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Assessment of an in vitro model of pulmonary barrier to
study the translocation of nanoparticles
Samir Dekali, Christelle Gamez, Thierry Kortulewski, Kelly Blazy, Patrice
Rat, Ghislaine Lacroix
To cite this version:
Samir Dekali, Christelle Gamez, Thierry Kortulewski, Kelly Blazy, Patrice Rat, et al.. Assessment
of an in vitro model of pulmonary barrier to study the translocation of nanoparticles. Toxicology
Reports, Elsevier, 2014, 1, pp.157-171. �10.1016/j.toxrep.2014.03.003�. �ineris-01855550�
ContentslistsavailableatScienceDirect
Toxicology
Reports
j o u r n al ho me p ag e : w w w . e l s e v i e r . c o m / l o c a t e / t o x r e p
Assessment
of
an
in
vitro
model
of
pulmonary
barrier
to
study
the
translocation
of
nanoparticles
Samir
Dekali
a,b,
Christelle
Gamez
a,
Thierry
Kortulewski
c,
Kelly
Blazy
a,
Patrice
Rat
b,1,
Ghislaine
Lacroix
a,∗,1aINERIS(InstitutNationaldel’EnvironnementindustrieletdesRISques),UnitédeToxicologieexpérimentale,
60550Verneuil-en-Halatte,France
bLaboratoiredechimieettoxicologieanalytiqueetcellulaire(C-TAC),FacultédesSciencesPharmaceutiquesetBiologiques,
UniversitéParisDescartes(PRESSorbonneParisCité),75270ParisCedex06,France
cCEA,DSV,iRCM,Plateformeimageriephotonique,92260Fontenay-aux-Roses,France
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received13January2014
Receivedinrevisedform10March2014 Accepted10March2014
Availableonline12May2014
Keywords: Alveolo-capillarybarrier Nanoparticles Polystyrene Calu-3 THP-1 HPMEC-ST1.6R
a
b
s
t
r
a
c
t
Asthelungisoneofthemainroutesofexposuretomanufacturednanoparticles,we devel-opedaninvitromodelresemblingthealveolo-capillarybarrierforthestudyofnanoparticle translocation.
In order toprovide arelevantand ethical invitro model, costeffective and easy-to-implementhumancelllineswereused.Pulmonaryepithelialcells(Calu-3cellline) and macrophages(THP-1 differentiatedcells) were cultivatedonthe apical sideand pulmonary endothelial cells (HPMEC-ST1.6R cell line) on the basal side of a micro-porouspolyestermembrane(Transwell®).Translocationofnon-functionalized(51and 110nm)andaminated(52nm)fluorescentpolystyrene(PS)nanobeadswasstudiedinthis system.
TheuseofCalu-3cellsallowedhightransepithelialelectricalresistance(TEER)values (>1000cm2)inco-cultureswithorwithoutmacrophages.After24hofexposureto
non-cytotoxicconcentrationsofnon-functionalizedPSnanobeads,therelativeTEERvalues (%/t0)weresignificantlydecreasedinco-cultures.Epithelialcellsandmacrophageswere
abletointernalizePSnanobeads.Regardingtranslocation,Transwell®membranesperse limitthepassageofnanoparticlesbetweenapicalandbasalside.However,small non-functionalizedPSnanobeads(51nm)wereabletotranslocateastheyweredetectedinthe basalsideofco-cultures.Altogether,theseresultsshowthatthisco-culturemodelpresent goodbarrierpropertiesallowingthestudyofnanoparticletranslocationbutresearcheffort needtobedonetoimprovetheneutralityoftheporousmembranedelimitatingapicaland basalsidesofthemodel.
©2014TheAuthors.PublishedbyElsevierIrelandLtd.Thisisanopenaccessarticleunder theCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).
∗ Correspondingauthorat:INERIS,ParctechnologiqueALATA,BP2, 60550,Verneuil-en-Halatte,France.Tel.:+33344556315;
fax:+33344556605.
E-mailaddress:ghislaine.lacroix@ineris.fr(G.Lacroix).
1 Theseauthorscontributedequallytothesupervisionofthestudy.
1. Introduction
Withtheirnew properties,nanoparticles(NPs) open abroadfieldofnovelopportunitiesforindustrials. How-ever,consumersorworkerscaneasilycomeintocontact with these NPs and their effects on human health are stillpoorlyunderstood[1].Inhalationisoneofthemajor routes of exposure to NPs present in ambient air and,
http://dx.doi.org/10.1016/j.toxrep.2014.03.003
2214-7500/©2014TheAuthors.PublishedbyElsevierIrelandLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/3.0/).
dependingon theirunique physico-chemicalproperties (size,shape,etc.),NPscanreachthealveolarregionofthe lung[2].Consequently,NPscouldtranslocatethroughthe alveolo-capillarybarrierintothebloodstream,and poten-tiallyreachanddamageotherorgans[3,4].
Thealveolo-capillarybarrieris vitalfortheorganism asitallowsgasexchangesinthecourseofbreathing.This barrierconsistsofallelementsbetweenalveoliandblood micro-vesselsincluding:surfactant,alveolarmacrophages, pneumocytes,abasallaminaandtheendotheliumofblood micro-vessels[5].Twotypesofpneumocytesconstitutethe alveolarepithelium.TypeIpneumocytesarelargeflattened cells covering roughly 90% of the alveolarsurface area, playingthemainroleinthehematosisprocess[6].TypeII pneumocytes(7%ofthealveolarsurfacearea)arecuboidal cellsinterspersedbetweentype Ipneumocytes, synthe-sizingthealveolarsurfactantandactingasprogenitorsof typeIpneumocytes[7,8].Thealveolarepitheliumishighly impermeable,essentiallyduetothepresenceof intercel-lulartightjunctionsbetweenepithelialcellsand,although lesssignificantly,tothedecreaseofsurfacetensionby sur-factantbetweentheairspaceandthealveolarliquid[9]. Alveolarmacrophagesarenormallyquiescent(weak pro-inflammatorysecretionandphagocytosisability)inorder topreventdamagingthealveoli[10].Thealveolar endothe-lium is continuous,non-fenestrated, and constitutedby micro-vascularendothelialcells[11].
As these cell types interact to form a functional alveolo-capillarybarrier,severalinvitroco-culturemodels usingdifferentcellularassociationshavebeendeveloped recentlytostudythecytotoxiceffectsofNPsclosetothe invivosituation[12–15].Rothen-Rutishauseretal.used theA549alveolarcellline(originatingfromhumanlung carcinoma),inassociationwithprimaryhumandendritic cellsandmacrophagesinTranswell®membranes,inorder tostudythelocal effects ofNPson theepitheliumand alveolarmacrophages,ontheonehand,andtheuptakeby dendriticcells(systemiceffects),ontheother[16,17]. How-ever,A549epithelialcells,whicharethemostwidelyused andcharacterizedalveolarepithelialmodelinliterature, arenot able toform a strongbarrier, and are inappro-priate for NP translocation studies [12,18]. NCI-H441 cells(originatingfromhumanlungpapillary adenocarci-noma)have bronchiolar morphology and develop more elevatedTEERvalues after incubation with dexametha-sone when co-cultured with endothelial cells [19,20]. Hermannset al.usedNCI-H441 cells in co-culturewith ISO-HAS-1 endothelial cells on Transwell® membranes to study the interactions with polyethyleneimine NPs
[21].However,Farcaletal.,byaddingPMA-differentiated THP-1macrophagesontheapicalsideofthesamemodel, showedadrasticdecreaseofthetransepithelialelectrical resistance (TEER) values in co-culture associated with a highrelease of TNF-␣ and IL-8 afterassociation with differentiatedTHP-1macrophages[4].
