Thermal annealing of carbon nanotubes reveals a strong toxicological impact of the structural defects

15 Pages·2017·1.21 MB·English

Thermal annealing of carbon nanotubes reveals a strong
toxicological impact of the structural defects
Agathe Figarol, Jérémie Pourchez, Delphine Boudard, Valérie Forest, Sarah
Berhanu, Jean-Marc Tulliani, Jean-Pierre Lecompte, Michèle Cottier, Didier
Bernache-Assollant, Philippe Grosseau
To cite this version:
Agathe Figarol, Jérémie Pourchez, Delphine Boudard, Valérie Forest, Sarah Berhanu, et al.. Thermal
annealingofcarbonnanotubesrevealsastrongtoxicologicalimpactofthestructuraldefects. Journalof
Nanoparticle Research, 2015, 17 (4), pp.article 194. ￿10.1007/s11051-015-2999-0￿. ￿emse-01148477v2￿
HAL Id: emse-01148477
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JNanopartRes(2015)17:194
DOI10.1007/s11051-015-2999-0
RESEARCH PAPER
Thermal annealing of carbon nanotubes reveals
a toxicological impact of the structural defects
Agathe Figarol . Je´re´mie Pourchez . Delphine Boudard . Vale´rie Forest .
. . .
Sarah Berhanu Jean-Marc Tulliani Jean-Pierre Lecompte
Miche`le Cottier . Didier Bernache-Assollant . Philippe Grosseau
Received:6January2015/Accepted:8April2015/Publishedonline:22April2015
(cid:2)SpringerScience+BusinessMediaDordrecht2015
Abstract The biological response to pristine and the annealed carbon nanotubes showed a significant
annealed multi-walled carbon nanotubes (MWCNT) increase of the pro-inflammatory response. We as-
was assessed on murine macrophages (RAW 264.7). sumed that this behavior was due to the reduction in
First,thephysicochemicalfeaturesoftheas-produced structural defects that may modify the layer of
MWCNTandannealedat2125 (cid:3)Cfor1 hwerefully adsorbed biomolecules. Surprisingly, the purification
characterized. A decrease in structural defects, hy- of metallic catalysts did not have any significant
drophobicity and catalytic impurities was detected impactontheoxidativestress.Wesuggestedthatthe
after annealing. Thereafter, their impact on cyto- structural improvements from the 2125 (cid:3)C treatment
toxicity, oxidative stress, and pro-inflammatory re- candecreasethecarbonnanotubescavengingcapacity
sponse was investigated at concentrations ranging andthusallowahigherfreeradicalreleasewhichmay
from 15 to 120 lg mL-1. No effect of the 2125 (cid:3)C counterbalancethedecreaseofoxidativestressdueto
treatmentwasdetectedonthecytotoxicity.Incontrast, alowercontentofmetallicimpurities.
Electronicsupplementarymaterial Theonlineversionof
thisarticle(doi:10.1007/s11051-015-2999-0)containssupple-
mentarymaterial,whichisavailabletoauthorizedusers.
A.Figarol(cid:2)P.Grosseau S.Berhanu
SPIN-EMSE,CNRS:UMR5307,LGF,EcoleNationale CentredesMate´riaux,CNRSUMR7633,Armines-
Supe´rieuredesMines,42023Saint-E´tienne,France MinesParisTech,91003Evry,France
A.Figarol(&)(cid:2)J.Pourchez(&)(cid:2)V.Forest(cid:2) J.-M.Tulliani
D.Bernache-Assollant DepartmentofAppliedScienceandTechnology,
CIS-EMSE,EA4624,SFRIFRESIS,LINA,Ecole PolitecnicodiTorino,10129Turin,Italy
NationaleSupe´rieuredesMines,42023Saint-E´tienne,
France J.-P.Lecompte
e-mail:fi[email protected] SPCTS,CentreEurope´endelace´ramiqueCNRS:UMR
7315,87068Limoges,France
J.Pourchez
e-mail:[email protected]
D.Boudard(cid:2)M.Cottier
EA4624,SFRIFRESIS,LINA,Universite´ JeanMonnet
Saint-Etienne,42023Saint-E´tienne,France
123
194 Page2of14 JNanopartRes(2015)17:194
Keywords Carbonnanotubes(cid:2)Annealing(cid:2)High cytotoxicity, pro-inflammatory response, and oxida-
temperaturetreatment(cid:2)Oxidativestress(cid:2) tive stress (Brown et al. 2007; Shvedova et al. 2009;
Inflammation(cid:2)Defects(cid:2)Environmentalandhealth Fro¨hlich et al. 2012). Nonetheless, available data are
effects still fragmentary and often contradictory. The use of
different biological models and toxicity assessment
methodologies, as well as the lack of full physico-
Abbreviations
chemical data in nanotoxicology studies can, at least
B.E.T. Brunauer–Emmet–Tellermethod
partially,explainthecontrastingfindings.Thus,oneof
CNT Carbonnanotubes
the main keys to improve our understanding of CNT
CNTa Annealedcarbonnanotubes
toxicity is to design specific toxicological works in
CVD Chemicalvapordeposition
order to accurately examine the biological impact of
DCF 20,70-Dichlorodihydrofluorescein
given physicochemical features (Jiang et al. 2009;
DMEM Dulbecco’sModifiedEagle’sMedium
Kayatetal.2011;SchrursandLison2012).
