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Copyright copy 2011 American Scientific PublishersAll rights reservedPrinted in the United States of America

Nanoscience andNanotechnology Letters

Vol 3 88ndash93 2011

Assessment of Human Lung MacrophagesAfter Exposure to Multi-Walled Carbon

Nanotubes Part I Cytotoxicity

Lin Zhu13 Amanda M Schrand1 Andrey A Voevodin4 Dong Wook Chang3Liming Dai2lowast and Saber M Hussain1lowast

1AFRL711 HPWRHPB Wright-Patterson AFB OH 45433-5707 USA2Department of Chemical Engineering Case Western Reserve University Cleveland OH 44106-7217 USA

3Department of Chemical and Material Engineering University of Dayton Dayton OH 45409 USA4AFRLRXBT Wright-Patterson AFB OH 45433-5707 USA

Due to the widespread production and use of carbon nanotubes in almost every area of science(ie drug delivery biosensors fuel cells and thermal management systems) they are receivingconsiderable attention for their novel mechanical electrical and chemical properties At this time ofhigh exposure potential it is critical to ascertain the biological impact of these materials on likelytarget organs tissues and cells such as those of the lung The aim of this study was to evaluate thedegree of cytotoxicity to human lung macrophage (U937) cells after exposure to unpurified or acid-purified multi-walled carbon nanotubes Cells were incubated with multi-walled carbon nanotubesand assessed for cytotoxicity generation of reactive oxygen species morphological changes anduptake The results demonstrate that multi-walled carbon nanotubes can accumulate in human lungmacrophage cells to different degrees based on their surface chemistry MWNT-COOH reduces cellviability in a dose-dependent manner under these experimental conditions (5ndash50 gml 2ndash24 h)However images of individual cells demonstrate morphological changes at low concentrationsTherefore before nanomaterials are fully accepted and integrated into biological systems they willcontinue to undergo further scrutiny at various stages of their processing (ie before and afterpurification) and with models ranging from simple to complex (ie cells vs whole animals) to gaina better understanding between their physicochemical properties and bio-effects

Keywords U937 Cell MWNTs MWNT-COOH ROS

1 INTRODUCTION

Carbon nanotubes consist of carbon hexagons arranged ina concentric manner with ends capped by fullerene-likestructures containing pentagons31 They can be further sub-classified based on the number of walls and are termedsingle-walled (SWNTs) double-walled (DWNTs) multi-walled (MWNTs) etc although many other forms andstructures such as carbon nanohorns nanodiamonds etcexist Due to the atomic arrangement of CNTs they arecharacterized by high aspect ratios high strengths highthermal and electrical conductivities and the ability to bechemically functionalized or filled It has been determinedthat the properties of CNTs are strongly dependent upontheir inherent semi-conductive or conductive structure thepresence of impurities such as residual catalysts or other

lowastAuthors to whom correspondence should be addressed

forms of carbon and defects from chemical or thermalprocessing for purification and functionalizationPhenomenon such as enhanced chemical reactivity per-

meability and the profound effect of surface chemistryare all expected to influence the cellular dynamics ofnanomaterials1 In the case of carbon nanotubes (CNTs)most studies have focused on organs such as the lungs2ndash7

Other studies have utilized macrophages in cell cul-ture which are responsible for removing foreign debrisand inflammatory responses8ndash11 Previous studies in ourlaboratory12 and others10 have shown that nanoparticlescan alter the phagocytic response of macrophages whichmay have implications in disease conditions Thereforefurther investigation into the factors that influence nano-material cytotoxicity such as purification procedures andresultant surface chemistry continue to be elucidated13ndash15

Alveolar macrophages are found in the alveolar sacsdeep within the lungs These cells are the first line of

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Zhu et al Assessment of Human Lung Macrophages After Exposure to Multi-Walled Carbon Nanotubes Part I Cytotoxicity

immunological defense against inhaled particles and serveas a good model to investigate how inhaled particlescan adversely affect cell function and lead to degrada-tion of health30 In this study we chose the human lungmacrophage cell line (U937) as a model to investigatethe potential cytotoxicity of unpurified multi-walled car-bon nanotubes (MWNTs) and acid purified multi-walledcarbon nanotubes (MWNT-COOH) by detecting the gen-eration of reactive oxygen species (ROS) morphologicalchanges and cell uptake

