomaterial. However, its nanoscale features may induce unknown biological responses. Limited studies with NFC are available and the biological impacts of its use have not been thoroughly explored. This study assesses the in vitro biological responses elicited by wood-derived NFC gels, when human dermal fibroblasts, lung MRC-5 cells and THP-1 macrophage cells are exposed to the nanomaterial. Furthermore, whether the presence of surface charged groups (i.e. carboxymethyl and hydroxypropyltrimethylammonium groups) on NFC can induce distinct biological responses is investigated. Results: The introduction of surface charged groups resulted in individual nanofibrils, while fibril aggregates predominated in the unmodified NFC gel suspensions as observed by transmission electron microscopy. In the presence of proteins, the surface modified NFCs formed compact agglomerates while the agglomeration pattern of the unmodified NFC was similar in the presence of proteins and in physiological buffer. Unmodified and modified NFC gels did not induce cytotoxicity in human dermal fibroblasts, lung and macrophage cells. No significant ROS production by THP-1 macrophages was found and no cellular uptake was observed. However, an inflammatory response was detected when THP-1 macrophages were treated with unmodified NFC as assessed by an increase in TNF-α and IL1-β levels, an effect that was absent when surface charged groups were introduced into NFC. Conclusions: Taken together, the data presented here show the absence of cytotoxic effects associated with the exposure to unmodified, carboxymethylated and hydroxypropyltrimethylammonium-modified NFCs. Unmodified NFC presented a pro-inflammatory effect which can be further moderated by introducing surface modifications such as carboxymethyl and hydroxypropyltrimethylammonium groups into the nanofibrils. The present findings suggest that the inflammatory response to NFC might be driven by the material surface chemistry, and thus open up for the possibility of designing safe nanocellulose materials.
Cytotoxicity; Inflammation; Nanocellulose; Surface chemistry
In recent years, an emerging demand for nano-based
products from sustainable and environmental-friendly
resources is placing nanocellulose on top of exciting
nanomaterials near commercialization [1–4].
Nanocellulose consists of cellulose fibrils or crystallites with at
least one dimension in the nanoscale and presents the
typical physicochemical properties of cellulose such as
hydrophilicity, mechanical strength and broad possibility
of chemical modifications together with specific
nanomaterial properties like high specific surface area and
high aspect ratio [3, 4]. It can be derived from a diversity
of sources, including wood, algae, bacteria and tunicates.
Nanocellulose from wood and other higher plants is
typically isolated as crystals (nanocellulose crystals) or
nanofibrils (nanofibrillated cellulose (NFC)) through
chemical and/or mechanical treatment of cellulose, while
fibres of bacterial nanocellulose are produced by
bacterial synthesis from low molecular weight sugars or other
carbon sources [3–5].
Due to its novel physicochemical and structural
features, nanocellulose has been proposed for a myriad of
applications in industry (paper, packaging, electronics,
cosmetics, etc.) and in the biomedical field, such as
sensors and scaffolds for tissue engineering [6–10].
Although cellulose is considered as non-toxic, the novel
physicochemical properties and nanoscale dimensions of
nanocellulose may imply different biological effects from
conventional cellulose [4, 5].
With the expected increase in the presence of
nanocellulose in consumer products it is critical to assess and
confirm the safety of the nanomaterial [8, 11].
Particularly, from an occupational point of view, potential
hazard effects have been recently identified during the
different life cycle stages of nanocellulose . During
production and manufacturing of nanocellulose, two
main potential hazards were identified, i) accidental
inhalation of nanocellulose released to air after drying it
and packaging it, and ii) dermal contact with slurry of
nanocellulose when it is being combined with other
materials, which can spill to the skin or clothes of the
There is a void of knowledge concerning the effect of
cellulose-based nanomaterials on human health . The
in vitro studies available for nanocellulose, particularly
for NFC, are mostly focus on exposure by inhalation
with data from exposure via the dermal and oral routes
still lacking . The few toxicological studies with NFC
have so far shown no indication of toxicity [12–15].
However, different raw materials, manufacturing process
and post-manufacturing chemical modifications may
alter the material’s physicochemical properties .
Properties like fibril dimensions, degree of crystallinity,
specific surface area, degree of branching of the
nanofibrils and modifications of the material chemical
properties may affect the interactions between the
cellulose nanofibrils and biological systems.