FEG-SEM Field-emissiongun-scanningelectron
Surfacetreatmentsoftheas-producedCNTchange
microscopy
their properties and enlarge their potential applica-
HR-TEM High-resolutiontransmissionelectron
tions.Hightemperaturetreatment,alsocalledthermal
microscopy
annealingorgraphitization,hasbeenwelldocumented
H DCF-DA 2070-Dichlorodihydrofluorescein
2 as a way to improve the purity (i.e. decrease of
LDH Lactatedehydrogenase
metallic impurity content) and structural order of
MWCNT Multi-walledcarbonnanotubes
multi-walledcarbonnanotubes(MWCNT).Common
ROS Reactiveoxygenspecies
production techniques of MWCNT such as chemical
TDS Thermaldesorption
vapor deposition (CVD) require the use of metallic
TNF-a Tumornecrosisfactora
nanoparticles as catalysts. As a result, metal residues
XPS X-rayphotoelectronspectroscopy
areclassicallyfoundentrappedinMWCNTorontheir
XRD X-raydiffraction
surface. Moreover, these MWCNT are non-perfect
cylindrical graphene sheets and exhibit structural
defectsincrystallinelatticesmainlyassociatedtosp3
Introduction carbon hybridization (Ebbesen and Takada 1995).
According to Bougrine et al. (2001), annealing
Carbonnanotubes(CNT)havereceivedagreatdealof MWCNT at 1600 (cid:3)C under argon reduces the level
attentionsincetheirfulldescriptionintheearly1990s of metallic impurities due to the vanishing of metal
(Iijima 1991). CNT have demonstrated high electric particles.Besides,over1600 (cid:3)C,thecarboninterlayer
and thermal conductivity, exceptional mechanical spacingstartstocontract,withbetterresultsobtained
resistance,andadsorptioncapacityleadingtonumer- at2400 (cid:3)C. BoncelandKoziol (2014)suggestedthat
ous studies in academic and industrial research metallicimpuritiesgraduallyescapeasvaporsthrough
(Baughman et al. 2002; De Volder et al. 2013). one CNT open end at a temperature for which a
However,CNTareclassifiedasinhalablefibersdueto sufficiently high vapor pressure of metal is achieved.
their small aerodynamic diameter. Common public Chen et al. (2007) confirmed this purification and
opinionassociatestheirmorphologicalresemblanceto structural enhancement with a threshold value of
asbestos fibers and this raises health concerns. Based 1800 (cid:3)C.Heattreatmenttimewasprolongedfrom2to
on the scientific literature, the biological impact of 60 minatthemaximumtemperature.Theystatedthat
inhaledCNTremainsuncertain,butithasbeenshown theeffectofdurationtimewasmuchlowerthanthatof
that this nanomaterial has the capacity to trigger annealing temperature. Enhancement of MWCNT
pathogenicpathways.Invivostudiesinratsandmice crystallinityappearstoimprovetheirelectricconduc-
havereportedevidencesofinflammation,formationof tivity,whichcouldbeofgreatinterestformicroelec-
granulomas, fibrotic structures, and oxidative stress tronicorbiomedicalapplications(Fioritoetal.2009).
(Mulleretal.2005;Shvedovaetal.2008;Porteretal. Similarly, their axial strength and Young’s modulus
2010). Similarly, cell exposure to CNT could trigger are improved, encouraging the development of high
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JNanopartRes(2015)17:194 Page3of14 194
performance composite material (Pinault et al. 2004; FeisoftenusedasacatalystforCNTproductionandis
Yamamotoetal.2013). implicated in the Fenton reaction: H O ? Fe2? ?