2 RESULTS AND DISCUSSION

The unpurified MWNTs and acid-purified MWNT-COOHwere characterized with transmission electron microscopy(TEM) air thermogravimetric analysis (TGA) Ramanspectroscopy and X-ray photoelectron spectroscopy (XPS)There was visual evidence for a reduction in nanotubelength after the acid purification and sonication treat-ment compared to the original unpurified sample (data notshown) However once the MWNTs were introduced intoaqueous solutions the nanotubes behaved in an agglom-erated fashion We previously demonstrated that largeMWNT agglomerates (microns in size) were present in1 mgml stock solutions which were only briefly soni-cated for 10 seconds16 In this study immediately afterdosing cells in culture with a low 5 gml concentra-tion of MWNTs or MWNT-COOH there was visual evi-dence for large agglomerated bundles of MWNTs withsizes gt50 microns (data not shown) in comparison to bet-ter dispersion of MWNT-COOH However differences inMWNT length (35 microns vs 12 microns measured withTEM) due to short or extended sonication times have notbeen shown to influence subsequent cytotoxicity7 There-fore in addition to visualization of primary MWNT struc-ture and overall dispersion metal catalyst impurity contentand surface chemistry were considered primary character-istics for each MWNT sample in this studyThe TGA data for the unpurified MWNTs (Fig 1)

revealed sim11 wt orange-colored residue after beingheated above 730 C most likely due to the presence ofFe catalyst residues that became converted to Fe2O3 uponthermal oxidation The MWNT-COOH sample showeda reduced Fe catalyst residue content of sim19 wtabove 730 C which indicates that the acid treatmentremoved most of the Fe catalyst residue These resultsfor impurity content were similar to our previous stud-ies using the same MWNTs which displayed 8 wtFe for unpurified MWNTs compared to 049 wt foracid-purified MWNTs16 when analyzed with inductively-coupled plasma-optical emission spectroscopy (ICP-OES)The Raman spectra of the MWNT sample before and

after acid purification showed an increase in the disor-der (D) band (1350 cmminus1 for the MWNT-COOH sam-ple suggesting that some surface damage due to oxidation

Fig 1 Characterization of MWNTs and MWNT-COOH with TGAshowing removal of Fe catalyst residues after acid purification

occurred (data not shown) X-ray photoelectron spectro-scopic (XPS) measurements performed on MWNT-COOHshow increased oxygen content after oxidation of the unpu-rified MWNTs through increased OmdashC bonds from 05 to8 wt and O C bonds from 07 to 85 wt (Table I)Therefore the physicochemical properties of the MWNTsample are distinctly different from the MWNT-COOHsample In this regard it is critical to maintain a carefulbalance between the surface chemical functionalization ofCNTs and their structural integrity in order to retain theirnovel propertiesIn order to assess if there is a differential response of

human lung macrophages to unpurified MWNTs comparedto acid purified MWNT-COOH changes in cytotoxicitymorphology and reactive oxygen species (ROS) generationwere investigated Changes in viabilities related to cytotox-icity were spectrophotometrically assayed with the MTSassay which is sensitive to mitochondrial function Thepositive control CdO was used to verify the validity of theMTS assay and shows a strong and significant decreasein cell viability at the low dose of 5 gml (Fig 2) Bycomparison cells exposed to MWNTs or MWNT-COOHat concentrations ranging from 5ndash50 gml for 24 h onlydisplayed a significant decrease in viability at the highestconcentration of 50 gml for MWNT-COOH comparedto no significant decreases in viability at the other con-centrations (5ndash25 gml) or at any of the concentrations(5ndash50 gml) for the unpurified MWNTs (Fig 2)One explanation for the decreased viability of the

cells after exposure to the 50 gml concentration of

Table I XPS results for oxygen (O) and iron (Fe) content (wt) ofMWNT and MWNT-COOH

O

Sample H2O OndashC O C ndashOH OndashFe Fe

MWNT mdash 05 07 02 02MWNT-COOH 08 80 85 mdash lt01

Nanosci Nanotechnol Lett 3 88ndash93 2011 89


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Assessment of Human Lung Macrophages After Exposure to Multi-Walled Carbon Nanotubes Part I Cytotoxicity Zhu et al