To thebest of our knowledge, noco-culture model, includingmacrophages, epithelial and endothelial cells, showing acceptable barrier properties have been pre-viously published. The objective of this work was to developaninvitroco-culturemodelwithbarrier proper-tiesmimickingtheinvivosituation,andusabletostudy
NPtranslocation.ThebronchialCalu-3epithelialcellline (originatingfromahumanlungadenocarcinoma)was cho-sendueitselevatedTEERvalues(600–1000cm2)when
cultivated on Transwell® membranes [22]. These cells wereco-cultivatedwithTHP-1differentiatedmacrophages on the apical side of a Transwell®, and with micro-vascular endothelial cells (HPMEC-ST1.6R) on the basal side.HPMEC-ST1.6R cells were selectedbecause oftheir micro-vascularmorphology,theirpulmonaryorigin,and duetotheconstitutiveorinducibleexpressionofspecific endothelialphenotypicmarkers(e.g.CD31,vonWillebrand factor,etc.)[23].ToassesstheeffectsofNPsizeand sur-facechemistry, non-functionalized(51and110nm)and aminated(52nm)fluorescentpolystyrene(PS)nanobeads weretestedonco-cultures.
2. Materialsandmethods
2.1. Nanoparticles
Non-functionalized (PS-NF, primary sizes of 51 and 110nm)and aminated (PS-NH2,primary sizeof 52nm)
orange fluorescent polystyrene nanobeads (Magsphere Incorporated) were used. All NPs were suspended at 1mg/mLinRPMI1640culturemediawithoutphenolred (Invitrogen)supplementedwith5%(v/v)heat-inactivated fetal calf serum (FCS) (Sigma–Aldrich) and 1% (v/v) penicillin/streptomycin called“completemedium” (Invi-trogen). PS-NH2 (52nm) nanobeads were vortexed in
the culture media just before use. Non-functionalized nanobeads were indirectly sonicated 2min (20s pause every20s)at30Wwithacuphorn(SonicatorS-4000, Mis-onix Incorporated)at room temperature. Then, particle sizedistributionandzetapotentialweremeasuredusinga Zetasizer®NanoZS(Malvern).Suspensionsand morphol-ogyof NPswerealsocharacterizedwitha transmission electronmicroscope(TEM)(STEMCM12,lab6,electrongun 120kV,Philips).
2.2. Cellculture
A549(ATCCnumber:CCL-185TM),Calu-3(ATCC
num-ber: HTB-55TM), NCI-H441 (ATCC number:HTB-174TM),
andTHP-1 (ATCC number: TIB-202TM) cell lines were
obtainedfromLGCStandards.Theywereroutinelygrown in RPMI 1640 medium supplemented with 10%(v/v) heat-inactivatedFCS (Sigma–Aldrich)and1% (v/v) peni-cillin/streptomycin(Invitrogen).MonocyticTHP-1cellsare non-adherentandroutinelymaintainedbetween105and
106cells/mLinorder to avoid anycellular stress.Before
theiruseinourstudy,theyweredifferentiated(not acti-vated)inadherentmacrophagesbyincubationwith50nM ofPMA(Phorbol12-myristate13-acetate)(Sigma–Aldrich, Ref.P1585)during24haspreviouslydescribed[24].The InstituteofPathologyofMainzinGermanykindlyprovided HPMEC-ST1.6R endothelial cells. According to Krump-Konvalikovaetal.[23],thesecellswereroutinelycultured ontissuecultureplasticwarepre-coatedwithgelatin(BD Biosciences) in M199 medium (Sigma–Aldrich) supple-mented with 20% (v/v) heat-inactivated FCS, 50g/mL endothelial cell growth supplements (Sigma–Aldrich),
25g/mL sodiumheparin (Sigma–Aldrich)and 1% (v/v) penicillin/streptomycin(Invitrogen).
2.3. Co-cultures
Transwell® membranes (polyester, 12mm diameter, 0.4m pores) (Corning Incorporated) were inverted in a Petri dish and coated by applying 250L/Transwell® ofa 50g/mLtype IVcollagensolution(Sigma–Aldrich) in sterile water on the under surface for 2h at 37◦C. Afterremoving thecollagensolution in excessby aspi-ration, the under surface was coated by the addition of100L/Transwell® ofa50g/mLfibronectinsolution (Sigma–Aldrich)inHBSS(withoutCaCl2andMgCl2)
(Invi-trogen) for 1hat roomtemperature. Excessfibronectin solution was then removed by aspiration,and HPMEC-ST1.6Rendothelialcells(2.5×104cells/cm2)wereseeded
inavolumeof250Landallowedtoattachfor2hat37◦C, 5%CO2and95%humidity.Thenthemediumwasremoved
byaspiration,andtheTranswell®membraneswererighted andplacedintothemedium-containingwellsofa12-well plate.Calu-3epithelialcells(5×104cells/cm2)werethen
seeded in theapicalside. The daythebi-cultures were seeded(withCalu-3andHPMEC-ST1.6Rcells)was consid-eredasday0.Bi-cultureswerethencultivatedinCalu-3 cell growthmedium.THP-1monocyteswere separately differentiatedatday6post-seedingofthebi-culturesin macrophage-likeadherentcellsaccordingtotheprotocol describedintheprevioussection.Atday7post-seeding, when confluent bi-cultures reached sufficient transepi-thelialelectricalresistance(TEER)values(≥1000cm2),
THP-1 macrophages were deposited on the apical side at a concentrationof2×104cells/mL. The mediumwas
changedinbothsideseverytwodays.Allco-cultureswere grownat37◦C,5%CO2and95%humidity.
2.4. TEERmeasurements
TEERmeasurementswereperformedoncellmono-and co-culturesusinganEVOMVoltohmmeter(World Preci-sionInstrument)equippedwithanEndohm®12chamber asdescribedby[25].TEERwasmeasuredfromday2today 11post-seeding.TheEndohm®12chamberwaspre-filled withpre-warmed(37◦C)RPMI1640mediumcontaining 10%(v/v)FCSand1%(v/v)penicillin/streptomycin.Then, Transwell®membraneswereplacedintothechamberand resistancemeasuredusingtheEVOMvoltohmmeter.Blank controls, consisting of coatedfilter membraneswithout
cellsweremeasured.TEERwascalculatedwiththe follow-ingformula:
TEER=(RM−RB)×A
whereRMistheexperimentalvalueofcellmono-or
co-cultures,RB theexperimentalvalueoftheblankcontrol
andAthesurfaceareaofthefiltermembrane(1.12cm2).
TEERwasexpressedincm2.
2.5. Immunofluorescence
Inimmunofluorescenceassays,cellswererinsedin pre-warmed(37◦C)HBSS(with CaCl2 and MgCl2)and fixed
for15minatroomtemperaturein4%(v/v) paraformalde-hyde(Sigma–Aldrich).Fixedcellswerepermeabilizedfor 3minwith0.5%(v/v)TritonX100(AcrosOrganics)atroom temperatureandthenincubatedfor30mininHBSS(with CaCl2andMgCl2)containing2%BSA(Sigma–Aldrich)
fol-lowedbya1hincubationtimewithrespectivelyprimary andsecondaryantibodies.Allantibodiesspecificationsand dilutionsaresummarized inTable1. Theywerediluted with2%BSA inHBSScontaining CaCl2 and MgCl2.
Pho-tomicrographswereacquiredusingaZeissAxioImager.Z1 fluorescencemicroscopewithanApoTome(CarlZeiss). 2.6. Permeabilitymeasurements
Permeability measurements were done in the api-caltobasolateraldirectioninTranswell® membranesas describedbyLemieuxetal.[26].Briefly, theTranswell® membranes were pre-incubated with a pre-warmed (37◦C) HBSS “transport buffer” (with CaCl2 and MgCl2,
pH 7.4) (Invitrogen) on both the apical and the baso-lateral sides. Lucifer Yellow CH dipotassium salt (LY) (Sigma–Aldrich)(0.5kDa) wasthendiluted in theHBSS and added to the apical side at a final concentration of 100g/mL. After 1h, aliquots from the basolateral side were collected in a black 96-well micro-plate for determination of the fluorescence leakage of the LY with a cyto-fluorometer adapted to the micro-plate (excitation=485nm, emission=530nm) (TECAN Infinite
M2000).Theapparentpermeabilitycoefficient(Papp,unit:
cms−1)wascalculatedasfollows: Papp=dQ
dt × 1 AC0
wheredQ/dtistheamountofcompoundtransportedper timepoint (mols−1), Ais thesurfaceareaofthefilter
Table1
Summaryofprimaryandsecondaryantibodiesusedinthisstudyforimmunofluorescenceapplication.