2 2
Nowadays, the availability of annealed MWCNT OH(cid:2) ? Fe3? ? OH- (Kagan et al. 2006; Shvedova
onthemarketisincreasingwhiletheireffectsonhealth etal.2012;Geetal.2012).Bussyetal.(2012)exposed
are still not fully understood. In fact, Fiorito et al. amechanismofreleaseofFeimpuritiesintheacidic
(2009)comparedMWCNTpurifiedbyacidfunction- lysosomes after cellular uptake of CNT in murine
alization or by annealing at 2400 (cid:3)C and concluded macrophages. Al, another metallic catalyst, is a non-
thatthelatterwerelesscytotoxicbutinducedahigher redox-activemetal;however,itcanhaveastrongpro-
pro-inflammatoryresponseonprimaryhumanmacro- oxidant activity (Exley 2004; Mahdi et al. 2010;
phages. Contradicting results from Simon-Deckers Ruipe´rez et al. 2012). Al-superoxide could indeed
etal.(2008)evidencednoimpactonthecytotoxicityof stabilizeferrousionbyreducingitsrateofoxidation.It
humanepithelialcells(A549)ofMWCNTannealedat thus indirectly promotes the production of ROS with
2000 (cid:3)C compared to raw MWCNT shortened by the use of the ferrous ions in the Fenton reaction.
ultrasonication. Cheng et al. (Cheng et al. 2009) Furthermore,Zhangetal.(2011)comparedthecellular
reached a similar conclusion when comparing responseofHeLacellsexposedtoMWCNTannealed
MWCNT annealed at 2600 (cid:3)C and raw MWCNT on at2400 (cid:3)C,oxidizedbyaHNO treatmentorirradiated
3
human monocyte-derived macrophages (HMM). A with60Co.AnnealedMWCNTexhibitedalowerlevel
morecompletestudywaspublishedastwocompanion of structural defects than oxidized or irradiated
papers (Fenoglio et al. 2008; Muller et al. 2008) and MWCNT. The induced oxidative stress and cyto-
assessedthephysicochemicalandbiologicaleffectsof toxicitywerealsolowerforannealedCNT.Liuetal.
annealingMWCNT at2400 (cid:3)C.Invitrogenotoxicity (2011) focused on the acellular activity of an an-
on lung epithelial cells (RLE) seemed to be slightly tioxidant, the glutathione (GSH). All the studied
reduced by this thermal treatment. A decrease in carbon-based nanomaterials, including CNT, were
cytotoxicity and inflammation was furthermore ob- linked with a dose-dependent decrease of GSH
served in vivo in Wistar rats after intra-tracheal concentrationintheacellularmedium.Withannealed
instillations(2 mg/rat).However,highlevelsofcyto- carbon black (at 2600 (cid:3)C), however, the final GSH
toxicity were observed again after a 6 h grinding. concentration was higher than with pristine carbon
Fenoglioetal.(2008)andMulleretal.(2008)suggest black. These two studies suggest a relationship
thus that the positive effect on cytotoxicity and pro- betweenstructuraldefectandoxidativestress.
inflammatoryresponsewasmainlyoccasionedbythe Further studies have assessed the biological re-
decrease in surface reactive sites due to the higher sponse toward annealed MWCNT but without any
structuralorder.Unfortunately,theeffectsonoxidative comparison to raw MWCNT. Hirano et al. (2008)
stress have not been directly studied as a cellular showed a high cytotoxicity of MWCNT annealed at
response. However, in cell-free conditions, annealed 2600 (cid:3)C which was not linked to apoptosis or
MWCNTdemonstratedalowerscavengingcapacityof oxidativestress,alongwithalowinflammation.Data
theoxygenradicals(Fenoglioetal.2008;Mulleretal. from Tsukahara and Haniu (2011) also indicated a
2008). Furthermore, no MWCNT directly generated dose-dependentcytotoxicitywithnosignofapoptosis
anyoxidativespecies.ThescavengingactivityofCNT and oxidative stress, although some cytokines levels
corresponds to their ‘‘antioxidant’’ capacity. Thus, it wereincreased(IL-6andIL-8butnotTNF-a,IL-1b,
lowers the concentration of reactive oxygen species IL-10, and IL-12) indicating a medium pro-inflam-
(ROS) of the surrounding medium. This activity has matoryresponse.