In the work presented here the focus is on NFC
derived from wood and the potential occupational hazards
derived from exposure to it. The aim is to evaluate the
in vitro biological responses elicited by NFC, having in
mind an occupational scenario and focusing on
inhalation and dermal exposure routes. Whether chemical
surface modifications of NFC could cause distinct
biological responses is further investigated. Typically,
surface charged groups are introduced during NFC
production to facilitate the fibrillation process by adding
repulsive charges . In this context, a side-by-side in
vitro toxicity assessment of wood-derived NFC gels with
different surface modifications (carboxymethylated and
towards human dermal fibroblasts (HDF), lung MRC-5
cells and THP-1 macrophage cells is presented here,
together with the evaluation of the cell responses to
Synthesis and surface modification of NFC
NFC was produced from commercial, never dried, bleached
sulfite softwood dissolving pulp (Domsjö Fabriker AB,
Sweden). Unmodified-NFC (U-NFC),
carboxymethylatedNFC and hydroxypropyltrimethylammonium-NFC, here
referred to as anionic NFC (A-NFC) and cationic NFC
(CNFC), in that order, were provided by Innventia AB
(Sweden). U-NFC was prepared by enzymatic pretreatment
of the pulp  while carboxymethylation and
epoxypropyltrimethylammonium chloride (EPTMAC)
quaternization pretreatments were used to prepare A-NFC and
CNFC respectively, as previously described . All samples
were biocide free and showed no bacterial contamination
when tested with 3 M™ Petrifilm™ bacteria tests (total
Transmission electron microscopy
The morphology of the fibres in phosphate buffer and in
cell culture medium was investigated by transmission
electron microscopy (TEM). Samples were prepared as
described by Usov et al. . Briefly, 5 μl of NFC stock
solution (in PBS) were deposited onto carbon-coated
copper grids (400 mesh), while 5 μl of NFC in RPMI cell
culture medium (500 μg/ml, prepared as described
below) were placed on formvar coated 200 mesh copper
grids. After adsorption, the sample grids were stained
with 2% uranyl acetate to achieve a noncrystalline film
of stain embedding the fibres. Following the staining
step, the excess moisture was drained along the
Table 1 Physico-chemical characteristics of the NFC samples under study
20–30 nm fibril aggregates −11.3 ± 1.3 −4.0 ± 0.2 −5.2 ± 0.6
4–5 nm individual fibrils
−34.1 ± 1.7 −12.7 ± 2.7 −12.6 ± 1.6
4–5 nm individual fibrils
−7.2 ± 2.9 −8.1 ± 2.0
dispersed with an ultrasonic probe (Vibracell 600 W,
20 kHz) during 12 min. The final stock solutions were
sterilized by autoclaving, except for C-NFC which was
subject to ultraviolet radiation (UV) treatment during
two cycles of 45 min each. Before each exposure, the
stock solutions were diluted in cell culture media
(concentration range 50 to 500 μg/mL) and sonicated for
30 min in a water bath sonicator (Bransonic 3510)
before being added to the cells.
The human dermal fibroblasts (HDF) from European
Collection of Authenticated Cell Cultures (ECACC), the
human MRC-5 lung fibroblast cell line (private
collection), and the human THP-1 monocytic cell line
(ECACC) were used in this study. MRC-5 and HDF cells
were cultured in DMEM/F12 medium (ThermoFisher
Scientific), and THP-1 cells in RPMI 1640 medium
(ThermoFisher Scientific) supplemented with 10% (v/v)
C-NFC EPTMAC quaternization 1.60 h
ζ – potential
Dispersions of 0.001% (w/w) of the NFC samples were
prepared in 10 mM NaCl and in cell culture media,
DMEM and RPMI1640 (ThermoFisher Scientific),
through ultrasonication for 30 s (Vibracell 600 W,
20 kHz). The electrophoretic mobility of the samples
was measured using a universal dip cell in a ZetaSizer
Nano instrument (Malvern Instruments) at 25 °C and
37 °C (in the case of cell culture media). The ζ-potentials
were determined from the electrophoretic mobility
applying the Smoluchowski equation .
Preparation of NFC exposure suspensions
Stock solutions of U-NFC, A-NFC and C-NFC were
prepared in phosphate buffer (PBS) at 5 mg/mL and
Fig. 1 Morphology of NFC suspended in phosphate buffer. Transmission electron microscopy images of U-NFC (a and d), A-NFC (b and e) and C-NFC
(c and f)
Fig. 2 Morphology of NFC suspended in cell culture medium. Transmission electron microscopy images of U-NFC (a and d), A-NFC (b and e) and
C-NFC (c and f)
heat-inactivated fetal bovine serum (FBS), 100 IU/mL
penicillin and 100 μg/mL streptomycin (both from
ThermoFisher Scientific). The cells were incubated in a
humidified atmosphere at 37 °C, 5% CO2 and sub-cultured
at 70–80% confluency.
For each experiment, HDF and MRC-5 cells were
seeded in 96 well-plates at a density of 6 × 103 cells/
well (200 μl/well) 1 day prior to NFC treatment.