Fig 2 Cell viability assessment with the MTS assay after 24 h of expo-sure to MWNTs or MWNT-COOH There was significant toxicity for thepositive control CdO at 5 gml and MWNT-COOH at 50 gml Thedata are represented as the average of three independent experiments plusmnthe standard deviation Stars indicate significant difference from controlp lt 005 Doses are 0 for the Control and 5 10 25 and 50 gml fortreatment groups

MWNT-COOH is the difference in surface chemistrybetween the two samples In support of this notion ourprevious research found that functionalization of MWNTswith sodium sulfuric acid salt (ndashSO3Na or ndashphenyl-SO3Na) increased CNT biocompatibility in neuroblastomacells compared to unfunctionalized MWNTs or carboxylicacid (ndashCOOH) functionalized MWNTs13 Further severalother groups have shown similar trends for acid-purifiedMWNTs where MWNT toxicity was highly dependentupon surface chemistry (ndashCOOH ndashOH)17ndash19 Magrez et al(2006) found that the cell viabilities of human lung tumorcells (H596) were decreased after incubation with MWNTscontaining acidic surface functionalities (ndashCOOH ndashOH)compared to MWNTs18 Bottini et al (2006) examinedMWNTs (diameter= 20ndash40 nm length= 1ndash5 m puritygt95) and acid-purified MWNT-COOH at concentrationsof 40 gml and 400 gml from 24ndash120 h in T-cells thenassessed cell viability and proliferation with Trypan Bluedye staining There were no significant decreases in viabil-ity for either material after 24 h However after 48 h via-bility was significantly decreased for cells incubated with400 gml concentration of MWNT-COOH compared tothe later time point of 96 h for the MWNTs suggestingthat MWNT-COOH were more toxic than the MWNTs Atthe concentration of 40 gml of MWNT-COOH west-ern blotting with an antibody against phosphotyrosinedemonstrated no detrimental effects on receptor-inducedT-cell activation Thus 40 gml of MWNT-COOH didnot seem to have toxic effects on the function of T-cellssupporting the notion that this amount does not mea-surably harm the cells17 Furthermore there is a strongrelationship between surface chemistry and nanomaterial

dispersion in solution For example acid treatment leadsto the oxidation of the CNT surface and the generationof carboxyl (ndashCOOH) and hydroxyl (ndashOH) groups whichincreases their hydrophilicity and dispersion18 To elimi-nate the potential that the CNTs were interfereing with theMTS assay an acellular MTS assay was performed Theresults indicated that both MWNTs and MWNT-COOH donot interfere with the chemicals (data not shown)We previously demonstrated that CNTs have the abil-

ity to generate reactive oxygen species (ROS) before andafter introduction into certain mammalian cells28 For thisreason we investigated the ROS response of U937 cells inaddition to the acellular response to MWNTs and MWNT-COOH with the fluorescent DCFH-DA assay The validityof the assay was assessed with the positive control hydro-gen peroxide (H2O2 which showed a significant increasein ROS at a 2 nM concentration after 24 h (Fig 3)By comparison cells exposed to MWNTs at concentra-tions gt25 gml produced a significantly greater ROSresponse than the controls or cells exposed to MWNT-COOH which had uniform and low ROS values at all ofthe concentrations tested (5ndash50 gml) (Fig 3) To deter-mine whether the ROS was directly produced by the cellswe performed an acellular ROS assay to assess the abil-ity of the MWNTs or MWNT-COOH to generate ROSin the absence of cells Neither MWNTs nor MWNT-COOH produced inherent ROS after 24 h at the high50 gml concentration (data not shown) Therefore inthe absence of cells there was no significant production ofROS and the results demonstrate a purely cellular responseto the MWNTs and MWNT-COOH as we have previ-ously demonstrated in other cells lines28 These results also

Fig 3 Assessment of reactive oxygen species (ROS) generation after24 h showing significant increase after dosing with positive controlhydrogen peroxide at 2 mM and MWNTs at 25 and 50 gml The dataare represented as the average of triplicate experiments plusmn the standarddeviation Stars indicate significant difference from control p lt 005Doses are 0 for the Control and 5 10 25 and 50 gml for treatmentgroups