beenshowntobeenhancedbylatticedefects(Fenoglio Overall, few toxicological studies are available
etal.2006,2008;Galanoetal.2010).Tothebestofour with full comparison of pristine and annealed
knowledge, few studies have been conducted to MWCNTandresultsarecontradictorywithveryfew
examine the impact of the oxidative stress of the studies on oxidative stress. In the present work,
CNTpurificationbyanannealingtreatment.Frequent- characterization and in vitro biological assessments
ly, a causal link between metallic impurities and oftheroleofmetallicimpuritiesandstructuraldefects
oxidativestresshasbeensuggested(Kaganetal.2006; were conducted on the as-produced and annealed
Shvedovaetal.2012;Geetal.2012;Bussyetal.2012). MWCNTat2125 (cid:3)C.
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194 Page4of14 JNanopartRes(2015)17:194
Experimentalprocedures of at least four measurements. Diameters and CNT
morphologyweremoreoveranalyzedbyhigh-resolu-
Powdersandthermalannealingtreatment tion transmission electron microscopy (HR-TEM).
ofMWCNT HR-TEM images were acquired on a Tecnai F20 ST
microscope(FEICompany)operatingat200 kVwith
MWCNT(NanocylNC7000,RuedelaVacherie236, a field-emission gun as the electron source. The
Auvelais, Belgique) were synthesized by CVD and samples were prepared by dispersion in ethanol and
have a diameter of 9.5 nm and a length of 1.5 lm subsequent ultrasonication. A small amount of the
according to the manufacturer. The as-produced dispersion was then deposited on a TEM copper grid
MWCNT were labeled CNT. For the annealing coated with a holey carbon film (Agar Scientific,
treatment, these CNT were allowed to degas under ElektronTechnologyUKLtd,Stansted,Essex,CM24
vacuum overnight then were heated at 10 (cid:3)C min-1 8GF, UK). The specific surface area (SSA) was
until200 (cid:3)Candremainedatthistemperaturefor5 h determinedbyN adsorptionat77 Kafterout-gassing
2
under vacuum and 18 h under argon. The annealed for 4 h at 110 (cid:3)C (Micromeritics ASAP 2000, Mi-
treatment started then, with a heating rate of cromeriticsCorporateHeadquarters,4356Communi-
10 (cid:3)C min-1 until the maximal temperature of cationsDr.,USA)usingtheBrunauer–Emmet–Teller
2125 (cid:3)C was reached and hold for 1 h. The resulting (B.E.T.) method. The purification from metallic
annealed carbon nanotubes were labeled CNTa. All impurities was assessed by inductively coupled plas-
weighing and the maximum of dried powder ma- ma atomic emission spectroscopy (ICP-AES) identi-
nipulationswereconductedunderanextractorhoodin fying trace metals after mineralization by nitro-
anenclosedlaboratoryatlowpressure. hydrochloric acid. Furthermore, to fully characterize
the MWCNT, X-ray photoelectron spectroscopy
PhysicochemicalcharacterizationofMWCNT (XPS) was used to analyze the surface chemistry.
The oxygen atomic percentages were obtained by
Field-emission scanning electron microscopy (FEG- analyzing the ratio between the intensities of the O
1s
SEM,JEOLJSM6500F,Akishima,Tokyo,Japan)at andC XPSpeaks(with20 %incertitude,monochro-
1s
2 kV was used to characterize the CNT morphology matized Al K source, 400 lm width analyzed,
a
and diameter. A few milligrams of MWCNT powder 1.3 9 10-7 Pa of residual pressure, Thermo VG
wereputonacarbon-coatedholeyfilm.Sampleswere ThetaProbe, Thermo Fisher Scientific Inc., Wyman
coated with a 3-nm gold layer before FEG-SEM Street,Waltham,USA).Sampleswerepreparedbythe
observations. 100 MWCNT diameters per picture sonication of a 30 lg mL-1 CNT suspension in
were measured by means of ImageJ software (three ethanol (5–15 min, 24 W mL-1, 3 mm probe, Bran-
pictures were used for a total of 300 measurements). son Sonifier SD450, Parc d’Affaires Silic, Rungis,
Theenhancementinstructuralorderwasmonitoredby France).Onedropwasdepositedanddriedona1 cm2
Ramanspectroscopy(XploRA,HoribaScientific,rue silicon wafer previously covered by a thin layer of
deLille,Villeneuved’Ascq,France)ondrypowder.A gold. The spectra were corrected for Shirley-type
laserat532 nm,a950objective,and20acquisitions backgrounds. Automatic search of the peak positions
of 20 s were used with, first, a 1200 T network for a was conducted from the fixed C1s peak at 284.5 eV.