THP-1 monocytes were seeded in 96 well-plates at a
density of 2.5 × 104 cells/well (200 μl/well) and
differentiated into macrophages using 5 ng/mL phorbol
myristate acetate (PMA) (Sigma-Aldrich) for 48 h
before NFC treatment .
Near confluent monolayers of HDF and MRC-5 cells,
and THP-1 differentiated macrophages were finally
exposed to U-NFC, A-NFC and C-NFC at concentrations
ranging from 50 to 500 μg/mL in 96-well plate (200 μl/
well) for 24 h. Food grade microcrystalline cellulose
(MCC, Avicel® CL-611, 100 nm in diameter
microcrystals mixed with soluble sodium carboxymethylcellulose,
FMC Biopolymer) was used as reference material. As a
positive control, 5% (w/w) dimethyl sulfoxide (DMSO)
(Sigma-Aldrich) in cell culture medium was used, while
non-treated cells served as negative control.
Cellular metabolic activity: Alamar Blue assay
The alamar blue (AB) assay was used to assess cellular
metabolic activity as a marker of cytotoxicity. After
exposure to NFC, cells were carefully washed with 100 μL
warm PBS, and 200 μL of Alamar Blue® reagent
(ThermoFisher Scientific) diluted (1:10) was added to each
well and incubated for 90 min at 37 °C. After
incubation, 100 μL of each well were collected and added to
a black 96-well plate. The fluorescence was measured
at 560 nm excitation and 590 nm emission
wavelengths using a plate reader (Tecan Infinite M200).
Results were expressed as percentage of cell viability
with respect to the negative control. The experiments
were performed at least three times in triplicate wells
for each dose. Interference of the NFC samples with
the assay was tested in an acellular system by
incubating different doses of NFC with the AB reagent
for 90 min at 37 °C in 96 well plates. Cell culture
media interference was also measured. Neither NFC
samples nor the cell culture media interfere with the
Cellular membrane integrity: Lactate Dehydrogenase assay
The release of the intracellular enzyme lactate
dehydrogenase (LDH), indicative of cell membrane damage, was
assessed by a LDH kit (Abcam) according to the
manufacturer’s guidelines. Briefly, after cell exposure to the
NFC samples, cell culture supernatants were collected
and adherent cells were lysated for 30 min with cell lysis
buffer diluted in cell culture medium (1:10) and the
lysates were collected. Lysates and supernatants were
centrifuged at 6800 g during 10 min to avoid any potential
interference of NFC. The enzyme activity in the lysates
and supernatants samples was measured by reading the
absorbance at 450 nm wavelength and reference
wavelength 650 nm using a plate reader (Tecan Infinite
M200). The experiments were conducted at least three
times in duplicate wells for each dose. LDH release
(LDH activity in cell culture supernatant) was
Fig. 3 Metabolic activity of cells after NFC exposure. Cell viability of
NFC-treated cells was assesed by evaluating cell metabolic activity
using the alamar blue assay. a HDF cells, b MRC-5 cells and c THP-1
macrophages exposed to increasing doses of NFC (50–500 μg/mL) for
24 h. MCC is a food grade nanocellulose used as a reference material.
The positive control was DMSO (5% v/v in cell culture media) an
inducer of cytotoxic effects. Data are expressed as percentage relative
to the negative control (untreated cells) and presented as mean ± SEM
of three independent experiments. Significant results as compared to
the negative control are marked with asterisks (** p < 0.01 and
**** p < 0.0001)
normalized by the total LDH activity (sum of LDH
activity in cell culture supernatants and lysates) which
correlates with the total number of cells in order to avoid any
underestimation of toxicity .
Cell morphology - Light microscopy
After 24 h exposure to NFC, cells were carefully rinsed
with warm PBS and observed under light microscopy
(Nikon Eclipse TE2000-U) to evaluate their morphology.
The inflammatory response was investigated by
quantifying the secreted levels of the cytokines tumor necrosis
factor α (TNF-α) and interleukin 1 beta (IL1-β). THP-1
monocytes were differentiated into macrophages and
treated with the NFC suspensions as described above.
After 24 h exposure, cell culture supernatants were
collected, centrifuged at 6800 g during 10 min and further
analyzed for the levels of cytokines using ELISA Kits
(human TNF-α and human IL1-β ELISA Kits, Thermo
Fischer Scientific) according to the manufacturer’s
protocol. As a positive control for TNF-α and IL1-β
induction, cells were treated with lipopolysaccharide (LPS)
from Pseudomonas aeruginosa at 1 ng/mL. The same
experiments were performed in the presence of polymyxin
B (PMB) at a final concentration of 25 μg/mL in order
to inhibit the potential effects of any endotoxin present
in the NFC samples . The experiments were
conducted at least three times in duplicate wells for each
dose. TNF-α and IL1-β concentrations were calculated
from a standard curve plotted for each experiment.