Primaryantibodies Secondaryantibodies Human epitopes Species Supplier (reference) Dilution used Species Fluorophore (exc/em) Supplier (reference) Dilutionused Occludin Rabbit Invitrogen
(40–4700)
1:125 Goat AlexaFluor®
(495nm/519nm)
Invitrogen (A11008)
1:2000 E-cadherin Mouse SantaCruz
Biotechnology Inc.(sc-73916) 1:50 Horse DyLight® (592nm/617nm) Vector Laboratories (DI2594) 1:300
VE-cadherin Mouse Hycult®biotech (HM2032)
(cm2)and C
0 theinitialdonorconcentration (M).The
massbalance(R)wascalculatedas: R(%)=100×A+D
D0
where A and D are the amounts of the compounds in theacceptoranddonorsides,respectively,andD0 isthe
amountintroducedatt0.Themassbalancesofallthe
com-poundswerebetween80and120%.
dQ,AandDweredeterminedusingastandardcurveof LY.
3. Electronmicroscopy
Fortransmission electronmicroscopy (TEM),cell co-cultures were rinsed on Transwell® membranes with pre-warmed (37◦C) HBSS (with CaCl2 and MgCl2) and
fixedwith2.5%(v/v)glutaraldehydeinaphosphatebuffer (0.1M,pH7.4)for1hatroomtemperature.Afterwashing, sampleswere post-fixed with1% (v/v) osmium tetrox-ide(Electron MicroscopySciences)indistilled waterfor 1h30minatroomtemperatureinthedarkandfurther dehydratedatdifferentethanolconcentrations.After car-ryingthefiltermembraneswithcellsthroughpropylene oxideasanintermedium,thesampleswereembeddedin Eponresinandsubmittedtopolymerizationat60◦Cfor 48h.Ultrathinsections80nmthickwerecut perpendicu-lartothefilterwithanultra-microtome(Ultracut,Reichert Jung)andplacedontocoppergrids.Samplesweredoubly stainedwith1%(v/v)uranylacetateandleadcitrate,and ultrastructuralanalysiswasperformedwitha transmis-sionelectronmicroscope(JEOL100S®,JeolLtd.,80kV). Forthepurposeofscanningelectronmicroscopy(SEM), thecellco-cultureswerefixedandpost-fixedasdescribed above. Afterdehydration in graded ethanol and drying usinghexamethyldisilazane,sampleswereplacedonstubs andanalysedunderascanningelectronmicroscope(FEI Quanta400®,FEICompany).
3.1. AlamarBlue®viabilityassay
After24hexposuretoPSnanobeads,AlamarBlue®assay (Invitrogen)wasperformed onmono-culturesas previ-ously described [27] and on co-cultures in Transwell® membranes.In mono-cultures,theassaywasperformed in96-wellmicro-plates.Inco-cultures,themediumwas discardedandfiltermembraneswerewashedtwotimes with a pre-warmed (37◦C) HBSS containing CaCl2 and
MgCl2.Then300Land 1mLofa mixcontainingRPMI
1640mediumwithoutphenolredsupplementedwith5% (v/v) FCS and 10% (v/v) AlamarBlue® (Invitrogen) were depositedintheapicalandbasolateralsides,respectively. After3hofincubation at 37◦C in anatmosphere of5% CO2 and95%humidity,theapicalandbasolateralmedia
werepooledona24-wellplateandthecellviabilitywas estimated by measuring the emitted fluorescence with acyto-fluorometer(excitation=555nm,emission=585nm)
(TECANInfiniteM2000, Switzerland).Thepercentage of cellviabilityforeachsamplewascalculatedcomparedto non-exposedcells(correspondingto100%viability).The potentialinterferencesofthePSnanobeadswiththeassay
Table2
SummaryofNPsconcentrationsstudiedandcorrespondencebetween g/cm2andg/mLunits.
Concentrations(g/cm2) 1.6 8.1 16.1 32.3
Concentrations(g/mL) 7.3 14.5 29.1 58.1
weresystematicallyassessed, andnosignificant modifi-cationinfluorescencewasobserved.NPsconcentrations tested and their correspondance between g/cm2 and
g/mLunitsaresummarizedinTable2. 3.2. CellularuptakeofPSnanobeadsbybi-and tri-cultures
Bi-culturesofCalu-3andHPMEC-ST1.6Rcells,and tri-cultureswithmacrophages,wereexposedto32.3g/cm2
of PS nanobeads for 24h. For tri-cultures, at day 6 post-seeding, THP-1 cells were differentiated into macrophage-likecellsaspreviouslydescribed.Then, THP-1 macrophagesweredetached usinga“cell dissociation bufferenzyme-freeandHank’sbased”(Invitrogen, Refer-ence13150-013),and stainedwiththeCellProliferation Dye eFluor® 450 (eBioscience)according tothe recom-mendationsoftheprovider.Theselabelledmacrophages werethenseededontheapicalsideofthebi-culturesfor 24h.Tri-culturesorbi-cultureswerethenexposedtoPS fluorescentnanobeadsfor24h.Thecells wererinsedin pre-warmed(37◦C) HBSS(withCaCl2 and MgCl2)
(Invi-trogen)and fixedfor15minatroomtemperaturein4% (v/v) paraformaldehyde (Sigma–Aldrich), washed twice withHBSSandpermeabilizedfor3minwith0.5%(v/v) Tri-ton X100(Acros Organics). Thecells werethen washed withHBSSandincubatedat37◦Cfor20minwithamixof water,1:40AlexaFluor® 635Phalloidin(Invitrogen),and propidium iodideat0.1mg/mL (Invitrogen).Finally,the Transwell®membraneswerewashedwithHBSS contain-ing0.025%(v/v)TritonX100beforemountinginProLong® Goldantifadereagent(Invitrogen)toproceedtoconfocal microscopyvisualization.
AllpictureacquisitionswereperformedusingaNikon A1Rconfocallaserscanningmicroscopesystemconnected toaninvertedECLIPSETi(NikonCorp.).Acquisitionswere performedinsequentialmodewitha100×objective(NA 1.45)byusingoptimalspatialresolutionsettings.Nuclear and cytoplasmic sides were identified with propidium iodidestaining(excitationwith561nmlaser,emission col-lectedwitha595/50nmfilterset)and AlexaFluor® 635 phalloidin (excitation with 640nm laser, emission col-lectedwitha700/50nmfilterset),respectively.Nanobead fluorescencewasexcitedusinga488nmlaser(emission collectedwitha525/50nmfilterset).Macrophage fluores-cencewasexcitedusinga403nmlaser(emissioncollected witha450/50nmfilterset).3Dimagereconstructionswere performedusingVolocity®software(PerkinElmer). 3.3. Acellulartranslocationassays
Non-functionalized (PS-NF, primary sizes of 51 and 110nm) and aminated (PS-NH2, primary size 52nm)
inRPMI1640culturemediawithoutphenolred supple-mented with5%(v/v) heat-inactivatedFCSand 1%(v/v) penicillin/streptomycin,ataconcentrationof16.1g/cm2.