spectra between 200 and 3000 cm-1, then a 2400 T Atomic percentages were calculated from the peak
network, giving a spectra between 1000 and areasandgivensensitivityfactors.Assessmentofthe
2000 cm-1 for the I /I characterization. Two usual surface chemistry was completed by thermal desorp-
d g
bandsareindeedtypicalofCNT.TheD-bandintensity tion spectroscopy (TDS, supplementary data—Fig.
(Dfordisorder)islinkedtothesp3hybridizedcarbon, S1). 10 mgofpowder was heated under vacuum at a
and increases with ill-organized graphite structure heating rate of 20 (cid:3)C min-1 until 850 (cid:3)C. Two
(BelinandEpron2005).TheG-band(Gforgraphite) turbomolecular pumps ensure a vacuum of less than
correspondstoasplittingoftheE stretchingmodeof 10-2 Pa from air atmosphere. A mass spectropho-
2g
graphite and is independent of the structural defects. tometer (Balzers QMG 112 quadrupole, Iramali 18,
Therefore,theratioofbothintensitiesI /I informson Balzers, Liechtenstein) collected and analyzed the
d g
thelevelofstructuraldefects.I /I valuesarethemean volatilized elements. Zeta potentials were measured
d g
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JNanopartRes(2015)17:194 Page5of14 194
usingaZetasizerNanoZS(MalvernInstruments)and Lactate dehydrogenase (LDH) released from cells
30 lg mL-1 carbon nanotubes suspensions in deion- with damaged membranes using the CytoTox-96(cid:4)
ized water or culture medium prepared by sonication Non-radioactive Cytotoxicity Assay (Promega,
(5 min,24 W mL-1,3 mmprobe,BransonSonifier). Woods Hollow Road, Madison, USA) according to
the manufacturer’s instructions. Detection was per-
PreparationofMWCNTsuspensions formed using a microplate reader (Multiskan GO,
forthetoxicityassays Thermo Fisher Scientific Inc., Wyman Street, Wal-
tham,USA)at450 nm.TheamountofreleasedLDH
0.80 mg of MWCNT was weighed and dispersed in wasreportedasapercentageofthetotalcellularLDH
5 mL of culture medium: Dulbecco’s Modified Ea- (measuredafterthecompletelysisofcontrolcells).
gle’s Medium (DMEM, Invitrogen, Carlsbad, CA,
USA) supplemented with 10 % of fetal calf serum Pro-inflammatoryresponseofmacrophages
(Invitrogen), and 1 % penicillin–streptomycin (peni- afterexposuretoMWCNT
cillin 10,000 U mL-1, streptomycin 10 mg mL-1,
Sigma-Aldrich, Saint-Louis, MO, USA). A 5-min In parallel to the cytotoxicity test, 100,000 cells/well
sonication was performed to fully disperse each were seeded in a second 96-well plate and incubated
MWCNTsample(3 mmprobe,24 W mL-1,Branson with the MWCNT for 24 h. Tumor necrosis factor
Sonifier S-450D). Serial dilutions were conducted to alpha(TNF-a)releasewasassessedusingacommer-
obtainconcentrationsof160,80,40,and20 lg mL-1 cial enzyme-linked immunosorbent assay kit (Quan-
ofMWCNTinsupplementedculturemediumDMEM. tikine(cid:4)MouseTNF-a/TNFSF1AImmunoassay,R&D
Forthe toxicity assays, 25 lL ofcellsuspensionwas systems Inc., McKinley Place NE, Minneapolis,
supplemented by 75 lL of the MWCNT suspension USA). Theoptical densitywas determinedaccording
(1/4 cells, 3/4 MWCNT). The final concentrations tothemanufacturer’sinstructions,usingamicroplate
were 120, 60, 30, and 15 lg mL-1. The suspension reader(MultiskanGO,ThermoScientific)at450 nm.
stabilityoveraweekwasestablishedbydynamiclight A standard curve was established and results were
scattering (ZetaSizer Nano ZS). It measured the expressedinpg mL-1ofTNF-a.
