Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro

Gasser Michael, Wick Peter, Clift, Blank Fabian, Diener Liliane, Yan Bing, Gehr Peter, Krug, Rothen-Rutishauser, Rothen-Rutishauser Barbara

Description

Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (−NH 2 ) and carboxyl (−COOH) surface modifications. Results Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. Conclusions The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies.

Sujets

• Macrophage
• Epithelium
• Dendritic cell

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 3
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Gasser
etal.ParticleandFibreToxicology
2012,
9
:17
http://www.particleandfibretoxicology.com/content/9/1/17

RESEARCH

OpenAccess

Pulmonarysurfactantcoatingofmulti-walled
carbonnanotubes(MWCNTs)influencestheir
oxidativeandpro-inflammatorypotentialinvitro
MichaelGasser
1,2
,PeterWick
2
,MartinJDClift
1
,FabianBlank
3
,LilianeDiener
2
,BingYan
4,5
,PeterGehr
6
,
HaraldFKrug
2
andBarbaraRothen-Rutishauser
1,3*

Abstract
Background:
Increasingconcernhasbeenexpressedregardingthepotentialadversehealtheffectsthatmaybe
associatedwithhumanexposuretoinhaledmulti-walledcarbonnanotubes(MWCNTs).Thusitisimperativethatan
understandingastotheunderlyingmechanismsandtheidentificationofthekeyfactorsinvolvedinadverseeffects
aregained.Inthealveoli,MWCNTsfirstinteractwiththepulmonarysurfactant.Atthisinterface,proteinsandlipids
ofthepulmonarysurfactantbindtoMWCNTs,affectingtheirsurfacecharacteristics.Aimofthepresentstudywas
toinvestigateifthepre-coatingofMWCNTswithpulmonarysurfactanthasaninfluenceonpotentialadverse
effects,uponboth(i)humanmonocytederivedmacrophages(MDM)monocultures,and(ii)asophisticatedinvitro
modelofthehumanepithelialairwaybarrier.BothinvitrosystemswereexposedtoMWCNTseitherpre-coated
withaporcinepulmonarysurfactant(Curosurf)ornot.TheeffectofMWCNTssurfacechargewasalsoinvestigated
intermsofamino(
−
NH
2
)andcarboxyl(
−
COOH)surfacemodifications.
Results:
Pre-coatingofMWCNTswithCurosurfaffectstheiroxidativepotentialbyincreasingthereactiveoxygen
specieslevelsanddecreasingintracellularglutathionedepletioninMDMaswellasdecreasesthereleaseofTumour
necrosisfactoralpha(TNF-
α
).Inaddition,aninductionofapoptosiswasobservedafterexposuretoCurosurf
pre-coatedMWCNTs.Intriplecell-coculturesthereleaseofInterleukin-8(IL-8)wasincreasedafterexposureto
Curosurfpre-coatedMWCNTs.EffectsoftheMWCNTsfunctionalizationswereminorinbothMDMandtriplecell
co-cultures.
Conclusions:
Thepresentstudyclearlyindicatesthatthepre-coatingofMWCNTswithpulmonarysurfactantmore
thanthefunctionalizationofthetubesisakeyfactorindeterminingtheirabilitytocauseoxidativestress,
cytokine/chemokinereleaseandapoptosis.Thusthecoatingofnano-objectswithpulmonarysurfactantshouldbe
consideredforfuturelunginvitroriskassessmentstudies.
Keywords:
Multi-walledcarbonnanotubes(MWCNTs),Pulmonarysurfactant(Curosurf),Macrophages,
Epithelialcells,Dendriticcells,Triplecellco-culture,Pro-inflammatoryandoxidativereactions

*Correspondence:[e-mail]
1
AdolpheMerkleInstitute,UniversityofFribourg,Marly,Switzerland
3
RespiratoryMedicine,DepartmentofClinicalResearch,InselspitalUniversity
Hospital,UniversityofBern,Bern,Switzerland
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Gasseretal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.

Gasser
etal.ParticleandFibreToxicology
2012,
9
:17
http://www.particleandfibretoxicology.com/content/9/1/17

Background
Theever-developingindustryofnanotechnologythelast
twodecadeshasculminatedinaplethoraofnewnano-
objects(definedasmaterialwithone,twoorthreeexter-
naldimensionsinthenanoscale)whicharebeingused
withinavarietyofconsumerandindustrialapplications.
Amongthemostprominentnano-objectsarecarbon
nanotubes(CNTs);hollownanofibresformedfromcarbon
[1].BasedontheirstructureCNTsareclassifiedaseither,
single-wallcarbonnanotubes(SWCNTs),whichcomprise
asinglelayerofcarbonatoms,ormulti-wallcarbonnano-
tubes(MWCNTs),comprisingofmultipleconcentric
tubes[2].Structuralandmechanicalcharacteristicssuch
asanextremestrength,stiffnessandrobustness[3]make
CNTsinterestingfortheuseinaninfinitenumberof
applicationssuchassportinggoods,automobileproducts
orhouseholditems.Additionally,CNTsholdgreatprom-
iseforapplicationwithinmedicine,particularlyasatool
intherapeuticsanddiagnostics[4].Withtheirincreasing
numberofapplications,CNTemissionsintotheenviron-
mentandhumanexposuremayincrease.MainlyCNT
production,processinganddisposalmaybehazardousfor
humans[5].MoreoverduringtheuseofCNTcontaining
products,CNTsmaybereleasedintotheenvironmentas
forinstancefromabrasionordegradationofCNTcon-
tainingproducts.PossibleportalsbywhichCNTsmay
enterthehumanbodyincludetheskin,thegastro-
intestinaltractandinjection(nanomedicine).However,it
iswellacceptedfrompreviousresearchusingnano-sized
particles[6]andCNTs[7,8]thatinhalationistheprimary
exposureroutetothehumanbodyifCNTsarereleased
intotheenvironmentalair.
ConcernsaboutthesafetyofCNTshavebeenraisedfora
numberofdifferentreasons[3,9,10](i)duetotheirsmall
aerodynamicdiameterCNTsarehypothesisedtoreachthe
lowerrespiratorytract,(ii)CNTspossess,likeothernano-
objects,ahighsurfacetomassratio,thusalargesurface
caninteractwiththebiologicalsurroundings,and(iii)some
CNTswhicharefibreshapedmay(ifstructureddimensions
aresimilar)behavelikeasbestos,orotherpathogenicfibres
whicharetoxicduetotheirneedle-likeshape.Moreover
(iv),numerousinvivostudies(e.g.)[11-13]haveshowndif-
ferenttypesofMWCNTstoremaininthelungforupto
severalmonthsafterdepositionindicatingthepotentialfor
prolongedbiopersistence.
RecentlythepotentialadverseeffectsofCNTshave
beenstudiedonvariousbiologicalsystems,usingdiffer-
entexposuremethodsbothinvivoandinvitro[14,15].
Despitetheunrealisticallyhighdoseswhichhavebeen
usedwithinsomeofthepreviousstudies(e.g.)[16],itis
knownthatsubpleuralfibrosis[17],granulomaforma-
tion[10]andmesothelioma[16]similartotheeffectsof
crocidoliteasbestosfibres,canappearafterinvivoexpo-
surestomainlystraight,stiffandextremelylongCNTs.

