Coating carbon nanotubes with a polystyrene-based polymer protects against pulmonary toxicity
- Lyes Tabet1, 7,
- Cyrill Bussy1,
- Ari Setyan2, 6,
- Angélique Simon-Deckers1,
- Michel J Rossi3,
- Jorge Boczkowski†1, 4, 5 and
- Sophie Lanone†1, 5Email author
© Tabet et al; licensee BioMed Central Ltd. 2011
Received: 14 September 2010
Accepted: 21 January 2011
Published: 21 January 2011
carbon nanotubes (CNT) can have adverse effects on health. Therefore, minimizing the risk associated with CNT exposure is of crucial importance. The aim of this work was to evaluate if coating multi-walled CNT (MWCNT) with polymers could modify their toxicity, thus representing a useful strategy to decrease adverse health effects of CNT. We used industrially-produced MWCNT uncoated (NT1) or coated (50/50 wt%) with acid-based (NT2) or polystyrene-based (NT3) polymer, and exposed murine macrophages (RAW 264.7 cell line) or Balb/c mice by intratracheal administration. Biological experiments were performed both in vitro and in vivo, examining time- and dose-dependent effects of CNT, in terms of cytotoxicity, expression of genes and proteins related to oxidative stress, inflammation and tissue remodeling, cell and lung tissue morphology (optical and transmission electron microscopy), and bronchoalveolar lavage fluid content analysis.
extensive physico-chemical characterization of MWCNT was performed, and showed, although similar dimensions for the 3 MWCNT, a much smaller specific surface area for NT2 and NT3 as compared to NT1 (54.1, 34 and 227.54 m2/g respectively), along with different surface characteristics. MWCNT-induced cytotoxicity, oxidative stress, and inflammation were increased by acid-based and decreased by polystyrene-based polymer coating both in vitro in murine macrophages and in vivo in lung of mice monitored for 6 months.
these results demonstrate that coating CNT with polymers, without affecting their intrinsic structure, may constitute a useful strategy for decreasing CNT toxicity, and may hold promise for improving occupational safety and that of general the user.
Carbon nanotubes (CNT) exhibit unique properties, including mechanical, thermal and electrical conductivity, as well as field emission properties. These properties are associated with many applications (car industry, sport accessories, ...), and lead to a steady increase in the industrial production of CNT. However, it is increasingly obvious that exposure to nanoparticles in general, and CNT in particular, can have adverse effects on human health, especially at the level of the pulmonary system, which is a primary route of exposure . This is raising considerable concern [2–9]. Therefore, minimizing the risk associated with CNT exposure is of crucial importance.
Among adverse health effects secondary to exposure to CNT, inflammation and oxidative stress are particularly worrisome because they can be associated with tissue remodeling and impaired function and/or carcinogenesis . Recent data show that the length and rigidity of the nanotubes influences the pro-inflammatory effect of CNT . However, the possible influence of other physicochemical properties remains incompletely understood . Since surface characteristics influence the pro-inflammatory effect of spherical nanoparticles , we hypothesized that embedding CNT in polymers, thus modifying the surface environment of those CNT, could modify their toxicity, and thus represent a useful strategy to decrease adverse health effects of industrially-produced CNT, without affecting their specific properties and further applications. The protective effect of the compatible solute Ectoine against carbon nanoparticle-induced lung inflammation has been nicely demonstrated recently . However, a strategy based on the production of intrinsically safer nanomaterials seems to be much more promising. Such an approach is essential to the further development and safe use of CNT, at an occupational level as well as at the level of the general user.
To assess our hypothesis, we utilized an original approach by coating industrially produced multi-walled CNT (MWCNT) with two different polymers (acid-based and polystyrene-based, respectively) that resulted in different surface environment but similar specific surface areas. To the best of our knowledge, although surface modifications is important to CNT toxicity [15–19], polymer coating has never been used before as an approach to modulate CNT toxicity. Coating could represent a useful tool, as it allows modifying CNT's external surface environment, without affecting their intrinsic structure. We investigated whether coating modified the toxicity of MWCNT in vitro in murine macrophages and in vivo in mice monitored for 6 months after intratracheal MWCNT instillation. Here, we report that MWCNT-induced cytotoxicity, oxidative stress, and inflammation in both models were increased by acid-based polymer coating and decreased by polystyrene polymer coating. These results demonstrate that surface characteristics play a major role in the biological response to CNT and that modifications of the surface environment by coating with specific compounds may constitute a useful strategy for decreasing CNT toxicity, without affecting their intrinsic structure, already at the time of their production. This may therefore hold promise for improving occupational safety as well as that of general users.