average hydrodynamic diameter of the equivalent
spherewithadensityof1withthesametranslational Oxidativestressofmacrophagesafterexposure
diffusioncoefficient. toMWCNT
Invitrotoxicityassays Acute oxidative stress was determined by measuring
the level of intracellular ROS by the OxiSelectTM
CellcultureofRAWmacrophages Intracellular ROS Assay Kit (Cell Biolabs). Briefly,
the cells were seeded at 2 9 106 cells/well and
RAW 264.7 macrophage cell line was provided by allowed to adhere in a black 96-well plate for 4 h. A
ATCC Cell Biology Collection (Promochem LGC, 1 h pre-treatment with the cell-permeant 2070-
Queens Road, Teddington, Middlesex, UK). It was dichlorodihydrofluorescein (H DCF-DA) was carried
2
derived from mice peritoneal macrophages trans- out before a 90-min exposure to the MWCNT. The
formedbytheAlbesonMurineLeukemiaVirus.Cells fluorescence intensities were read at 480 nm excita-
were grown in 10 % fetal calf serum supplemented tion and 530 nm emission (Fluoroskan Ascent,Ther-
DMEMandmaintainedat37 (cid:3)Cundera5 %carbon moscientific).Apositivecontrolwithcellsexposedto
dioxidehumidifiedatmosphere. 1 mM H O was added. Biases were found to result
2 2
from the fluorescence absorption by the black
Cytotoxicityofmacrophagesafterexposure MWCNT. Results were thus corrected by the mean
toMWCNT ofcell-freestandardcurves.Theyweretracedafterthe
variation of the fluorescent intensity of known
The cells were seeded in a 96-well plate at 100,000 concentrations of DCF probe (20,70-dichlorodihy-
cells/well and incubated with the MWCNT for 24 h. drofluorescein) in complemented medium with and
Cell viability was then evaluated by quantifying the without MWCNT and without cells. Corrections of
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artifacts were calculated and applied for each CNT diameter given by the producer, the entanglement of
typeandeachdose(Supplementarydata—Fig.S2and the tubes unfortunately did not allow to confirm the
TableS1). given length of 1.5 lm. The nanotube structure was
more specifically assessed by HR-TEM before and
Statisticalanalysis after the annealing treatment (Fig. 2). HR-TEM
revealedsomesignificantvariationbetweennanotubes
Resultswereexpressedasthemeanofthreeindepen- comingfromthesamesample,bothforuntreatedand
dentexperiments,eachcarriedoutintriplicates,with annealedCNT.Forinstance,thenumberofinnerwalls
standard errors of the mean value. Statistical sig- ranged from 3 to more than 20 depending on the
nificancewasdeclaredwhenp\0.05usingaStudent individual nanotube under consideration. The crys-
testwithTanagrasoftware(Rakotomalala2005). talline quality also varied significantly between nan-
otubes forming one sample. Typical defects such as
kinks, wavy tubes, surface roughness, or ‘‘bamboo-
Results type’’ inner structures were observed for both CNT
and CNTa. However, the number of metallic impuri-
Physicochemicalfeatures ties due to catalyst particles located inside the tubes
greatly decreased after the annealing procedure.
MorphologyandspecificsurfaceareaoftheMWCNT Moreover, CNTa tended to exhibit less surface
roughness, indicating that the annealing treatment
A thorough characterization of the nanomaterials is hadslightlyimprovedthenanotubecrystallinequality.
crucial.FEG-SEMimagesarepresentedinFig. 1.The No significant change due to the thermal annealing
morphology of the MWCNT remained alike after areawasdetectedintheSSA.