Reactive oxygen species production
The levels of intracellular reactive oxygen species
(ROS) were measured using the
dichlorodihydrofluorescein diacetate (DCFH-DA) assay (Abcam)
according to the manufacturer’s guidelines. DCFH-DA
is a lipophilic cell permeable compound that is
deacetylated in the cytoplasm by cellular esterases, and
later oxidized by ROS to a highly fluorescent
molecule. THP-1 monocytes were differentiated into
macrophages and loaded with 20 μM DCFH-DA in
PBS for 30 min at 37 °C. Thereafter, cells were
treated with the NFC suspensions (50, 100, 250
500 μg/mL) and fluorescence was recorded every
30 min over 120 min (excitation 485 nm, emission
535 nm) at 37 °C using a plate reader (Tecan
Infinite M200). Tert-butyl hydroperoxide (TBHP, 50 μM)
was used as positive control.
Cellular uptake of NFC - Transmission electron
TEM was used to investigate if the NFC materials were
uptaken by THP-1 macrophages. THP-1 macrophages
were exposed to the different NFC samples (500 μg/ml)
for 24 h and then fixed in 2.5% (v/v) glutaraldehyde
overnight at 4 °C. Samples were washed with sodium
cacodylate buffer and subsequently post-fixed with 1%
osmium tetroxide in sodium cacodylate buffer.
Afterwards, the cells were dehydrated in ascending ethanol
series, embedded in epon and finally polymerized at 60 °
C for 48 h. From the embedded cells, ultrathin sections
(50–60 nm) were cut parallel to the vertical axis of the
inserts, mounted on copper grids and stained with
uranyl acetate and lead citrate. Imaging was done with a
Technai G2 microscope (FEI, Netherlands) LaB6
filament at 80 kV.
Fig. 4 Lactate dehydrogenase (LDH) activity of cells after NFC exposure. Cytotoxicity of NFC-treated cells was evaluated by measuring total and
extracellular LDH activity. a HDF cells, b MRC-5 cells and c THP-1 macrophages were treated with a range of NFC concentration from 50 to
500 μg/mL during 24 h. MCC is a food grade nanocellulose used as a reference material. The positive control was DMSO (5% v/v in cell culture
media) an inducer of cytotoxicity. Data are presented as mean ± SEM of three independent experiments. Significant results as compared to the
negative control are marked with asterisks (* p < 0.05 and ** p < 0.01)
Data analysis was conducted using GraphPad Prism 6,
version 6.07 (GraphPad Software Inc., La Jolla, USA)
by one-way or two-way analysis of variance (ANOVA)
followed by Dunnett’s multiple comparison post-hoc
tests. p-values lower than 0.05 were considered
statistically significant. Results are presented as the mean
± standard error of the mean (SEM).
The successful introduction of carboxymethyl and
hydroxypropyltrimethylammonium groups on A-NFC
and C-NFC, respectively, was previously verified by
Hua et al. . Results showed a higher surface
group density for C-NFC than for A-NFC. No
specific surface groups were introduced during the
production of U-NFC (mild enzymatic pretreatment of
the wood pulp) and thus only low levels of carboxyl
group content could be expected due to the presence
of residual hemicellulose. Furthermore, the degree of
crystallinity, as previously determined , was
similar between both unmodified and modified NFCs
indicating that the chemical modifications did not
considerably alter the crystallinity of NFC.
Fig. 5 Cell Morphology after NFC exposure. Morphology of HDF, MRC-5 and THP-1 macrophages after direct contact with NFC. Top images show HDF,
MRC-5 and THP-1 cells untreated (negative control). For all other conditions, HDF, MRC-5 and THP-1 cells were treated with the highest concentration of
U-NFC, A-NFC and C-NFC (500 μg/mL) for 24 h. Black arrows indicate agglomerates of fibres. Images of cells treated with the positive control (DMSO 5%)
are given in Additional file 1: Figure S1. Scale bars represent 100 μm
Zeta potential was measured for all NFCs. At pH 7.4,
A-NFC and C-NFC suspended in 10 mM NaCl
presented zeta potential values of − 34.1 and 24.2 mV,
respectively, confirming the presence of the negatively
and positively charged groups in the modified NFCs.
The high absolute values of zeta potential for the
charged samples implies a good stability of the
suspensions . However, U-NFC formed an unstable
dispersion, showing tendency to aggregate, with a
slightly negative surface charge (−11.3 mV). When
incubated in cell culture media and at 37 °C, all NFCs
samples showed negative zeta potential values in the
range of − 4 and − 12 mV, independently of the cell
culture medium used.