90 Nanosci Nanotechnol Lett 3 88ndash93 2011


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suggest that the metal catalyst content is directly propor-tional to the cellular ROS generation by the cells withMWNTs (8ndash11 wt Fe) displaying significant ROS lev-els at 25ndash50 gml compared to a low ROS responseby the cells exposed to the acid-purified MWNT-COOH(02ndash049 wt Fe)28

Indeed elemental impurities such as residual metalcatalysts (Fe Co Ni etc) from the CNT synthesisprocess have been implicated in the ROS and toxicresponse of CNTs1620ndash23 Impure SWNTs (amorphous car-bon lt5 wt Co 28 wt Cu 003 wt Fe 0009 wtMo 42 wt) induced ROS after interaction with A549cells thereby inhibiting cell growth2024 This ROS induc-tion was absent in metal-reduced CNT samples (HNO3

acid treatment reduced metal content from 8 wt to25 wt) at any time (10 min 24 h) or concentration(5 10 50 100 gml)20 However the actual amountof residual catalyst may be more important For exam-ple the present of metal catalyst impurities in MWNTs(impurities 424 wt of Fe purified 008 wt of Fe) didnot influence cytotoxicity (MTT assay LDH assay XTTassay) on human lung alveolar epithelial cells (A549)7 Inthis case the CNTs demonstrate good biocompatibility inmacrophages when highly purified8

In many of our previous studies the production of ROShas been directly linked to nanomaterial toxicity2526 How-ever in this study U937 cells maintained high viability(MTS assay) even after exposure to high concentrations(50 gml) of unpurified MWNTs while concurrently pro-ducing significant levels of ROS The presence of the Fecatalyst particles likely plays a role in the MWNT-inducedROS response as mentioned above Another possibleexplanation for the reduced viability in the cells incu-bated with the high dose (50 gml) of MWNT-COOHis that the macrophages are able to directly encounterand engulf a greater quantity of the acid-purified andhydrophilic MWNTs-COOH compared to the unpurifiedand hydrophobic MWNTs due to better dispersion ofMWNT-COOH in the cell culture media compared toMWNTs Support for this mechanism was readily apparent

Fig 4 Light microscope morphological and uptake assessment of cells exposed to 5 gml MWNTs or MWNT-COOH for 24 h (A) Control(B) 5 gml MWNTs or (C) 5 gml MWNT-COOH for 24 h Notice that the cells dosed with MWNT-COOH show greater uptake and more irregularmorphologies than the cells dosed with MWNTs Original images taken at 20times magnification Scale bars are 50 m

even at low doses (5 gml) from light microscope imagesof the cells and CNTs (Fig 4) After 24 h evidence foruptake of both MWNTs (Fig 4(B)) and MWNT-COOH(Fig 4(C)) and subsequent morphological alterations wereapparent However the cells dosed with MWNT-COOHdisplayed greater uptake and more irregular borders andenlarged morphologies than the cells dosed with MWNTsThis suggests that indeed better dispersion can lead tocellular alterations such as increased uptake and toxic-ity which may or may not be detectable by biochem-ical assays (ie MTS ROS) which rely on large cellnumbers and averages In support of our results a recentstudy by Magrez et al (2006) demonstrated with lightmicroscopy and cytopathological analysis that human lungtumor cells (H596) after 24 h of incubation with verylow doses (002 gml) of MWNTs lose their mutualattachments retract their cytoplasm and have smaller andmore condensed nuclei However these distinct morpho-logical changes would not have been noticed from the veryslight differences at this same time point and concentra-tion when measured with the MTT cell viability assayA different study demonstrated that grinding MWNTs canlead to increased dispersion and cell availability leadingto a higher cytotoxic and pro-inflammatory response inrat peritoneal macrophages suggesting that dispersion canhave a direct impact on cytotoxicity4 Therefore thoroughphysicochemical characterization (size morphology sur-face chemistry etc) of the materials prior to cell culturestudies in conjunction with examination of the macro-scopic properties of nanomaterials under cell culture con-ditions and at the level of individual cells provides acritical dimension for understanding the results27