We investigated the effects of industrially-produced MWCNT that were either uncoated (NT1) or coated with carboxylic polyacid polymer (NT2) or a hydrophobic polystyrene polybutadiene polymethylmethacrylate polymer (NT3). These MWCNT were provided by ARKEMA-France (Colombes, France). The two coated MWCNT were composed of 50% MWCNT and 50% coating polymer by weight (determined by Thermo Gravimetric Analysis). Coatings were performed as follows; both coated MWCNT were produced by impregnation of NT1 powder (50% weight) with a solution of polymer (carboxylic polyacid polymer for NT2, and polystyren polybutadiene polymethylmethacrylate for NT3), the solvent being thereafter eliminated by drying under vacuum conditions. Solvent was water for NT2, and methyl ethyl cetone for NT3. NT2 remained coated after dispersion in the culture medium, as revealed by the Toluidine Blue (TB) assay, which measures the stability of polymer grafting  (data not shown). Briefly, a 5.10-4 M TB aqueous solution was prepared and 0.2 ml of a buffer solution of 2-amino 2-methyl propanol was added to reach and maintain the solution pH value at 10. Each MWCNT sample was placed in TB solution at 30°C up to 24 hours. At different time points, uncomplexed TB molecules were removed by rapid washing (30 minutes) of the sample by a basic aqueous solution (NaOH 5.10-4 M, pH 9). Then, each sample was in 10 ml of an aqueous acetic acid solution (50% v/v) for 24 hours to obtain complete decomplexation of TB from the sample. The decomplexation solution was analyzed by UV/visible spectroscopy at 633 nm. There was no chemical link between the polymers and the nanotubes.
Both coated NT (NT2 and NT3) appear as powders, as the pristine one (NT1), and were dispersed in culture medium for both in vitro and in vivo experiments. The two polymers were also dispersed in culture medium in order to assess their intrinsic effects.
The effects of the three MWCNT were compared to those of crocidolite asbestos fibres (80 nm in diameter) kindly donated by Dr. Ghislaine Lacroix (INERIS, Verneuil-en-Hallatte-France) and of nanosized carbon black (CB, FR103, 95 nm in diameter; Degussa, Dusseldorf, Germany).
We chose to study industrially-produced MWCNT to obtain results relevant to occupational health, in the context of increasing industrial development and use of CNT.
Physicochemical characterization of multi-walled carbon nanotubes
MWCNT dimensions were measured using transmission electron microscopy (TEM), and MWCNT morphology was observed using scanning electronic microscopy (SEM). Chemical composition and carbon content were determined using inductively coupled plasma mass spectroscopy (ICP-MS), electron spectroscopy for chemical analysis (ESCA). Specific surface area was measured using Brunauer Emmett Teller (BET) adsorption isotherms of nitrogen at 77 K. Surface functional groups were identified by surface titration using six probe gases flowing across a Knudsen flow reactor [21–23]. The relative uncertainty for both values (substrate/probe gas pair) is typically 25%.
All particles were suspended at 10 mg/ml in Dulbecco's modified Eagle medium (DMEM). We choose not to disperse CNT using any particular agent because our aim was to examine the biological effects of surface properties of coated and uncoated CNT without any other interference. The suspensions were then vortexed for 1 min, and sonicated (Elma S30H, 50-60 Hz) for 30 min under cooling conditions, with a 30-s interruption every 10 min with the vortex at maximum speed. Immediately after the end of sonication, the particle solutions were vortexed again for 1 min at maximum speed and diluted to various concentrations in culture medium (see below). CNT dispersion was quantified by dynamic light scattering using a Zetasizer Nano S (Malvern Instruments Ltd, Worcestershire, UK). Under control conditions, cells were cultured in serum-free DMEM without the particles (see below).