The morphology of the nanocellulose fibres suspended
in PBS was observed by TEM (Fig. 1). Small
agglomerates were observed in U-NFC, while A-NFC and C-NFC
suspensions showed dispersed fibres (Fig. 1, upper
panels). High magnification images (Fig. 1, lower panels)
depict the dimensions of the individual fibrils or fibril
aggregates. U-NFC showed bundles of several μm long
fibres, forming 20–30 nm in diameter fibre aggregates
(Fig. 1d). The presence of surface charges on A-NFC
and C-NFC resulted in better dispersion of the
individual fibres, as shown in Fig. 1e and f, where
individual nanofibrils (4–5 nm in diameter) with slight
aggregation can be observed.
When the NFC suspensions in cell culture medium
were observed under TEM, compact agglomerates 10–
50 μm in diameter were observed in the A-NFC and
CNFC samples, while smaller agglomerates (1–2 μm in
diameter) were found in the U-NFC suspension (Fig. 2,
upper panels). When taking a closer look at the
agglomerates (Fig. 2, lower panels), it was observed that A-NFC
and C-NFC still presented individual nanofibrils and the
U-NFC sample had fibre aggregates similar to the ones
observed when suspended in PBS.
NFCs are not cytotoxic for immune, dermal and lung cells
The cytotoxic effect of the different NFCs was evaluated
by using two different assays, AB and LDH assays, i.e. by
investigating the effect on cell metabolic activity and on
cell membrane integrity, respectively (Figs. 3 and 4).
The AB assay showed that after 24 h exposure, the
metabolic activity of the treated cells was comparable to
the activity of the non-treated cells (negative control) for
the HDF and MRC-5 cells (Fig. 3a and b). Interestingly,
THP-1 cells showed a significantly higher metabolic
Fig. 6 Cytokine production after NFC exposure. a TNF-α and b IL1-β concentration in the supernatants of THP-1 macrophages exposed to
increasing doses of NFC (50–500 μg/mL) for 24 h. For the U-NFC sample cytokine secretion was assessed in the presence of PMB in order to
supress the contribution of endotoxin contamination to the secreted cytokine levels. MCC is a commercial grade nanocellulose used as a
reference material. Negative control represents untreated cells. Cells treated with LPS (1 ng/mL), an inducer of cytokine production, represent the
positive control of the assay. The data are presented as mean ± SEM of three independent experiments. Significant results as compared to the
negative control are marked with asterisks (*p < 0.05 and **** p < 0.0001)
activity of the NFC-treated cells compared with the
negative control (Fig. 3c), an effect that has been also
observed by other authors when exposing macrophages
to NFC .
No signs of toxicity were observed when the LDH
assay was used to evaluate cell membrane damage
following 24 h treatment with NFCs (Fig. 4). HDF
cells exposed to NFCs with or without surface
modifications did not significantly change the LDH release
when compared to the negative control (Fig. 4a).
Moreover, no significant differences in total cell
number (total LDH) between treated and non-treated cells
were found. The MRC-5 cells and THP-1
macrophages showed a similar pattern of LDH release and
total LDH to the results found for HDF cells (Fig. 4b
and c, respectively). The reference material MCC was
also not cytotoxic for the studied cells under the
conditions of the present work.
Accordingly, we did not find altered morphology
for any of the three cell types treated with NFC gels
after 24 h exposure when compared to the negative
control (Fig. 5). In the positive control, 5% DMSO,
cells presented unhealthy and round morphology,
and loss of attachment, as expected (Additional file
1: Figure S1).
U-NFC induces the release of pro-inflammatory cytokines
Whether the NFC samples trigger an inflammatory
response in THP-1 macrophages was evaluated by
measuring the levels of two pro-inflammatory cytokines,
TNF-α and IL1-β in cell culture supernatants
following 24 h exposure. First, the possible input of NFC
endotoxin contamination to cytokine production was
investigated by measuring the cytokine levels in the
presence of PMB. Results showed that the levels of
cytokines secreted by cells treated with U-NFC were
significantly higher in the absence of PMB than when
the experiments were performed in the presence of
the LPS inhibitor (Additional file 2: Figure S2A and
S2B). Thus, indicating a contribution of endotoxin
contamination to the inflammatory response trigger
by U-NFC. Therefore, the inflammatory potential of
such sample was further investigated by conducting
the experiments in the presence of PMB in a
concentration that was shown to inhibit cytokine secretion
in LPS-stimulated THP-1 macrophages (Additional
file 2: Figure S2C). In this way we assure that the
secreted levels of TNF-α and IL-1β are solely a
consequence of the material-cell interactions and not due
to endotoxin contamination. A significant release of
TNF-α by cells treated with high dose of U-NFC
Fig. 7 Cellular reactive oxygen species (ROS) production after NFC exposure. Kinetic study of ROS production of THP-1 macrophages treated with
increasing doses (50–500 μg/mL) of (a) U-NFC, (b) A- NFC and (c) C-NFC. ROS assessed with the ROS-specific fluorescent probe DCFDA-DA every
30 min during 120 min. Negative control represents untreated cells. Tert-butyl hydroperoxide (TBHP), an inducer of oxidative stress, represents the
positive control. Data are expressed as relative fluorescence units (RFU) and presented as the mean ± SEM of three independent experiments
(500 μg/mL) compared with the negative control was
observed after 24 h exposure. THP-1 macrophages
treated with A-NFC or C-NFC did, however, not
secrete significant levels of TNF-α compared to the
negative control (Fig. 6a).