3 SUMMARY AND CONCLUSIONS

Although MWNTs and MWNT-COOH can accumulatein human lung macrophage cells to different degreesthey do not produce overt cell toxicity under theseconditions (5ndash50 gml MWNT or MWNT-COOH2ndash24 h) However there are morphological alterations at



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Table II Summary of results

ObservationAssay Description MWNTs MWNT-COOH

Extent ofagglomera-tion

Lightmicroscopy

High Low

Residual metalcatalyst (Fe)Content

Materials char-acterization

High (11 wt) Low (19 wt)

Surfacechemistry

Degree ofoxidation

Low (07 wtO C andndashOH)

High (85 wtO C andndashOH)

Macrophageuptake

Lightmicroscopy

Yes Yes (greaterthan MWNTs)

MTS Cell viability24 h

No significance Significantdecrease at25 50 gml

ROS Reactiveoxygenspeciesgeneration24 h

Concentration-dependentincrease from25ndash50 gml

Low at all concen-trations from5ndash50 gml

low doses of MWNT-COOH and significant ROS pro-duction for MWNTs at high doses indicating a potentialalthough different cellular stress response for both mate-rials (Table II) Therefore before nanomaterials are fullyaccepted and integrated into biological systems they willcontinue to undergo further scrutiny at various stages oftheir processing (ie before and after purification) andwith models ranging from simple to complex (ie cellsvs whole animals) to gain a better understanding betweentheir physicochemical properties and bio-effects

4 MATERIAL AND METHODS

41 Carbon Nanotube Characterization

The commercially-available multi-walled carbon nanotubes(MWNTs) were synthesized by pyrolysis of propyleneusing iron-based catalyst were purchased from Tsinghuaand Nanfeng Chemical Group Cooperation China andwere previously used for DNA damage and nanotoxicitystudies162829 In order to remove the catalyst residues andgenerate carboxylic acid surface functional groups the as-received multiwall carbon nanotube (MWNT) was refluxedwith vigorous stirring in a sulfuric acid (H2SO4nitricacid (HNO3 mixture (31 VV) for 3 hrs at 100 CAfter cooling to room temperature the acidic solution waspoured into ice water The aqueous black suspension wasfiltered through a 045 m nylon membrane and washedrepeatedly with excess water Finally the purified MWNTswere dried under vacuum overnight Transmission electronmicroscopy was performed on a Hitachi H-7600 TEM at100 kV after spotting the MWNTs onto formvarcarboncoated grids The air thermogravimetric analysis was per-formed on a TGA TA500 X-ray photoelectron spectro-scopic (XPS) measurements were performed on a VG

Microtech ESCA 2000 using monochromatic Mg K radia-tion at a power of 300 W) Raman spectra were obtained onan inVia micro-Raman spectrometer (Renishaw) recordedwith a 514 nm laser for nanotubes MWNT stock solutionswere prepared at concentrations of 1 mgml in water thenbriefly sonicated for 2ndash3 minutes with intermittent rest forsim3 minutes and repeated 3ndash4 times

42 Cell Culture

Human alveolar macrophage cells (U937 ATCC) wereculture in T-75 flasks and incubated with 5 CO2 at37 C The U937 cells are a monocyte cell line that canbe stimulated to mature into macrophages which playthe major role in both non-specific and specific defensemechanisms in the body The U937 cells were maintainedin RPMI media supplemented with 10 Heat Inacti-vated FBS (Invitrogen) and 1 penicillin and streptomycin(Invitrogen) The cells were stimulated with 100 ngmlphorbol 12-myristate 13-acetate (PMA) for 48 hours toallow differentiation into human alveolar macrophage cellsbefore dosing

43 MTS Assay

The MTS assay (Promega) was used to investigate mito-chondrial function Yellow MTT (3-(45-dimethyliazol-2-yl)-(25-diphenyltetrazolium bromide) is reduced toaqueous insoluble purple formazan by a mitochondrialenzyme showing intact functional mitochondria Mito-chondrial function of the cells was spectrophotometricallyinvestigated after 24-h exposure to MWNTs and MWNTs-COOH Briefly the culture media was removed and freshmedia without FBS containing a 110 dilution (20 lMTS solution 200 l exposure medium) was added andincubated for 2 hours A spectrophotometer (BioTek Syn-ergy HT USA) was used to determine the absorbance at490 nm according to the manufacturerrsquos procedure Thedata are represented as the average of triplicate plusmn the stan-dard deviation