TheapicalsideoftheTranswell® membraneswasfilled with 300L of particle suspensions. The basolateral side was filled with 1mL of RPMI 1640 culture media without phenol red supplemented with 5% (v/v) heat-inactivatedFCSand1%(v/v)penicillin/streptomycin.The Transwell® plateswerethenincubatedfor24hat37◦C, 5%CO2 and95% humidity.After24h,samplesfromthe
apical and basolateral sides were collected in a black 96-well micro-plate for determination of the fluores-cencewithacyto-fluorometeradaptedtothemicro-plate (excitation=488nm, emission=550nm) (TECAN Infinite
M2000).Therelativefluorescenceineachsidewas calcu-latedinrelationtotheinitialamountofparticlesused. 3.4. Translocationstudyofnanobeadsacrossco-cultures
ThesamePSnanobeadswereusedfortransport exper-imentsin bi-and tri-cultures.The cells wereincubated with 1.6, 8.1, 16.1 and 32.3g/cm2 of NPs dispersed
in RPMI 1640 culture media without phenol red sup-plemented with 5% (v/v) heat-inactivated FCS and 1% (v/v)penicillin/streptomycinfor24h.Someofthe exper-imentswereperformedat37◦Candothersat4◦C.After 24h,samplesfromtheapicalandbasolateralsideswere collectedin a black 96-wellmicro-plate for determina-tionofthefluorescencewithacyto-fluorometeradapted tothemicro-plate(excitation=488nm,emission=550nm)
(TECANInfiniteM2000).Therelativefluorescenceineach sidewascalculatedinrelationtotheautofluorescenceof theculturemedium.
3.5. Statisticalanalysis
Allquantitativedataarerepresentedbythemeanof threeindependentexperiments±SD.Thedatawere ana-lysedbyone-wayanalysisofvariance(ANOVA)followed byDunnett’st-testtocomparethedifferenttreatedgroups tothecontrol(˛risk=0.05).Toexaminethedifferences betweenpairs ofgroups, Student’st-tests (˛risk=0.05) wereused.
4. Results
4.1. Choiceofthepulmonaryepithelialcellline
Onthealveolo-capillarybarrier,tightjunctionsplayan importantfunctionalrole.Apreviousstudyreportedthat mono-culturesofhumantype Ipneumocytesdeveloped TEERvaluesof2180±62cm2after8dayspost-seeding
on Transwell® membranes [28,29]. Thus, we measured the TEER development in different pulmonary epithe-lial celllines across time, toselect cells presenting the highest values (Fig. S1). A549 cells reached maximum TEER values of roughly 18.7±2.5cm2 after 11 days
post-seeding on 12-well Transwell® plates. NCI-H441 cellsdevelopedsignificantlyhigherTEERvaluesfromdays 8 to11 post-seeding (approximately 100.2±25.4cm2
at day 11 post-seeding). Treatment of NCI-H441 cell
mono-culturesby1MDEXfromdays3to11post-seeding significantlyincreasedtheTEERvaluestoamaximumof 263.9±12.7cm2 at day 11 post-seeding. Calu-3 cells
developed significantly higher TEER values from days 6 (592.5±0.5cm2) to 11 (1233.5±58.1cm2)
post-seedingcomparedtoothercelllinesandweretherefore selectedfortheco-culturemodelofthealveolo-capillary barrier.
Supplementary Figure S1 related to this article can befound, in theonline version, atdoi:10.1016/j.toxrep.
2014.03.003.
4.2. Barrierpropertiesofbi-andtri-cultures
TheendothelialcelllineHPMEC-ST1.6Rdidnotdevelop significantTEERvalues,showingamaximumTEERvalueof 11.8±0.8afterday8post-seeding(Fig.1A).Theco-culture withCalu-3epithelialcellsshowedsignificantlyincreased TEERvaluesfromdays4to11post-seedingsimilartothose observedinCalu-3cellmono-cultures.
Bi-cultures were examined 8 days post-seeding by immunofluorescence for the expression of intercellular
Fig.1.Barrierpropertiesofmono-andbi-cultures.Arepresentsthetime courseofTEERdevelopmentinmono-andbi-culturesofCalu-3epithelial cellsandHPMEC-ST1.6Rendothelialcells.Allcellswereculturedin 12-Transwell®filterplatesusingRPMI1640mediasupplementedwith10%
(v/v)FCSand1%(v/v)penicillin/streptomycin.Mono-culturesofCalu-3 andHPMEC-ST1.6Rcellsarerepresentedbygreenandredcurves, respec-tively,andbi-culturesbythebluecurve.Datarepresentthemean±SDof fourindependentexperiments.Immunofluorescentlabellingofadherent (E-/VE-cadherin)andtight(occludin)junctionproteinswasperformedin bi-culturesonCalu-3andHPMEC-ST1.6Rcells(inwhite,B–E),atday8 post-seeding(scalebars=20m).
Fig.2. InfluenceofTHP-1differentiatedmacrophageseedingonTEERof bi-cultures.Priortoaddingtobi-cultures,THP-1monocyteswere dif-ferentiatedduring24husing30ng/mLofPMA.Atday7post-seeding bi-cultures,THP-1differentiatedmacrophageswereaddedatthe api-calsideofCalu-3cells.TEERmeasurementswerethenperformedat2, 4,6and24haftermacrophageseeding.Datarepresentthemean±SDof threeindependentexperiments.One-wayANOVAandDunnett’spost-test (tri-culturesvs.bi-culturescomparisons)wereperformed(**p<0.01).
proteins involved in tight junction (TJ; Occludin) and adherent junction (AJ; E-/VE-cadherin for Calu-3 and HPMEC-ST1.6Rrespectively)formation.Ontheapicalside of theTranswell® membranes,E-cadherin and occludin appearedintensivelyanduniformlylabelledbetween Calu-3 epithelial cells (Fig. 1 B and C respectively). On the basalside,VE-cadherinappearedintensivelybutnot uni-formlylabelledbetweenHPMEC-ST1.6Rendothelialcells duetoadevelopmentofmulti-layeredstructures(Fig.1
D).Occludinwasnotlabelledonendothelialcells(Fig.1E). PMA pre-differentiated THP-1 macrophages were addedatday7post-seedingontheapicalsideoftheCalu-3 cells in bi-cultures. After 24h in co-culture, no signifi-cantdecreaseof the TEERvalues wasnoted (Fig. 2).In ordertoverifythatthesemeasurementswereperformed correctly,wealsocontrolledtheinfluenceofhigher THP-1 macrophages concentration (2×105cells/mL), which
inducedsignificantTEERdecreaseintri-culturescompared tobi-cultures (datanotshown).This resultis also con-sistentwiththestatisticalanalysisoftheLYPapp values
oftri-cultures(Fig.S2).SignificantlyincreasedPappvalues
oftheLYacrossHPMEC-ST1.6Rcellmono-cultures com-paredtoCalu-3cells alsoconfirmedresultsobtainedby TEERmeasurementsinthecellcultures (Fig.1A). How-ever,assessment oftheLY transportshowedsignificant differencesbetweentheCalu-3mono-culturesand bi-/tri-cultures,contrarytotheTEERmeasurementsthatdidnot revealanydifferences.
Supplementary Figure S2 related to this article can be found, inthe onlineversion, at doi:10.1016/j.toxrep.
2014.03.003.
4.3. Macrophageschangethesurfacemorphologyof epithelialcellsinco-cultures
Atday8post-seeding,SEManalysiswasperformedon both theapical and basolateral sides of the bi-cultures and tri-cultures in order to observe the morphological changesinducedbytheadditionofmacrophages(Fig.3). Ontheapicalsideofbi-cultures,numerousmicrovilliwere observed (Fig.3A). On tri-cultures,THP-1 differentiated macrophageswereobservedontheapicalsideofCalu-3 cells(Fig.3B),butnotontheendothelialside.Macrophages wereadherentandpresentednumerouspseudopodia.SEM analysis also revealed epithelial morphological changes withthedisappearanceofmicrovillionthecellapex,and ahillysurface.