In Vitro Studies
a. RAW 264.7 macrophage culture and exposure
RAW 264.7 murine macrophages were purchased from the American Type Culture Collection (Manassas, VA), and were cultured as previously described . Cells were exposed for 6 or 24 hours to 0.1 to 100 μg/ml (0.02-20 μg/cm2) of MWCNT or to 100 μg/ml (20 μg/cm2) of crocidolite fibres or CB nanoparticles in serum-free medium, as it is known that serum can interact with nanomaterials and modify cellular response .
b. Light microscopy
Cell morphology was assessed by light microscopy after cell staining with Harris haematoxylin-phloxin. The size and number of the agglomerates present on the cells were determined as previously described . Light microscopy studies were done in blinded fashion by two independent observers (LT and CB). The coefficient of variation for each measurement was <5%.
c. Cell viability
Cell viability was assessed using three methods, namely the MTT and Neutral Red assays and quantification of DNA content. These tests were performed as previously described . Results were expressed as the mean of at least three independent experiments, each having six replicates, given as the ratio of the mean under each condition over the mean under the control condition (cells exposed to DMEM). Since nanomaterials may interfere with cytotoxicity tests , we performed the assays with and without 100 μg/ml of NT1, NT2, or NT3 during incubation with the dye, and we measured absorbance. No interference of NT1, NT2, or NT3 with any of the assays was observed (data not shown).
d. Transmission Electron Microscopy
Cells exposed for 24 h to 10 μg/ml of NT1, NT2, or NT3 were examined by TEM as previously described . The percentage of cells with MWCNT-containing vacuoles after 24 hours' exposure to 100 μg/ml was determined on semi-fine slides. Under each stimulation condition, five fields were randomly selected from top to bottom across the vertical diameter of the sample. The measurements were made in blinded fashion by two independent observers (LT and CB). The coefficient of variation for the measurement was <5%.
e. Reverse Transcription and Quantitative PCR (Q-PCR)
Sequences of primers used for quantitative PCR assays
In Vivo Studies
a. Animal exposure
The experiments were approved by the local Institutional Animal Care and Use Committee, and the experimental protocol complied with French legislation about animal studies. Male Balb/C mice aged 7-9 weeks and weighing 22 ± 0.23 g, were purchased from Janvier (Le Genest-St-Isle, France) and housed in standard wire-topped cages in temperature-controlled units with food and water ad libitum.
NT1, NT2, or NT3 were suspended in DMEM as for the in vitro studies. The suspension was instilled intratracheally after anaesthesia of the animal with 1.6 mg ketamine (Merial, Lyon, France) plus 300 mg xylazine (Bayer, Puteaux, France). A single dose (10 or 100 μg/mouse) of NT1, NT2, or NT3 was given to each animal. The highest dose was relevant to the 20 μg/cm2 concentration used in the in vitro studies . The mice were sacrificed 1, 7, 30, 90, or 180 days post-instillation. In a subset of experiments, animals were exposed to 200 μg of NT2 or NT3, in order to allow adequate comparison with 100 μg NT1.
b. Bronchoalveolar Lavage (BAL) and Lung Recovery
The mice were anesthetized with an intramuscular injection of ketamine/xylazine and sacrificed by exsanguination. BAL fluid analysis and lung tissue recovery were performed as described previously . The percentage of macrophages with MWCNT-containing vacuoles was determined as for the in vitro studies. The mRNA expression of the genes evaluated in vitro was measured using Q-PCR on homogenates, with the same set of primers (Table 1). In addition, we measured the expression of collagen-1 and -3, used as markers for interstitial fibrosis.
c. Histological study of lung samples
Lung histology was examined in a subset of animals different from that used for investigating BAL fluid and lung gene expression determination. The lungs were fixed with 0.8 ml of a 1:1 mix of 0.9% saline with Tissue-Teck ornithine carbamoyltransferase fluid (Sakura, Zoetervoude, The Netherlands) and were snapfrozen in liquid nitrogen. Lung histology studies were performed as described previously .
The number and size of MWCNT agglomerates in the lungs of animals exposed for 24 hours, 1 month, or 6 months was measured in five representative animals per group. For each animal, five fields were randomly selected from top to bottom across the vertical diameter of the section. Calculations were performed using ImageJ software (http://rsbweb.nih.gov/ij/), and the analysis was performed in blinded fashion by two independent observers (LT and CB). The coefficient of variation for each measurement was <5%.