44 Light Microscopy

Cells were observed with light microscopy (Olympus phasecontrast microscope) to assess overall changes in morphol-ogy and MWNT uptake U937 cells were cultured in sixwell dishes at a density of 42times 104 cellml then stim-ulated with PMA (100 ngml) for 48 hours After wash-ing with 1xPBS (Invitrogen) the solution was changed toRPMI medium dosed with MWNT and MWNT-COOHand MWNTs at final concentrations of 5 gml

45 Reactive Oxygen Species (ROS) Generation

For the ROS assay-DCFH-DA U937 cells were seeded atdensity 150times103 ml in black 96 well plates U937 cells



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were stimulated with PMA at 100 ngml for 48 hoursthen the media was removed the cells were washed with1xPBS (Invitrogen) twice 200 l of 100 M DCFH-DA(Invitrogen) (in culture medium) was added to each welland incubated at 37 C and 5 CO2 culture conditions for30 minutes After the aspiration of the DCFH-DA 200 lof dosing solution (5 10 25 50 gml) was added to eachwell The positive control hydrogen peroxide (Fisher Sci-entific) was applied at a concentration of 2 nM The platewas covered with aluminum foil to block light and placedin an incubator for 24 hours then the intensity of the ROSprobe was measured on a spectrophotometer (BioTek Syn-ergy HT) with excitation at 48520 nm and absorbanceat 52820 nm according to the manufacturerrsquos procedureThe data are represented as the average of triplicate plusmn thestandard deviation

Acknowledgments Thanks to Dr Laura Braydich-Stolle Mr Jonathan Lin and Ms Christin Grabinski fortechnical assistance Lin Zhu received funding througha Dayton Area Graduate Studies Institute fellowship andORISE fellowship Amanda M Schrand received fund-ing from the National Research Council (NRC) Fellow-ship program funded by the Joint Science and TechnologyOffice for Chemical and Biological Defense (JSTO-CBD)a program administered by the Defense Threat ReductionAgency (DTRA)

References and Notes

1 L Dai (ed) Carbon Nanotechnology Recent Developments inChemistry Physics Materials Science and Device ApplicationsElsevier Amsterdam (2006)

2 B Warheit B R Laurence K L Reed D H Roach G A MReynolds and T R Webb Tox Sci 77 117 (2004)

3 C W Lam J T James R McCluskey and R L Hunter ToxicolLett 77 126 (2004)

4 J Muller F Huaux N Moreau P Misson J F Heilier M DelosM Arras A Fonseca J B Nagy and D Lison Toxicol Appl Phar-macol 207 221 (2005)

5 A Huczko and H Lange Fuller Nanotub Car N 9 247 (2001)6 A Huczko H Lange M Bystrzejewski P Baranowski H Grubek-

Jaworska P Nejman T Przybyowski K Czumiska J GlapiskiD R M Walton and H W Kroto Fuller Nanotub Car N 13 141(2005)

7 A Simon-Deckers B Gourget M Mayne-LrsquoHermite and N Herl-Carriegravere Toxicology 253 137 (2008)

8 S Fiorito A Serafino F Andreola and P Bernier Carbon 44 1100(2006)

9 K Sato Y Imai N Higashi Y Kumamota N Mukaida andT Irimura Int Immunol 17 559 (2005)

10 G Jia H Wang L Yan X Wang R Pei T Yan Y Zhao andX Guo Environ Sci Technol 39 1378 (2005)

11 L Murr K Garza K Soto A Carrasco T Powell and D RamirezJ Environ Res Public Health 2 31 (2005)

12 A J Wagner C A Bleckmann R C Murdock A M Schrand J JSchlager and S M Hussain J Phys Chem B 111 7353 (2007)

13 A M Schrand J Johnson L Dai S M Hussain J J SchlagerL Zhu Y Hong and E Osawa Cytotoxicity and Genotoxicityof Carbon Nanomaterials in Safety of Nanoparticles From Manu-facturing to Clinical Applications edited by T Webster SpringerScience+Business Media LLC New York (2008) pp 159ndash187