4.4. Ultrastructuralmorphologyoftri-culturemodels TEManalysiswasalsoperformed onboth theapical andbasolateralsidesofthebi-andtri-culturesinorderto observetheultrastructuralchangesinducedbythe addi-tionofmacrophages(Fig.4).Onbi-cultures,longmicrovilli (lengthofapproximately500nm),desmosomes,andTJ/AJ (Tight junctions/Adherent junctions) were observed on the apex of the Calu-3 epithelial cells (Fig. 4A and B). On tri-cultures,macrophagescontaining numerouslipid inclusionswereobserved(Fig.4C),butnotonthe endothe-lialside.Thesmallestmicrovillicomparedtobi-cultures were also observed, with desmosomes and TJ/AJ still existing in Calu-3 cells of these models (Fig. 4D). On
Fig.3.Representativescanningelectronmicroscopypicturesoftheapicalsideofbi-cultures(A,scalebar=2m)andtri-cultures(B,scalebar=5m).On pictureB,theblackstarindicatesthepresenceofanactivatedTHP-1macrophageadherentontheapicalsideoftheCalu-3cells.
Fig.4. UltrastructuralmorphologyofCalu-3cellsonbi-cultures(AandB)andtri-cultures(CandD).ThesecellswereculturedonTranswell®membranes andthenobservedatday8post-seedingbytransmissionelectronmicroscopy(TEM).Onbi-cultures(Fig.AandB,scalebar=500nm),Calu-3epithelialcells areobservedwithapicalmicrovilli(m),desmosomes(Des)andtightoradherentjunctions(Tj/Aj).Ontri-cultures,macrophages(M)withlipidinclusions (i)arelocatedontheapicalsideofCalu-3epithelialcells(Ep)showingsmallmicrovilli(m)(C,scalebar=5m).Desmosomesandtight/adherentjunctions arealsopresentontheapicalsideoftheepithelialcells(Fig.D,Cyt=cytoplasm,N=nucleus,n=nucleolus,scalebar=2m).Fig.EandFarephotographsof theendothelialcellslocatedonthebasalsideoftheTranswell®(v=vesicles,salebars=2and1mrespectively).
thebasalside, longandflattened endothelialcellswere observedwithahighnucleo-cytoplasmicratio(Fig.4E),and numerous perinuclear organelles suchas mitochondria. Cytoplasmicextensionsoftheseendothelialcellswerealso observedsuperposed,formingthinmulti-layers(Fig.4F). Thepresenceofmicro-vesiclesontheapicalsideofthese cellsalsoindicatesthepresenceofmolecularexchanges occurringontheplasmamembrane.
4.5. Fluorescentpolystyrenenanobeadscharacterization The size distributions of aminated (primary size of 52nm)andunmodified(primarysizesof51and110nm) PSnanobeadswerecharacterizedafterdispersioninthe complete culture medium. All suspensions were well mono-dispersed with low polydispersity indexes (<0.7; Malvern’s reference value) (Fig. 5). 99.80% of PS-NH2
(52nm)nanobeadsand91.85%ofPS-NF(51nm)nanobeads werebelow100nm(Fig.5AandB).90%ofPS-NF(110nm) nanobeadswerebelow200nm(Fig.5C).Allnanobeadshad negativezetapotentials.Despiteabsolutevaluescloseto zero,wemeasuredstablesizedistributionsforupto48h atroomtemperatureafterdispersion(datanotshown).NP suspensionswerefurthercharacterizedbyTEMand simi-larsizeswereobservedwithslightlyagglomeratedround NPs(Fig.S3).
Supplementary Figure S3 related to this article can befound, inthe onlineversion, at doi:10.1016/j.toxrep.
2014.03.003.
4.6. Cytotoxicityofpolystyrenenanobeads
AlamarBlue® assays showed that unmodified PS nanobeads(51and110nm)didnotinducea significant decreaseofthecellviabilityonmono-andco-cultures(data notshown).AminatedPSnanobeads(52nm)induced sig-nificantdose-dependentdecreasesofcellviabilityatdoses of16.1and32.3g/cm2onCalu-3epithelialcells(Fig.6A;
60.5±3.6%and46.5±10.4%ofviablecells,respectively). Significantdecreasesofcellviabilitywerealsoobserved at32.3g/cm2 onTHP-1differentiatedmacrophagesand
HPMEC-ST1.6Rendothelialcells(Fig.5BandC;50.2±11.6% and48.9±15.5%ofviablecells,respectively).No signifi-cantcytotoxicitywasobservedonthebi-andtri-cultures (Fig.5D–F).Inordertoverifythatmeasurementswere per-formedcorrectly,0.01%TritonX-100wasusedasapositive control.After24hexposureto0.01%TritonX-100,cell via-bilitywassystematicallysignificantlydecreasedcompared tocontrol cells inmono-, bi- and tri-cultures(datanot shown).
4.7. TEEReffectsdependonPSnanobeadsurface chemistryandsize
ToassesstheeffectsofPSnanobeadsontheintegrity of bi-and tri-cultures,all co-cultureswereexposed for 24handTEERwasmeasuredattimes0,2h,4hand24h. AllresultswereexpressedinrelativeTEERtothetime0, which correspondstothefirst TEERmeasurement after
Fig.5.Sizedistribution,zetapotential()andpolydispersityindexes (PDI)ofpolystyrenenanobeads.Aminated52nm,unmodified51nmand 110nm nanobeads(primarysizes)weredispersedintheRPMI1640 medium(withoutphenolredsupplementedwith5%(v/v)FCSand1% penicillin/streptomycin),andallparametersweremeasuredbydynamic lightscattering(DLS)(Fig.A–C,foreachnanobeadrespectively).Data representthemean±SDofthreeindependentexperiments.
theequilibrationtime.Inallco-culturesandforalltested nanobeadsnosignificantdecreasesofrelativeTEERvalues wereobservedafterexposuretonanobeadsfor2and4h (datanotshown).However,aslightandtransient signifi-cantdecreaseofrelativeTEERvalueswasobservedafter 4hexposureto PSnanobeadsin control cells, and was attributedtothemediumchange(datanotshown).Only PS-NFofbothsizeswasabletodecreasetherelativeTEER valuesinthebi-andtri-cultures(Fig.7).Thedecreasewas morepronouncedforthesmallerPS-NF(51nm)thanfor thelargerones(110nm)(Fig.7BandC).
4.8. PSnanobeaduptakedependsofthesurface chemistry
Inordertodeterminetheroleofmacrophagesin tri-cultures and the physico-chemicalproperties of NPs in co-cultureuptake, we carried out confocalfluorescence microscopyandthree-dimensionalreconstructionsofthe apicalandbasal sidesofthebi-andtri-cultures.Dueto theretentionofseveralfluorophoresusedtoperformthe multi-labelling, thefilter autofluorescence, and the low workingdistanceoftheobjectiveused(0.13mm),itwas
notpossibletoperformwholeacquisitionsfromtheapical celllayerstothebasalendothelialcells.
Inbi-cultures,allPSnanobeadswereadsorbedor inter-nalizedbyCalu-3epithelialcells(Fig.S4A–D).Onthebasal side,onlyPS-NH2(52nm)andPS-NF(110nm)nanobeads
wereobservedadsorbedonplasmamembranesor inter-nalizedbyHPMEC-ST1.6Rcells(Fig.S4E–H).
Supplementary Figure S4 related to this article can be found, inthe onlineversion, at doi:10.1016/j.toxrep.
2014.03.003.
Intri-cultures,allPSnanobeadswereinternalizedby Calu-3 epithelial cells (Fig. 8A–D). PS-NH2 (52nm) and
PS-NF(110nm)nanobeadswerenotablyinternalizedby macrophages(Fig.8BandD).Onthebasalside,onlyPS-NF (110nm)nanobeadswerefoundadsorbedontheplasma membranesofHPMEC-ST1.6Rcells(Fig.8H).