U-NFC triggered a significant release of IL1-β by
THP-1 macrophages at the two high doses, 250 and
500 μg/mL (Fig. 6b). Both modified NFCs, A- and
CNFC, did not induce IL1-β secretion. MCC did not
No intracellular ROS increase upon treatment with NFCs
For assessing the oxidative potential of NFCs, a kinetic
study of intracellular ROS production was performed
using the fluorescent marker DCFH-DA. No significant
ROS increase was observed during the first 120 min for
THP-1 macrophages treated with NFCs compared with
the negative control (Fig. 7). The positive control, TBHP,
induced a significant increase compared with the
negative control. The reference material MCC displayed a
similar cellular ROS profile as the investigated NFCs
(see Additional file 3: Figure S3).
THP-1 macrophages do not uptake the NFC materials
The TEM analysis of the cells after exposure to the
different NFC materials confirmed that there were no
alterations in cell morphology and showed that none of the
NFC samples were uptaken by the THP-1 macrophages.
No signs of phagocytosis attempts were found.
Representative TEM images of THP-1 macrophages after 24 h
exposure to the different NFC materials at a
concentration of 500 μg/ml, together with images of the
nontreated cells can be found in the supplementary
information (Additional file 4: Figure S4).
In this study, the toxicity impacts of unmodified and
surface modified NFC gels (i.e. carboxymethylated and
hydroxypropyltrimethylammonium-substituted NFC) on
dermal, lung and macrophage cells were compared for
the first time. The selected human cell types represent
cells most likely to be impacted by the potential
exposure to NFC in an occupational setting, including
immune surveillance and epithelial cells covering the
respiratory tract and the skin.
Surface charges are introduced during the preparation
of nanocellulose where adding repulsive charges
facilitates the defibrillation process. As expected, the
presence of surface charged groups in A-NFC and C-NFC
and the subsequent electrostatic repulsion between
fibres resulted in individual nanocellulose fibrils, while
fibres tended to aggregate in the low carboxyl group
content U-NFC. The zeta potential further confirmed
the different surface charges of the studied samples,
however in the presence of cell culture medium all
samples showed slightly negative charge (zeta potential
values between − 4 and − 12 mV). As described for other
nanomaterials, the nanofibres may be rapidly covered by
the biomolecules present in the cell culture medium,
forming a biomolecular corona that partially or fully
covers the nanofibers and mask the surface charge and,
consequently, changes the zeta potential [26, 27].
The effect of protein adsorption was also reflected in
the agglomeration pattern of A-NFC and C-NFC, which
drastically changed from dispersed fibres in phosphate
buffer to compact agglomerates in cell culture medium.
As also observed by Tomic et al. , the agglomeration
of U-NFC did not significantly change when comparing
cell culture medium and phosphate buffer suspensions,
showing the presence of small agglomerates in both
conditions. The type, amount and conformation of the
adsorbed proteins will be influenced by the nanofibre
surface chemistry among other nanomaterials properties.
In turn, the different protein adsorption patterns may
promote distinct agglomeration states of the
nanomaterial , as reflected here.
The assessment of potential toxic effects showed that
the NFC materials under study did not have any impact
on the metabolic activity or on the membrane integrity of
the treated cells following 24 h exposure. Overall, the
NFCs under study did not impair the cell viability of
dermal, lung or macrophage cells. This is in accordance with
previous in vitro studies showing that NFC gels are
noncytotoxic for a wide range of cells, including dendritic
cells, macrophages, fibroblasts, keratinocytes, human
cervic carcinoma and hepatoma cell lines [13–15, 28].
Moreover, NFC gels, aerogels and membranes were proven to
be biocompatible when evaluated for diverse tissue
engineering and biomedical applications [12, 18, 30–33].