14 A M Schrand J J Schlager L Dai and S M Hussain Nat Prot5 744 (2010)

15 S K Smart A I Cassady G Q Lu and D J Martin Carbon44 1034 (2006)

16 A M Schrand K Szczublewski J J Schlager L Dai and S MHussain Int J Neuroprot Neuroregen 3 115 (2007b)

17 M Bottini S Bruckner K Nika N Bottini S BellucciA Magrini A Bergamaschi and T Mustelin Toxicol Lett 160 121(2006)

18 A Magrez S Kasas V Salicio N Pasquier J W Seo M CelioS Catsicas B Schwaller and L Forro Nano Lett 6 1121(2006)

19 F Tian D Cui H Schwarz G G Estrada and H Kobayashi Tox-icol In Vitro 20 1202 (2006)

20 K Pulskamp J M Woumlrle-Knirsch K Kern and H F Krug Carbon45 2241 (2007a)

21 K Pulskamp S Diabateacute and H F Krug Toxicol Lett 168 58(2007b)

22 S Garibaldi C Brunelli V Bavastrello G Ghigliotti andC Nicolini Nanotechnology 17 391 (2006)

23 M Y Kagan A V Klaptsov I V Brodsky R CombescotX Leyronas A V Andriyash P A Loboda V A Lykov V YPolitov M N Chizhkov A K Murtazaev and V A CherepeninJ Phys-Usp 49 1079 (2006)

24 E Herzog A Casey F M Lyng G Chambers H J Byrne andM Davoren Toxicol Lett 174 49 (2007)

25 A M Schrand L K Braydich-Stolle J J Schlager L Dai andS M Hussain Nanotechnology 19 104 (2008)

26 C Carlson S M Hussain A M Schrand L K Braydich-StolleK L Hess R L Jones and J J Schlager J Phys Chem B112 13608 (2008)

27 S M Hussain L K Braydich-Stolle A M Schrand R CMurdock K O Yu D M Mattie J J Schlager and M TerronesAdv Mater 21 1 (2009)

28 A M Schranda L Dai J J Schlager S M Hussain and E OsawaDiam Relat Mater 16 2118 (2007a)

29 L Zhu D W Chang L Dai and Y Hong Nano Lett 7 3592(2007)

30 M T Kleinman C Sioutas M C Chang A J F Boere and F RCassee Toxicol Lett 137 151 (2003)

31 L Dai (ed) Intelligent Macromolecules for Smart DevicesSpringer-Verlag London (2004)

Received 13 May 2010 Accepted 4 August 2010



IP 12922124186Wed 09 Nov 2011 225439


immunological defense against inhaled particles and serveas a good model to investigate how inhaled particlescan adversely affect cell function and lead to degrada-tion of health30 In this study we chose the human lungmacrophage cell line (U937) as a model to investigatethe potential cytotoxicity of unpurified multi-walled car-bon nanotubes (MWNTs) and acid purified multi-walledcarbon nanotubes (MWNT-COOH) by detecting the gen-eration of reactive oxygen species (ROS) morphologicalchanges and cell uptake

2 RESULTS AND DISCUSSION

The unpurified MWNTs and acid-purified MWNT-COOHwere characterized with transmission electron microscopy(TEM) air thermogravimetric analysis (TGA) Ramanspectroscopy and X-ray photoelectron spectroscopy (XPS)There was visual evidence for a reduction in nanotubelength after the acid purification and sonication treat-ment compared to the original unpurified sample (data notshown) However once the MWNTs were introduced intoaqueous solutions the nanotubes behaved in an agglom-erated fashion We previously demonstrated that largeMWNT agglomerates (microns in size) were present in1 mgml stock solutions which were only briefly soni-cated for 10 seconds16 In this study immediately afterdosing cells in culture with a low 5 gml concentra-tion of MWNTs or MWNT-COOH there was visual evi-dence for large agglomerated bundles of MWNTs withsizes gt50 microns (data not shown) in comparison to bet-ter dispersion of MWNT-COOH However differences inMWNT length (35 microns vs 12 microns measured withTEM) due to short or extended sonication times have notbeen shown to influence subsequent cytotoxicity7 There-fore in addition to visualization of primary MWNT struc-ture and overall dispersion metal catalyst impurity contentand surface chemistry were considered primary character-istics for each MWNT sample in this studyThe TGA data for the unpurified MWNTs (Fig 1)