The values from at least four different experiments (in vitro study) and 6-8 animals (in vivo study) are represented as box and whisker plots. In vitro experiments were performed in triplicate. The data were analysed using the non-parametric Kruskal-Wallis test, followed by Dunn's multiple comparison test where appropriate. For all tests, p values smaller than 0.05 were considered significant.
Physicochemical characterization of multi-walled carbon nanotubes
The NT1 used in this study have been extensively characterized . NT2 and NT3, the two coated MWCNT were composed of 50% MWCNT and 50% coating polymer by weight. They were produced by chemical vapour deposition on a supported catalyst in a fluidized bed that yielded spheric heaps of MWCNT entangled around the supported catalyst, measuring about few hundred microns in diameter, and forming a free-flowing powder.
Physicochemical characteristics of the multi-walled carbon nanotubes (MWCNT) used in the study
Coating polymer (50/50 wt%)
Carboxylic polyacid polymer
PMMA a polymer
12 ± 1
12 ± 1
12 ± 1
Size distribution (diameter)
Specific surface (m 2 /g)
Agglomerate size - mean diameter (%<10 μm)
Characterization using a Knudsen flow reactor of the surface functional groups present on the multi-walled carbon nanotubes (MWCNT)
[gas-phase probe molecules]
N(CH 3 )3
CF 3 COOH
NH 2 OH
3.0 · 1015
8.1 · 1016
2.4 · 1016
2.4 · 1017
3.9 · 1018
1.2 · 1016
N°/cm 2 b
1.32 · 1012
35.6 · 1012
10.6 · 1012
105.5 · 1012
1715 · 1012
5.3 · 1012
6.4 · 1015
4.4 · 1016
5.9 · 1015
3.4 · 1017
1.8 · 1017
1.2 · 1015
N°/cm 2 b
11.8 · 1012
81.3 · 1012
1.1 · 1012
628.5 · 1012
332.7 · 1012
2.2 · 1012
2.6 · 1014
1.4 · 1016
5.8 · 1014
2.0 · 1017
1.1 · 1018
1.9 · 1015
N°/cm 2 b
0.8 · 1012
41.2 · 1012
1.7 · 1012
588 · 1012
3235 · 1012
5.6 · 1012
In vitro studies
Size and number of carbon nanotube agglomerates found in vitro 24 hours after exposure to multi-walled carbon nanotubes (MWCNT)
Surface area (μm2)
30.23 ± 7.32
18.5 ± 1.5
12.87 ± 3.4*
37.3 ± 3.7*
4.97 ± 1.61#
9.3 ± 1.9#
We next quantified the expression of several genes involved in inflammation and oxidative stress. Expression of the mRNAs for the antioxidant gene HO-1 and the pro-inflammatory gene CXCL2 was significantly increased in cells exposed to crocidolite, NT1, or NT2, compared to control cells exposed to medium alone (Figures 4b and 4c). NT1 induced a significantly greater increase in CXCL2 expression than did NT2 (p < 0.05, Figure 4c). No such modifications occurred after cell incubation with NT3, CB or the polymers alone (data not shown). Finally, with none of the nanomaterials studied were changes seen in mRNA expression of the antioxidant genes GPX-1, SOD-1, or SOD-2, or of the inflammatory gene TNF-a (data not shown).