4.9. PSnanobeadsareretainedbyTranswell® membranes
TodeterminewhetherornotTranswell® membranes prevent nanobead translocation from the apical to the basolateralsideacellularassayswereperformedwithall PS nanobeads. After24hincubation, significantly more elevatedamountsoffluorescenceweremeasuredinthe apical sides compared to the basal sides for all tested nanobeads (Fig. 9A). There was no significant differ-ence between the amounts of fluorescence of PS-NH2
(52nm) and PS-NF(51nm) nanobeads(Student’st-test, p=0.0640),whereastheseamountsincreasedvery signif-icantly for PS-NF (110nm) compared to PS-NF (51nm) nanobeads(Student’s t-test,**p=0.0068). Therefore,the surfacechemistryofnanobeadsdoesnotsignificantly mod-ifytheirtranslocation,whereastheirsizedoes.Thesumof therelativefluorescenceofthePS-NF(51nm)nanobeads measuredinthetwosides(approximately31%)wasbelow 100%, indicating a possible adsorptionof nanobeadson the Transwell® membranes. Three-dimensional acquisi-tionsperformedonmembranesexposedfor24htoeach nanobead revealed that some of the nanobeads were retainedbytherandomlydistributedporesofTranswell® membranes(Fig.8B–D).
Other acellular translocation experiments were performed under identical conditions to compare polyester (1×106pores/cm2) to polycarbonate
mem-branes (4×108pores/cm2). We did not find significant
differencesbetweenthesefiltermembranetypes(datanot shown).
4.10. Physico-chemicalpropertiesofPSnanobeads influencetheirtranslocationacrossco-cultures
Translocationassayswereperformedfromtheapical tothebasolateralside inthebi-andtri-culturesduring 24hwitheachnanobead.Takingintoaccountthe reten-tionofnanobeadsintheapicalside,weonlyperformed asemi-quantitativeevaluationoftheresults,whichwere expressedintermsoftherelativefluorescencecompared tothebasalautofluorescenceofthemedium.No signifi-cantincreaseoftherelativefluorescencewasmeasuredon thebasolateralsideofallco-culturesforPS-NH2(52nm)
Fig.6.CytotoxicityofPS-NH2nanobeadsonmono-andco-cultures.Mono-cultures(A–C),bi-culturesofCalu-3andHPMEC-ST1.6Rcells(D),and
tri-cultures(E),wereexposedfor24htoPS-NH2nanobeads,respectively.CellviabilitywasthenmeasuredwiththeAlamarBlue®assay.Datarepresentthe
mean±SDofthreeindependentexperiments.One-wayANOVAandDunnett’spost-test(comparisonsvs.controlcellsnotexposedtoNPs)wereperformed (**p<0.01).
and PS-NF(110nm)nanobeads(Fig.10A andB). Signif-icantincreases(concentrationdependent)oftherelative fluorescenceweremeasuredinthebasolateralsidesofthe bi-andtri-cultures(Fig.10C).
4.11. TranslocationofPS-NF(51nm)nanobeadsrequires passiveandactivemechanisms
To assess whether or not PS-NF (51nm) nanobeads translocationisrelatedtopassive(paracellularpathways) ortoactivemechanisms(e.g.endocytosis), translocation assays were performedat 4◦C or 37◦C on thebi- and tri-cultures. Significant decreases of fluorescence were measured in all co-cultures exposed to PS-NF (51nm) nanobeadsfor24hat4◦Ccomparedto37◦Catall concen-trationstested(Fig.11).Therefore,translocationofthese nanobeadsrequiresinparticularactivemechanisms,such asendocytosis,butalsopassivemechanismsaspreviously described(Figs.S4and7).
5. Discussion
In this work an in vitro model containing the three maincelltypesconstitutingthealveolo-capillarybarrier invivowasdeveloped(epithelialcells[Calu-3cellline]and macrophages[PMA-differentiatedTHP-1cells]inthe api-calside ofaTranswell®,andmicro-vascularendothelial cells[HPMEC-ST1.6Rendothelialcellline]inthebasalside). ToaddresstheroleofthesecellsintermsofNP translo-cation,well-characterizedfluorescentPSnanobeadswere testedonbi-culturesofepithelial/endothelialcellsandon tri-culturesincludingmacrophages.Variousparticlesizes
andsurfacefunctionalizationswerealsotestedto deter-minetheroleofthephysico-chemicalpropertiesinterms ofNPtranslocation.
Alveolarepithelialcells playanimportantpartinthe establishmentofthehighlyimpermeablealveolo-capillary barrier[28].MeasurementsoftheTEERvaluesinvarious epithelialcelllinesconfirmedthatA549failedtodevelop sufficientTEERvaluesaspreviouslydescribedinliterature
[12].NCI-H441cellsalsofailedtodevelophighTEER val-ueseven inthepresenceof dexamethasone,which was previouslyshowntoincreasetheTEERvaluesinthiscell
line[19,20].Calu-3bronchialepithelialcellsdeveloped
sig-nificantlymoreelevatedTEERvalues(1233.5±58.1cm2
atday11post-seeding),closetothosereportedfor pri-maryhumanalveolarepithelialcells(2180±62cm2at
day8post-seeding)[29].Theywerethenchosenasa sur-rogateforalveolarepithelialcellsinourmodel.Bi-cultures of Calu-3 and HPMEC-ST1.6R cells showed TEERvalues notsignificantlydifferentfromCalu-3cellmono-cultures, indicatingthecentralroleplayedbyCalu-3cells in bar-rierestablishment.ThepermeabilityoftheLuciferyellow significantlydecreasedinthebi-culturescomparedtothe Calu-3mono-culturesatday8post-seeding.Itisunlikely thatthesedifferencesbetweenTEERmeasurementsand Pappvalues areduetoanymodificationofbarrier
prop-ertiesinHBSSduringtheLYassay,asTEERvalues were checkedtobeunmodifiedduringandaftertheassay(data notshown).AsdidVanItallieetal.,wepostulatedthat thepermeabilityoftheLYisproportional totight junc-tionporenumber,whilesmallelectrolytesaresubjectto further selectivity by the profile of tight junction pro-teins(e.g.occludin)[30].Thismayexplainthedifferences
Fig.7.RelativeTEERofbi-andtri-culturesincubatedwithpolystyrene nanobeads.Co-culturesseededonTranswell®membraneswere
incu-batedwithPS-NH2(52nm)(A),PS-NF(51nm)(B),orPS-NF(110nm)
nanobeads(C)during24hinRPMI1640medium(withoutphenolred supplementedwith 5%(v/v)FCSand1%penicillin/streptomycin). Bi-cultures(bluebars)andtri-cultures(yellowbars)weretested.TheTEER isexpressedrelativetothetime0(timeafter1hequilibrationintheRPMI 1640mediumwithoutphenolredsupplementedwith5%(v/v)FCSand1% penicillin/streptomycin).Datarepresentthemean±SDofthree indepen-dentexperiments.One-wayANOVAandDunnett’spost-test(comparisons vs.controlcellsnotexposedtoNPs)wereperformed(*p<0.05,**p<0.01). (Forinterpretationofthereferencestocolorinthisfigurelegend,the readerisreferredtothewebversionofthearticle.)