Furthermore, no significant ROS production by THP-1
macrophages was found under the conditions tested in
the present work. However, it was shown that U-NFC
promoted an inflammatory response in terms of
secretion of TNF-α and IL-1β, a response that was suppressed
when surface charges were introduced on the
It is well-known that the inhalation of toxic airborne
particulates, particularly carbon nanotubes and other
fibre-like nanomaterials can cause pulmonary
inflammation . Macrophages play a critical role in the
recognition, and clearance of pathogens and foreigner
particulates. The acute phase responses to inhaled
particulates are described by a pulmonary inflammation set
by the release of a number of inflammatory mediators,
such as TNF-α and IL1-β. These two cytokines, TNF-α
and IL1-β, acting synergistically, are involved in the
pathogenesis of various acute and chronic respiratory
The secretion of pro-inflammatory cytokines by
macrophages under the influence of NFC has been
previously investigated in vitro and in vivo. While some
authors showed that NFC gels were non
proinflammatory in vitro [13, 15, 24, 31], Catalán and
co-workers found a pro-inflammatory response to
NFC in vivo . However, the authors declared that
the possible role of microbial contamination on the
inflammatory effect could not be ruled out.
Interestingly, when we have previously studied the
proinflammatory response of THP-1 monocytes cultured
on NFC films we found a pro-inflammatory effect
with U-NFC and A-NFC, while C-NFC behaved as
an inert material . Thus, a pro-inflammatory
effect was found when carboxymethyl groups (A-NFC)
were introduced on the nanofibrils, an effect that
was absent in the present work. Such difference in
the inflammatory response of A-NFC could be
partially due to the variations in the structures of the
nanocelluloses under study, i.e. gels versus
membranes, and in how the cells were exposed to the
Hypothetical cellular mechanisms of inflammation
caused by fibres include i) frustrated phagocytosis and
the subsequent production of ROS and oxidative stress
or ii) a direct effect of the fibres on the membrane
receptors . ROS production was not found when
macrophages were incubated with the NFC gels studied here
and TEM analysis of the exposed cells revealed no sings
of phagocytosis attempts, with untreated cells and
NFCexposed cells showing comparable morphologies.
Besides, it was not possible to detect the presence of any of
the NFC materials inside the cells. Therefore, we
hypothesize that the observed secretion of
proinflammatory cytokines is likely a consequence of
fibrereceptor interactions, where surface chemistry plays a
key role. Surface chemistry influences the type,
conformation and amount of adsorbed proteins which in turn
mediate the cell-nanomaterial interactions . It can be
speculated that the protein corona of U-NFC might
promote the interaction with THP-1 membrane receptors
resulting in the secretion of pro-inflammatory cytokines.
However, the presence of carboxymethyl and
hydroxypropyltrimethylammonium groups on A-NFC and
CNFC, respectively, most probably result in protein
coronas that do not promote the signalling for an
inflammatory effect. Besides, surface chemistry and the
subsequent protein adsorption might also indirectly
affect the cellular response to the nanomaterial by
impacting on its agglomeration pattern . The
difference in surface chemistry, particularly in surface charges,
was indeed reflected in the agglomeration patterns of
the NFC materials and therefore an effect of nanofibre
dispersion on the observed inflammatory response
cannot be dismissed. Authors have described the influence
of fibre agglomerates in in vivo cellular responses of
nanofibres such as carbon nanotubes and carbon fibres
[38, 39]. Mutlu et al. showed that the aggregation of
single-walled carbon nanotubes accounted for its
pulmonary toxicity, an effect that could not be seen when
the carbon nanotubes were nanoscale dispersed .
Nevertheless, in the present study, U-NFC with smaller
agglomerates compared with A-NFC and C-NFC was
the material that promoted a pro-inflammatory response
and macrophages did not react differently to the diverse
agglomerate sizes in terms of frustrated phagocytosis or
The cellular uptake of NFC has recently been
investigated by other authors [28, 36]. Catalán et al.
administrated TEMPO-oxidized NFC (negatively charged NFC)
to mice by pharyngeal aspiration and reported
doserelated accumulation of the material in the cytoplasm of
macrophages . Conversely to our work, in vitro
studies with dendritic cells showed partial internalization of
NFC . The authors stated that the interaction of
NFC and the dendritic cells depended on the thickness
and length of the material and highlight the need of
further studies to investigate the mechanism that
predominate in the NFC-dendritic cells interactions .
In summary, the findings presented here suggest that
the inflammatory response to NFC gels might be driven
by the surface chemistry. Furthermore, the fact that
UNFC induces an inflammatory response under no signs
of toxicity may pose a concern and indicates that, as also
observed by others, cytotoxicity and activity (e.g.
inflammatory response) do not necessary correlate .
Understanding the effect of NFC features, in this case, the
surface chemistry on their biological reactivity will
contribute towards safer industrial applications of such
nanomaterial. More studies are needed, especially for
prolonged exposure times and in vivo tests, to further
investigate the effect of NFC on the immune response
and enhance our present knowledge about the safety of
No indication of cytotoxicity or significant ROS
production were found when cells were exposed to the
unmodified and surface modified NFC gels. Besides, no cellular
uptake was observed. A pro-inflammatory response with
U-NFC in terms of cytokine secretion was found and
this effect was suppressed when surface charged groups
were present on the nanofibrils. This finding suggests
that the inflammatory response to NFC gels might be
driven by surface chemistry opening up the possibility
for the design of safe nanocellulose materials.