heatingat2125 (cid:3)C,presentingentangledtubeswithno
orveryfewtracesofamorphouscarbon.Asshownin
Table 1, where all physicochemical features are LatticedefectsoftheMWCNT
summarized, the mean diameter was not impacted,
norwasthediameterdistribution(seeSupplementary Raman spectroscopy was used to assess the lattice
Data—Fig. S3). The results were confirmed by HR- defectsofthetwoMWCNT.Thespectraexhibitedthe
TEM measurements: 17 ± 5 nm for CNT by SEM two typical bands of carbon material: the D-band
and HR-TEM, 17 ± 5 nm and 18 ± 4 nm for CNTa (1340 cm-1) and the G-band (1575 cm-1), with two
by SEM and HR-TEM, respectively (Fig. 2). While overtonepeaks:theD’-band(2685 cm-1)andtheG’-
the measured mean diameter is almost twice the band (2930 cm-1) (Fig. 3 is an illustration of one
Fig.1 FEG-SEMimagesofmulti-walledcarbonnanotubes:as-produced(CNT)andafteranannealingtreatmentat2125(cid:3)CunderAr
(CNTa)
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JNanopartRes(2015)17:194 Page7of14 194
Table1 Physicochemicalcharacteristicsofthetwocarbonnanotubes
Sample CNT CNTa
Diameter(nm) 17±5 17±5
Structuraldisorder(I /I ) 1.18±0.10 0.77±0.13
d g
SSA(m2g-1) 212±3 209±3
Catalyticimpurities(wt%) Fe0.19 Fe0.01
Al4.23 Al0.00
Co0.12 Co0.00
O(atomicproportion%) 8.7 3.5
Zetapotentialinwater(mV) -23.3±7.0 -36.4±4.3
Zetapotentialinculturemedium(mV) -127.0±8.6 -156.0±6.6
CNT as-produced MWCNT, CNTa MWCNT annealed at 2125(cid:3)C under Ar. Mean diameters were determined by FEG-SEM
observationsandconfirmedbyHR-TEM.I /I correspondstotheintensityratiooftheRamanDandG-bands.SSAspecificsurface
d g
area, measured according to Brunauer Emmett and Teller protocol. Catalytic impurity level was assessed by ICP-AES, levels
\0.01wt%werefoundforCu,Cr,Ni,andTi.O/CatomicproportionistheintensityratiooftheO andC XPSpeaks
1s 1s
Fig.2 HR-TEMimagesofmulti-walledcarbonnanotubes:as-produced(CNT)andafteranannealingtreatmentat2125(cid:3)CunderAr
(CNTa)
Ramanmeasurement).TheI /I ratiowassignificantly PurificationafterannealingtreatmentofCNTa
d g
lowered by the annealing treatment from 1.24 to 0.77
whichnotifiesofahighercrystallinitydegreewithouta As expected, the 2125 (cid:3)C treatment removed all
complete disappearance of the structural defects how- metallic impurities. It was quantitatively confirmed
ever.X-raydiffraction(XRD)wasperformedtofurther by ICP-AES since all traces of Fe, Al, and Co
analyzethecrystallinityofthesamples.Nochangewas, disappeared(Table 1).Alwasthemaincatalystused
however,detectablebetweenthetwoXRDdiffraction to produce those MWCNT and its level fell from
patterns(seeSupplementaryData—Figs.S4andS5). 4.23 wt%toanundetectablevalue(\0.01 wt%).The
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culture medium are presented in Table 1. The ob-
served decrease in zeta potential in water is quite
surprising. The pH of the suspensions was close
enough (4.9 ± 0.6 and 4.8 ± 0.1) so that the differ-
ence should not contribute to the difference in zeta
potentials.Ontheotherhand,suspensionsinbuffered
culturemediumwerestable.Zetapotentialswerevery
low,butthezetapotentialofthisculturemediumalone
(complementedDMEM)wasalready-100 mV ±12.
The difference in zeta potentials in culture medium
could be due to the difference in the profile of the
proteinsattheCNTsurface.
Fig.3 Ramanspectraofcarbonnanotubes:as-produced(CNT)
andafteranannealingtreatmentat2125(cid:3)CunderAr(CNTa) Biologicaltoxicityassessedinmacrophages
HR-TEMimagesallowedtoqualitativelyconfirmthe TheinteractionoftheMWCNTandthemacrophages
presence of metal content before the annealing was confirmed by microscopic observations (Supple-
treatmentandtheirdisappearanceafterit(supplemen- mentary data—Fig. S10). These pictures allow to
tarydata—Fig.S6). assessthefinalsizeofCNTandCNTaagglomeratesin
culture medium. CNT tended to exhibit less and
Chemicalmodificationsafterannealingtreatment smaller agglomerates than CNTa: average of
ofCNTa 2.2 lm ± 0.7 for CNT and 3.5 lm ± 1.9 for CNTa
(withsomeoftheagglomeratesasbigas10 lm).