Page2of13

Observedadverseeffectshavefurtherbeenexplained
bytheoxidativestressparadigm[18].Innumerousstud-
ies(e.g.)[19-21]anincreasedoxidativestressresponse
invivoandinvitrohasbeenreportedcausingasubse-
quent(pro-)inflammatoryreactionafterexposuresto
bothstraightandtangledCNTs.
Althoughnumerousstudiesaddresstheadversepoten-
tialofCNTs,theircomparabilityisoftenlimitedand
resultsarecontradictory.Explanationsforthesediscrep-
anciesincludedifferencesinadministereddose,the
physico-chemicalcharacteristics(e.g.agglomeration/ag-
gregationstate,metalimpurities,stiffness,length)ofthe
CNTsstudied,theexposuremethodofCNTs,ordiffer-
encesinthebiologicalsystememployed[15,22,23].Thus
conditions/characteristicshavetobemanipulatedsys-
tematicallyinordertoidentifykeyfactorsfortheirpo-
tential(adverse)biologicaleffects.Apromisingwayto
modifythepropertiesofCNTsisthefunctionalization
ofthesurface[24,25].FunctionalizationofCNTscanbe
usedtopromotethebindingofspecificbiomolecules
(suchassiRNA)[26]butalsotoimprovetheirbiocom-
patibility.TheadverseeffectpotentialofCNTscanbe
significantlydrivenbytheparticular(surface)modifica-
tionemployed[22]whereasstudieshaveshownboth,
increasesanddecreasesintoxicityafterexposuresto
CNTswithdifferentsurfacefunctionalizations[27,28].
FunctionalizationcanfurtheraffecttheCNTsdispersity
whichcanhavesubsequentconsequencesontheircell
uptakeandagglomerationintissue[29].
Itisnotonlyartificialsurfacemodificationsthatplaya
roleinregardstothepotentialadverseeffectsofCNTs.
ThesurfacecharacteristicsofMWCNTsmayalsobe
modifiedbytheadsorptionofbiomoleculesfollowingin-
halation,andthesubsequentinteractionwiththelung.
Specifically,aninitialcoatingoftheMWCNTswilltake
placewhentheyinteractwithpulmonarysurfactant
whichismainlyproducedbyepithelialtypeIIcellsand
whichislocatedattheair-liquidinterface.Surfactant
consists85-90%ofphospholipids[30],thespecificsur-
factantproteins(SP)-A,-B,-C,and-D(~10%)andits
mainfunctionisthereductionofthealveolarsurface
tensionandkeepingthegasexchangesurfaceatoptimal
sizeduringthemovementsofbreathing[31].Thus,dur-
ingdeposition,surfactantorsurfactantcomponentswill
bindtothesurfaceofMWCNTs[32,33].Previously,this
initialcoatinghasnotbeensufficientlyconsideredinre-
specttoinvitrolungtoxicitystudies.Themodulationof
theadversepotentialfromsurfactantbindingismainly
describedformicroparticles[34],howevertothebestof
ourknowledge,notforCNTsandothernano-objects.
AfterinhaledCNTsarecoatedwithpulmonarysurfac-
tant,theymaybedisplacedintotheaqueoushypophase
[35-37]andcomeincontactwithcellsoftheimmune
systemsuchasmacrophagesanddendriticcells,which

Gasser
etal.ParticleandFibreToxicology
2012,
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mayengulftheMWCNTsandclearthemfromthisarea
ofthelung[38].AlsoepithelialtypeIcellscaninteract
withCNTs,asthesecellsmainlycoverthealveolarsur-
face[31].
Objectivesofthestudyandmethodologicalapproach
Theprimaryaimofthisstudytherefore,wastoinvesti-
gatehowapre-coatingofMWCNTswithpulmonary
surfactantmayaffecttheirpotentialadverseeffectson
cellsoftheairbloodtissuebarrierinvitro.Inorderto
simulatethepulmonarysurfactantcoating,MWCNTs
werepre-coatedwithCurosurf,awellcharacterizednat-
uralporcinesurfactantpreparation[39-41].
Monocytederivedmacrophage(MDM)monocultures
aswellasasophisticated3Dinvitrotriplecel