Additional file 1: Figure S1. Light microscopy images of untreated
(top panels) and DMSO-treatedd (bottom panels) cells. Top images show
HDF, MRC-5 and THP-1 macrophage cells untreated (negative control)
and bottom images show cells treated with DMSO 5% in cell culture
media (positive control). Scale bars represent 100 μm. (TIF 4130 kb)
Additional file 2: Figure S2. Cytokines production after NFC exposure in
the presence and absence of PMB. (A) TNF-α and (B) IL-1 β concentration in
the culture supernatants of THP-1 macrophages exposed for 24 h with
increasing doses of NFCs (50–500 μg/mL) with and without PMB-treatment.
(C) Effect of PMB (25 μg/mL) on TNF-α and IL1-β production by THP-1
macrophages stimulated with LPS (1 ng/mL). Note that when PMB was added
to the LPS treated cells, the cytokine secretion was reduced to a level
comparable to that found for the negative control. MCC is a food grade
nanocellulose used a as reference material. Negative control represents untreated
cells. Data are presented as mean ± SEM of three independent experiments.
Statistically significant differeces in cytokine secretion between PMB treated
and untreated cells are marked with asterisks (** p < 0.01 and **** p <
0.0001). (TIF 389 kb)
Additional file 3: Figure S3. Cellular ROS production after the addition
of MCC to THP-1 macrophages. Kinetic study of ROS production of cells
treated with increasing doses (50–500 μg/mL) of MCC, a food grade
nanocellulose. ROS assessed with the ROS-specific fluorescent probe DCFDA-DA
every 30 min during 120 min. Negative control represents untreated cells.
Tert-butyl hydroperoxide (TBHP), an inducer of oxidative stress, represents
the positive control. Data are expressed as relative fluorescence units (RFU)
and presented as mean ± SEM of three independent experiments.
Significant results as compared to the negative control are marked with
asterisks (*** p < 0.001 and **** p < 0.0001). (TIF 65 kb)
Additional file 4: Figure S4. Transmission electron microscopy analysis
of THP-1 macrophages. Representative images of (a and b) untreated
cells, (c and d) cells exposed to U-NFC, (e and d) cells exposed to A-NFC,
(g and h) cells exposed to C-NFC. Cells were treated with 500 μg/ml of
NFC for 24 h. The arrow indicate the presence of NFC agglomerates in
the vicinity of the cells. (TIF 2251 kb)
AB: Alamar blue; A-NFC: Anionic nanofibrillated cellulose; ANOVA: Analysis of
variance; C-NFC: Cationic nanofibrillated cellulose; DCF: Dichlorodihydrofluorescein;
DCFH-DA: Dichlorodihydrofluorescein diacetate; DMEM/F12: Dulbecco’s Modified
Eagle Medium: Nutrient Mixture F-12; DMSO: Dimethyl sulfoxide;
ECACC: European Collection of Authenticated Cell Cultures; IL1-β: Interleukin 1
beta; LDH: Lactate Dehydrogenase; LPS: Lipopolysaccharide; NFC: Nanofibrillated
cellulose; PBS: Phosphate buffer; PMA: Phorbol myristate acetate; PMB: Polymyxin
B; ROS: Reactive oxygen species; RPMI 1640: Roswell Park Memorial Institute (RPMI)
1640 Medium; SEM: Standard error of the mean; TBHP: Tert-butyl hydroperoxide;
TEM: Transmission electron microscopy; TNF-α: Tumor necrosis factor α;
UNFC: Unmodified cellulose nanofibrillated
The authors would like to thank Ida-Maria Sintorn and Lars Haag (Vironova),
Vironova AB (Stockholm) and SciLifeLab BioVis Facility, Uppsala University for the TEM
images acquisition. Monica Johnson is acknowledged for assistance in the
experimental work with MRC-5 cells. Eva Ålander and Tom Lindström at Innventia
AB are acknowledged for supplying the nanocellulose materials and for valuable
discussions. Alex Basu at the Division of Nanotechnology and Functional Materials,
Uppsala University is acknowledged for imaging processing.
VRL participated in the design of the study, carried out all the cellular
experiments (except AB assay for HDF cells), performed the statistical
analysis, data interpretation and wrote the manuscript. CS performed part of
the in vitro experiments with HDF cells and the initial experiments with
THP1 cells. MS participated in the planning of the study and the evaluation of
the results. NF participated in the design of the study, carried out the cellular
uptake studies, did the data interpretation and wrote the manuscript
together with VRL. All authors read and approved the final manuscript.
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