observed between Papp values in Calu-3 cells and
co-cultures. The protein occludin was detected on Calu-3 epithelial cells but not on endothelial cells. Occludin expressionbyCalu-3cellswaspreviouslydescribedin lit-erature,andthisproteinismainlyimpliedintightjunction (TJ)formationinprimo-culturesoftypeIIalveolar epithe-lialcells [31,32].Adherent junction(AJ) proteinsE-and VE-cadherinwerealsoexpressedinbi-culturesby Calu-3and HPMEC-ST1.6R,respectively, asalready described
[32,33].ThepresenceofTJ/AJanddesmosomeswas
con-firmed by TEM analysis on the apex of Calu-3 cells in bi-cultures, associated with numerous microvilli. Addi-tion of pre-differentiated THP-1 macrophages at day 7 post-seedingontheapicalsideofthebi-culturesdidnot
induceanysignificantchangein TEERvalues, norinLY Pappvalues.Thisresultdiffersfromthatrecentlyobtained
by Farcal et al., who also seeded THP-1 differentiated macrophagesontheapicalsideofbi-culturesconstitutedof NCI-H441epithelialcellsandISO-HAS-1endothelialcells andco-incubatedwithdexamethasone[4].Theseauthors showeda drasticdecrease ofTEERvaluesafteraddition ofPMA-differentiatedTHP-1macrophagesassociatedwith substantialsecretionofTNF-␣andIL-8,showingthat NCI-H441 cells are more sensitive than Calu-3 cells to the addition of PMA-differentiated THP-1macrophages. We confirmedthis byperformingco-cultureexperimentsof Calu-3, NCI-H441, or dexamethasone-treated NCI-H441 cells with PMA-differentiated THP-1 macrophages. Sig-nificantdecreasesofTEERvalueswereonlyobservedin NCI-H441/THP-1co-culturesindependentlyof dexametha-sonepre-treatment.Kleinandcolleaguesalsoreporteda higherpermeabilityoftheirtetraculturesystemmimicking thealveolo-capillarybarrierafteraddinginnateimmune systemcellsTHP-1macrophagesandHMC-1mastocytes
[13].TEManalysisof tri-culturesshowedthatTJ/AJ and desmosomes were alwayspresent but that morpholog-ical changes were observed with length reduction and disappearanceof microvilli ontheapicalside of Calu-3 cells. As SEM analysisrevealed that macrophages seem tobeinanactivatedshape(numerouspseudopodia),we hypothesizethatthesecellscouldtriggerslightlyelevated pro-inflammatorycytokinereleasemodifyingthealveolar morphology.WealsosuggestthattracesofPMA,knownto modifyproteinexpressionontheapicalsurfaceof intesti-nalCaco-2cells,mayalsoaffectCalu-3cellsmicrovilli[34]. Consequently,asusingmacrophagestendstocausesome epithelialcellularmorphologicalchanges,theymightnot behaveastheydointhelunginvivo.Itisapossible lim-itationof theirusein co-culturecellsystems.However, furtherexperimentsareneededtoaddressthecausesand consequencesofthismodifiedepithelialmorphology.
In a secondstep, the translocation of fluorescentPS nanobeadsofvarioussizesandsurfacechemistrieswere tested on bi- and tri-cultures. These NPs were chosen becausetheyaretraceable,easilysynthesizableand com-mercially available in various sizes and with different surface functionalizations [35]. Nanobeads were firstly characterizedinthecompleteculturemedium.Weshowed thatsuspensionswerewellmono-dispersedandthatsize distributionswerestableforatleast48hatroom temper-ature(datanotshown).OnlyPS-NH2(52nm)nanobeads
were foundto becytotoxic onmono-cultures, whereas unmodifiednanobeads(51and110nm)werenot.These results,indicatingmorepotentcytotoxiceffectsofPS-NH2
(52nm)nanobeadscomparedtonon-functionalizedNPs, areconsistentwithpreviousresultsobtainedwithseveral celltypesinliterature[36–38].Surprisingly,no cytotox-icitywasobservedonbi-orontri-culturesforalltested nanobeads.Wepreviouslyreportedthisphenomenonwith cytotoxiceffectsofSiO2 andTiO2 NPsobservedafter6h
exposureonmonoculturesofTHP-1macrophagesbutnot onco-cultureswithA549orNCI-H441epithelialcells[27]. Several other authors also recentlyobserved decreased cytotoxicity on co-cultures compared to mono-cultures after exposure to diesel exhaust particles or silica NPs
Fig.8.Internalizationofpolystyrenenanobeadsbytri-cultures.Three-dimensionalmodelswith10×104THP-1macrophages/mL,seededonTranswell®
membranes,wereincubatedduring24hwith32.3g/cm2ofPS-NH
2(52nm),PS-NF(51nm),orPS-NF(110nm)nanobeads.Nuclearandcytoplasmic
sideswererespectivelyidentifiedbyusingpropidiumiodidestainingandAlexaFluor®635phalloidin(inblueandpinkonthepictures,respectively).
Macrophageswereidentifiedusingacellproliferationdye(eBioscience;inwhiteonthepictures).FluorescentPSnanobeadscorrespondtotheorange fluorescenceonthepictures.Three-dimensionalimagereconstructionsandmeasurementswereperformedusingVolocity®software(PerkinElmer)on boththeapical(A–D)andbasolateral(E–H)sidesoftheTranswell®.Thesepicturesarerepresentativeofseveralobservations.Scalebars=10m.
[39,40]. We postulate that this decrease of cytotoxicity
inco-culturesmayhavebeenduetothehighernumber ofcellsinco-culturecomparedtomono-culturesandthe consequentreducedcellsurfaceareaaccessibletoNPsin co-cultures.However,cytotoxicitywasalsonotobserved inbi-culturesapicallyexposedtoPS-NH2nanobeads.One reasonmaybethatintercellularsignallingcouldalsoplay aroleincytotoxicityprotection.Oneexperimentto con-firm this hypothesis couldbe to expose epithelialcells previouslycultivatedwithendothelial-conditionedmedia orviceversa.
Generally, TEER measurements after 24h exposure toPSnanobeadsrevealeddifferentsensitivitiesbetween co-cultures, which can be classified as follows: tri-cultures>bi-cultures. PS nanobeadsmay modifybarrier properties by acting on junction protein expression, as was previously demonstrated with modulation of the occludinmRNAin16HBE14o-bronchialcells[41].Dueto possibleinterferencesbetweenLYand PSnanobead flu-orescence,we performedonly TEERmeasurements,but complementaryinvestigations on themRNA expression orimmunofluorescenceofTJproteinsshouldbedoneto
Fig.9. AcellulartransportofpolystyrenenanobeadsacrossTranswell®membranes.Therelativefluorescence(comparedtotheinitialamountof
fluores-cencedeposited)ofthenanobeadswasmeasuredinbothsidesoftheTranswell®membraneswithoutcellsafter24hincubationinthecompleteculture
medium(A).Datarepresentthemean±SDofthreeindependentexperiments.Student’st-testswereperformed(*p<0.05)foreachgroupbetweenthe fluorescenceamountsintheapicalandbasolateralsides.Three-dimensionalimagereconstructionsofmembranesafterincubationwithPS-NH2(52nm),
PS-NF(51nm),orPS-NF(110nm)nanobeads(16.1g/cm2)wereperformedusingIMARISsoftware(B,CandD,respectively). confirmthis hypothesis.Arelationwithoxidativestress
and barrier properties has been described by various authors.Increasedtranslocationofquantumdotsacrossrat alveolarepithelialcells withdecreased TEERvalues was shownonlyin thepresence oftert-butylhydroperoxide (knownasanoxidativestressinducer)[42].Exposureof aco-cultureofepithelial(NCI-H441)andendothelial (ISO-HAS-1)cellstocarboxylatedPSnanobeadsunder mechani-calstraininducedROSgenerationandincreasednanobead translocation[43].Inotherexperiments,wedemonstrated thatallcellmono-cultureswereabletoreleasethe super-oxideanionafterexposuretoeachnanobead(unpublished data). Therefore, with an increased cell number in tri-culturescomparedtobi-cultures,anamplifiedreleaseof ROS may affect TEER values. Uptake experiments per-formed on bi- and tri-cultures revealed that NPs were internalizedoradsorbedbyCalu-3 epithelialcellsin bi-cultures,andsystematicallyinternalizedbymacrophages intri-cultures.SimilarresultswereobtainedbyBlanketal. whoreportedinatriplecellco-cultureofmacrophagesand epithelialcellsontheapicalside,anddendriticcellsonthe basolateralside,thatpolystyreneparticles(1m)wereless internalizedbymacrophagesandmorebydendriticcells whenA549epithelialcells (exhibitinglow TEERvalues) wereemployed,andinverselywhen16HBE14o-epithelial cells(withhigherTEERvalues)wereused[44].