The surface oxygen content was assessed by XPS
analysis.First,generalXPSspectrawereanalyzedfor Cytotoxicity
both samples (supplementary data—Fig. S7). Three
mainpeakswereidentified:theC1speakat284.5 eV, LDHreleasedfromcellswithdamagedmembranes,if
theO1speakat533 eV,andtheAu4fpeaksfromthe significantly higher than the negative controls, is an
substrateat84and87.5 eV.Theabsenceofcontribu- indicator of cytotoxicity. For both MWCNT, a
tionfromtheSiO substrate(nosignalfromtheSi2p significant LDH release was uniquely observed at
2
peak) was verified. A more detailed spectrum was the maximum dose of 120 lg mL-1 (Fig. 5). No
recorded for each of the four peaks. The O/C atomic significant difference was found between the cyto-
proportions(inpercentage)aregiveninTable 1.The toxicityinducedbyCNTorCNTa.
amount of oxygen decreased after the annealing
treatment. The heating has probably desorbed and
decomposed the oxygen-containing groups which
werepresentatthenanotubessurface.Furtherassess-
ment of the surface chemistry was conducted by the
analysis of zeta potentials and TDS. TDS results
showed a decrease in overall oxygenated groups
especially with the disappearance of the peak due to
physisorption(inthe0–200 (cid:3)Crange)(Fig. 4).Apeak
due to chemisorption of oxygenated groups (in the
400–600 (cid:3)Crange,Xiaetal.2007)appearedafterthe
annealingtreatmentandseemedtohavebeenmasked
bytheoverallhigherlevelofoxygenatedgroups.CO
2
andH Odesorptionsconfirmedthedecreaseofthose
2 Fig.4 Thermaldesorptionofcarbonnanotubes,followingthe
oxygenated groups (see Supplementary data—Figs.
M=28 related to CO. CNT: MWCNT as-produced, CNTa:
S8 and S9). Zeta potentials in deionized water and MWCNTafteranannealingtreatmentat2125(cid:3)CunderAr
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JNanopartRes(2015)17:194 Page9of14 194
Pro-inflammatoryresponse
TNF-a is one of the cytokines indicators of the pro-
inflammatory response for macrophage cells. The
annealing treatment seemed to significantly increase
the TNF-a production (Fig. 6). It was found sig-
nificant only at the highest concentration of CNT,
whilethelevelsweresignificantfrom60 lg mL-1of
CNTawithadose-dependentresponse.
Oxidativestress
Measurement of ROS production demonstrated a
significanthighdoseresponseforbothMWCNT,sign
of a high oxidative stress (Fig. 7). No significant
Fig.6 Pro-inflammatoryresponseofmacrophagesaftera24h
differencewasobservedaftertheannealingtreatment. contactwiththeas-producedMWCNT(CNT)andannealedat
Thelevelswerealreadysignificantbeforecorrections 2125(cid:3)C MWCNT (CNTa). Significantly different from the
of the bias induced by the CNT absorption of negativecontrol,**p\0.001,#significantlydifferentbetween
CNTandCNTa,p\0.01at30lgmL-1,p\0.001at60and
fluorescence (at comparable levels with the positive
120lgmL-1,andnotsignificantlydifferentat15lgmL-1
control).Aftercorrection,theresponsewasmagnified
withROSlevelsreachingthreetimesthelevelofthe
positivecontrol.
Discussion
Impactofthethermalannealing
onthephysicochemicalfeatures
Themainoriginalityofthisstudywastobespecifical-
lydesignedinordertoassesstheimpactofthelattice
defects and metallic impurities on the macrophage
Fig.7 Reactive oxygen species (ROS) production bymurine
macrophagesaftera90minexposuretoMWCNTas-produced
(CNT) and annealed at 2125(cid:3)C (CNTa) after correction of
fluorescentdecreasebyMWCNT.Significantlydifferentfrom
negative control, **p\0.001. NS no significant difference
betweenCNTandCNTa
in vitro response. First, the physiochemical features
wereevaluatedbeforeandaftera2125 (cid:3)Ctreatment.
Themeasuredmeandiameterswerefoundtobetwice
thevaluefromtheproducer.Detectingthediameterof
Fig.5 Cytotoxicity induced by a 24h exposure to MWCNT
CNT bundles rather than isolated CNT could modify
as-produced (CNT) and annealed at 2125(cid:3)C (CNTa). Sig-
themeasurement(Peigneyetal.2001).However,HR-
nificantlydifferentfromnegativecontrol,**p\0.001.NSnot
significantlydifferentbetweenCNTandCNTa TEM measurements allowed a better precision by
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