In vivo studies
Taken together, these results show that coating MWCNT with an acidic polymer reduced their specific surface five-fold but enhanced their dispersion in culture medium and their internalization by macrophages both in vitro and in vivo, compared to uncoated MWCNT. These results agree with earlier data  showing that the presence of acidic groups such as phenolic or carboxyl groups on the surface of carbon materials facilitates the internalization of these materials by mouse macrophages. The marked internalization of NT2 by mouse macrophages in our study was associated with the induction of a cytotoxic, oxidative, and inflammatory response despite the marked decrease in specific surface, compared to NT1. A similar direct relationship between the degree of internalization and the severity of the inflammatory response was described with metallic nanoparticles . In contrast to the in vitro experiments, in which the oxidative and inflammatory effects of NT1 and NT2 were similar, the in vivo experiments showed stronger oxidative and inflammatory responses to NT2 than to NT1, suggesting that cells other than macrophages were involved in the response to NT2 in vivo. This hypothesis is supported by the smaller size and larger number of NT2 agglomerates in the alveolar wall, suggesting enhanced contact of alveolar cells with NT2 compared to NT1. Accordingly, Shvedova and coworkers  demonstrated that exposure of mice to relatively well-dispersed single-walled CNT by inhalation produced a stronger inflammatory response than did pharyngeal aspiration of an agglomerated particle suspension (micrometer-size agglomerates). A stronger inflammatory response to NT2 could result from their acidic surface, in addition to their better internalization. Indeed, we recently obtained data showing that the inflammatory response of murine macrophages exposed to MWCNT generated by catalyst chemical vapor deposition was more marked when acidic groups were present on the MWCNT surface, compared to identically produced MWCNT of similar length that did not have acidic groups on their surface (Cyrill Bussy, unpublished data). Moreover, Hamilton and colleagues  recently showed that titanium dioxide nanoparticles induced inflammasome activation and inflammatory cytokine release through a cathepsin B-mediated mechanism involving lysosomal disruption and, therefore, acidification of the intracellular compartment, thus linking acidification to inflammatory response. It should be noted, however, that the inflammatory response to NT2, evaluated based on BAL fluid cellularity and lung gene expression, resolved between 1 week and 1 month post-instillation, which probably explained the absence of persistent histological abnormalities. The absence of fibrosis may also reflect the strain of mice used in this study as compared to others .
In contrast to the acidic polymer, the polystyrene polymer suppressed the cytotoxicity, oxidative stress, and inflammation observed both in vitro and in vivo when the nanotubes were not coated. Several mechanisms may explain this result. First, the coated MWCNT contained 50% polymer by weight and therefore contained half the amount of nanotube material supplied by NT1. To investigate the potential role for this difference, we performed in vivo experiments, comparing the effects of 100 μg of NT1 to those of a "double dose" of NT2 and NT3 (200 μg, thus containing the same amount of MWCNT than 100 μg of NT1). These experiments showed that BAL fluid cellularity 24 h after exposure to 200 μg NT2 or NT3 was similar to that seen with the original dose of 100 μg (data not shown), excluding a role for reduced MWCNT mass in the absence of NT3 toxicity. Second, the specific surface area was smaller with the coated MWCNT than with NT1. However, NT2 and NT3 had similar specific surface areas, yet different toxicities. Third, cell internalization was less marked with NT3 than with the other MWCNT. Similarly, Pan and coworkers  showed that coating TiO2 nanoparticles with a dense grafted polymer brush that exhibited hydrophobic properties, also exhibited by our NT3, prevented adherence to the cell membrane and hence penetration into the cell and induction of oxidative stress. Indeed, cells adhere less well to hydrophobic than to hydrophilic biomaterials . However, the reduced internalization of NT3 was probably not the only mechanism explaining the absence of toxicity of this material, since the in vivo experiments showed similar numbers of alveolar macrophages with MWCNT-containing vacuoles in the NT1 and NT3 groups. Fourth, NT3 may have a decreased ability to induce an oxidative and inflammatory response once internalized, compared to the other two MWCNT. This mechanism is consistent with the presence on the NT3 surface of large numbers of ozone oxidizable groups, compared to the other two MWCNT. These oxidizable groups may act as free radical scavengers  thereby decreasing the oxidative stress and consecutive inflammation in NT3-exposed cells and animals. Further studies are needed to determine whether such a mechanism is at play.
We administered MWCNT intratracheally to investigate their effects on the lung. We are aware that inhalation should be preferred over intratracheal instillation for toxicological investigations  but we believe our study is relevant to the analysis of the importance of surface characteristics as a determinant of MWCNT toxicity, as we compared the effects of the three materials administered to mice in the same way and using the same experimental set-up. Furthermore, the consistency of the results from our simultaneous in vitro and in vivo investigations supports the validity of our findings.
In conclusion, to the best of our knowledge, this study supplies the first evidence that using polymer-coating is a useful strategy to diminish adverse effects of CNT. Such tool to modify the surface environment of industrially produced CNT may hold promise for improving occupational safety, as well as general user's safety.
Lyes Tabet was a recipient of a joint grant from ADEME (Agence de l'Environnement et de la Maitrîse de l'Énergie) and ARKEMA. The authors thank Olivier Thibaudeau (Plate-Forme de Morphologie, Hôpital Bichat) for his assistance in preparing the anatomic specimens and Hélène Desqueyroux (ADEME) for her helpful comments.
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