Transwell®membranesarethemostusedandtheonly commerciallyavailable culturesupports allowing trans-portstudiesacrosscellularbarriers[4,16,19].Contraryto smallmolecules, thestudy ofNP translocation requires
ideallynointerferencewithmembranes.Consequently,we checkedwhetherornotpolyesterTranswell®membranes (0.4mpores)retainedPSnanobeads(ofdifferentsizes andsurfacechemistries).WefoundthatPSnanobeadswere mainlyretainedintheapicalsideoftheTranswell® mem-branesandadsorbedinthemicro-porousmembrane.Small quantitiesofPSnanobeadsweremeasuredin the baso-lateralsides,withsignificantly largeramountsof PS-NF (110nm)comparedtothesmallestNPs,indicatinga poten-tialeffectofparticlesize.Cartwrightetal.recentlyfound thatPSnanobeads(50and100nm)havegreateradherence topolyesterthantopolycarbonateTranswell®membranes
[10].Differentresultswereobtainedwithmono-dispersed nanobeadsusedinthisstudy,withnodifferencesbetween PSnanobeadtranslocationacrosspolyesterand polycar-bonate Transwell® membranes. We also compared 0.4 and3mporosities,buttheresultsremainedunchanged (datanotshown).Wehypothesizethatthethicknessofthe membranes (approximately 10m),and theshape and randomdistributionofthepores,mayplaymoreimportant roles. Consequently,translocation resultsacrosscellular modelswereanalysedcarefully.
After 24h of exposure of bi- and tri-cultures, only PS-NF(51nm)nanobeadsweresignificantlymeasuredin thebasolateralsidesbyspectrofluorometry,whilePS-NH2
(52nm)and PS-NF(110nm)nanobeadswerenot.Same experimentswereperformedonCalu-3cellmonocultures cultivated either on Transwells with 0.4m or 3m pores,indicatingafter14–16hofincubationwithPS-NH2
Fig. 10.Translocation of polystyrene nanobeads acrossbi- and tri-cultures.Bi-andtri-cultureswereexposedtoPS-NH2(52nm)(A),PS-NF
(110nm)(B),andPS-NF(51nm)(C)nanobeadsfor24hinRPMI1640 medium(withoutphenolredsupplementedwith5%(v/v)FCSand1% penicillin/streptomycin).Therelativefluorescence(comparedtothe auto-fluorescenceoftheculturemedium)ofthenanobeadswasthenmeasured inthebasolateralsidesoftheco-cultures.Datarepresentthemean±SD ofthreeindependentexperiments.One-wayANOVAandDunnett’s post-test(comparisonsvs.controlmediumwithoutcells)wereperformed (**p<0.01).
respectively[45].Consequently,wecouldhypothesizethat theretentionofPS-NH2(52nm)nanobeadsintheapical
compartment may bedue essentiallyto cellco-culture, andalsoslightlytotheporesize.Wealsoshowedthatthe observedtranslocationwaslargelyduetoactivepathways, butalsotoparacellularpathways,astraffickingdecreased significantly but still remained after 24h exposure to PS-NF(51nm)at4◦C comparedto37◦C. Similarresults were observed after exposure of rat alveolar epithelial cells tocarboxylated PSnanobeads(20nm)[46]. Aswe haveshown thatTHP-1cells internalizedPSnanobeads, wecouldexpectahighertranslocationinbi-cultures com-paredtotri-culturesbutnodifferencewasseen.Itwould have beeninterestingtoperform kineticmeasurements bystereologyoftheNPsdoseretainedbymacrophagesin tricultures,inordertoknowiftheydifferentiallyuptake eachtypeofNPs.Althoughnosignificantfluorescencewas detectedinthebasolateralsidefor PS-NH2 (52nm)and
PS-NF(110nm)nanobeads,confocalmicroscopyrevealed internalizedPS-NH2(52nm)nanobeadsintheendothelial
cellsofbi-cultures,butnotintri-cultures.PS-NF(110nm) nanobeadswerealsodetectedintheendothelialcellsin
Fig.11.TranslocationmechanismsofPS-NF(51nm)acrossbi-and tri-cultures. Bi-cultures(A)and tri-cultures(B) wereexposed toPS-NF (51nm)nanobeadsfor 24hin RPMI1640medium(without phenol redsupplementedwith5%(v/v)FCSand1%penicillin/streptomycin),at 37◦Corat4◦C(pinkandbluebars,respectively).Therelative fluores-cence(comparedtotheautofluorescenceoftheculturemedium)ofthe nanobeadswasthenmeasuredinthebasolateralsidesoftheco-cultures. Datarepresentthemean±SDofthreeindependentexperiments. Stu-dent’st-testswereperformed(*p<0.05)foreachgroupbetweenthe fluorescenceamountsinthebasolateralsidesafterexposuretoPS-NF (51nm)at4◦Cand37◦C.(Forinterpretationofthereferencestocolorin thisfigurelegend,thereaderisreferredtothewebversionofthearticle.)
bi-andtri-cultures.However,duetothelackofaccuracy of translocation studies by fluorescence measurements, wecannotaffirmthatPS-NH2(52nm)andPS-NF(110nm)
nanobeadscompletely moveacrossco-culturesin basal side.Theseresultsemphasizetherelevancyofmicroscopy methodstostudynanoparticleuptakeacrossco-cultures, andfurtherresearcheffortsstillneedtobedonetoadapt supportmembranestowardtranslocation studies. Inter-estingly,PS-NF(51nm)nanobeadswerenotdetectedin HPMEC-ST1.6Rendothelialcells,whereastheywereable tosignificantlytranslocateinthebasolateralsidesacross bi-andtri-cultures.FurtherexperimentsofTJlabellingare necessarytodetermineiftheseNPstranslocatebetween cells.Asonlythetime24hwastestedtoobservenanobead translocation, we can hypothesize that PS-NF (51nm) couldbeinternalizedearlierbyendothelialcellsandthen eliminatedin the basal side. However, theretention of nanobeads in the Transwell® insert may have affected the accuracy of the measurements (Figs. 10 and 11), andit mightthereforebedifficulttoascertainprecisely the effects of the physico-chemical properties and the differences between active and passive transport using thissystem.Duetoverylimitednanoparticletranslocation
observedinvivoinsecondaryorgansafterinhalationand possibleadsorptionofnanobeadsinmembranes,itseems difficult to quantitatively compare with translocation rates obtainedin this invitro study[3]. A recent study showsthat ultrathinporousSi membranesmightbean interestingalternativetoTranswell® membranesforthe studyofnanoparticletranslocation[47].
Inconclusion,wedevelopedtri-culturesresemblingthe alveolo-capillarybarrier,andexhibitingacceptablebarrier propertiesforthestudyofNPtranslocation.Macrophages, which play an important role in NP clearance in vivo, seemalsotoplay animportantrolein thesemodelsby internalizingNPsandprobablyaffectingbarrierintegrity. Thus,co-cultureswithmacrophages,epithelialcells,and endothelialcellsareappropriateforstudyingNP translo-cation.However,theTranswell®membranescommercially availabledonotallowsensitiveassessmentofNP transloca-tion,andfurtherresearcheffortsmustbemadetodevelop noveladaptedculturesupportstoaddressthisissuemore accurately.
Conflictofintereststatement
Theauthorsreportnoconflictofinterests.
Acknowledgements
WethankDrVera Krump-KonvalinkovaandProf.C.J. KirkpatrickatTheInstituteofPathology,Johannes Guten-bergUniversity,Mainz,Germany,fortheirkindgiftofthe HPMEC-ST1.6Rcell lineused inthis study.The authors alsothankRenéLai-KuenforhishelpwithTEManalysisat the“Plateformed’imageriecellulaireetmoléculaire”atthe FacultyofPharmacy,ParisDescartes,andPatriceDelalain forSEManalysisatINERIS.ThanksarealsoduetoDr Vin-centPagetforhisvaluableassistance.
ThisworkhasbeensupportedbyagrantoftheFrench MinistryforEcology,SustainableDevelopment,andEnergy (MEDDE),National Research ProgramNANOTRANS, and UTCFoundationforInnovation.
AppendixA. Supplementarydata
Supplementary data associated with this article can befound, in theonline version,at doi:10.1016/j.toxrep.
2014.03.003.
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