revealed sim11 wt orange-colored residue after beingheated above 730 C most likely due to the presence ofFe catalyst residues that became converted to Fe2O3 uponthermal oxidation The MWNT-COOH sample showeda reduced Fe catalyst residue content of sim19 wtabove 730 C which indicates that the acid treatmentremoved most of the Fe catalyst residue These resultsfor impurity content were similar to our previous stud-ies using the same MWNTs which displayed 8 wtFe for unpurified MWNTs compared to 049 wt foracid-purified MWNTs16 when analyzed with inductively-coupled plasma-optical emission spectroscopy (ICP-OES)The Raman spectra of the MWNT sample before and

after acid purification showed an increase in the disor-der (D) band (1350 cmminus1 for the MWNT-COOH sam-ple suggesting that some surface damage due to oxidation

Fig 1 Characterization of MWNTs and MWNT-COOH with TGAshowing removal of Fe catalyst residues after acid purification

occurred (data not shown) X-ray photoelectron spectro-scopic (XPS) measurements performed on MWNT-COOHshow increased oxygen content after oxidation of the unpu-rified MWNTs through increased OmdashC bonds from 05 to8 wt and O C bonds from 07 to 85 wt (Table I)Therefore the physicochemical properties of the MWNTsample are distinctly different from the MWNT-COOHsample In this regard it is critical to maintain a carefulbalance between the surface chemical functionalization ofCNTs and their structural integrity in order to retain theirnovel propertiesIn order to assess if there is a differential response of

human lung macrophages to unpurified MWNTs comparedto acid purified MWNT-COOH changes in cytotoxicitymorphology and reactive oxygen species (ROS) generationwere investigated Changes in viabilities related to cytotox-icity were spectrophotometrically assayed with the MTSassay which is sensitive to mitochondrial function Thepositive control CdO was used to verify the validity of theMTS assay and shows a strong and significant decreasein cell viability at the low dose of 5 gml (Fig 2) Bycomparison cells exposed to MWNTs or MWNT-COOHat concentrations ranging from 5ndash50 gml for 24 h onlydisplayed a significant decrease in viability at the highestconcentration of 50 gml for MWNT-COOH comparedto no significant decreases in viability at the other con-centrations (5ndash25 gml) or at any of the concentrations(5ndash50 gml) for the unpurified MWNTs (Fig 2)One explanation for the decreased viability of the

cells after exposure to the 50 gml concentration of

Table I XPS results for oxygen (O) and iron (Fe) content (wt) ofMWNT and MWNT-COOH

O

Sample H2O OndashC O C ndashOH OndashFe Fe

MWNT mdash 05 07 02 02MWNT-COOH 08 80 85 mdash lt01



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Lightmicroscopy

High Low




Surfacechemistry

Degree ofoxidation



Macrophageuptake

Lightmicroscopy












42 Cell Culture


43 MTS Assay


44 Light Microscopy






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IP 12922124186Wed 09 Nov 2011 225439









IP 12922124186Wed 09 Nov 2011 225439












IP 12922124186Wed 09 Nov 2011 225439





Lightmicroscopy

High Low




Surfacechemistry

Degree ofoxidation



Macrophageuptake

Lightmicroscopy












42 Cell Culture


43 MTS Assay


44 Light Microscopy






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IP 12922124186Wed 09 Nov 2011 225439












IP 12922124186Wed 09 Nov 2011 225439





Lightmicroscopy

High Low




Surfacechemistry

Degree ofoxidation



Macrophageuptake

Lightmicroscopy












42 Cell Culture


43 MTS Assay


44 Light Microscopy






IP 12922124186Wed 09 Nov 2011 225439







































IP 12922124186Wed 09 Nov 2011 225439





Lightmicroscopy

High Low




Surfacechemistry

Degree ofoxidation



Macrophageuptake

Lightmicroscopy












42 Cell Culture


43 MTS Assay


44 Light Microscopy






IP 12922124186Wed 09 Nov 2011 225439







































IP 12922124186Wed 09 Nov 2011 225439






































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