Selective adsorption of L1210 leukemia cells/human leukocytes on micropatterned surfaces prepared from polystyrene/polypropylene-polyethylene blends

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ContentslistsavailableatScienceDirect

Colloids

and

Surfaces

B:

Biointerfaces

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b

Selective

adsorption

of

L1210

leukemia

cells/human

leukocytes

on

micropatterned

surfaces

prepared

from

polystyrene/polypropylene-polyethylene

blends

Nevin

Atalay

Gengec

a

_,

_Hilal

_Unal

_Gulsuner

b

_,

_H.

_Yildirim

_Erbil

a,∗

_,

_Ayse

_Begum

_Tekinay

b

a_Gebze_Institute_of_Technology,_Department_of_Chemical_Engineering,₄₁₄₀₀_Gebze,_Kocaeli,_Turkey

b_Institute_of_Materials_Science_and_{Nanotechnology,}_National_{Nanotechnology}_Research_Center_(UNAM),_Bilkent_University,₀₆₈₀₀_Ankara,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27June2013

Receivedinrevisedform21August2013 Accepted18September2013

Available online 26 September 2013 Keywords:

Micropatterning L1210leukemiacells Leukocytes

Selectivecelladhesion Contactangle

a

b

s

t

r

a

c

t

TheobjectiveofthisstudyistopreparepolymericsurfaceswhichwilladsorbL1210leukemiacells

selectivelymorethanthatofhealthyhumanleukocytesinordertodevelopnewtreatmentoptionsfor

peoplewithleukemia.Chemicallyheterogeneousandmicropatternedsurfaceswereformedonround

glassslidesbydipcoatingwithaccompanyingphase-separationprocesswhereonlycommercial

poly-merswereused.Surfacepropertiesweredeterminedbyusingopticalmicroscopy,3Dproﬁlometry,SEM

andmeasuringcontactangles.Polymer,solvent/nonsolventtypes,blendcompositionandtemperature

werefoundtobeeffectiveincontrollingthedimensionsofsurfacemicroislands.MTTtestswereapplied

forcellviabilityperformanceofthesesurfaces.Polystyrene/polyethylene-polypropyleneblendsurfaces

werefoundtoshowconsiderablepositiveselectivitytoL1210leukemiacellswhereL1210/healthy

leuko-cytesadsorptionratioapproachedto9-foldinvitro.Effectsofwettability,surfacefreeenergy,microisland

sizegeometryontheadsorptionperformancesofL1210/leukocytespairsarediscussed.

1. Introduction

Cell–polymer surface interactions which are important for diseasetreatmentordesignofbiomedicalproductsarenotwell understood at present. These interactions were related to ﬁve parameterssuchastypeandpercentdistributionofthechemical groupsonthesurface[1],surfaceroughness[1–5],surface wetta-bility[6–8],surfacefreeenergy(SFE)ofthesubstrate[6,9],andsize andshapeofthecellusedfortesting[10,11].Thedistancebetween surfacepatternsandfocaladhesionclustersonthecellmembrane isalsoanimportantparameterforthecelltoformastrong adhe-siononasubstrate[1–3,12,13].Shapes,functionsandviabilityof thecells maychangeasaresultoftheirinteractionwitha pat-ternedsubstrate[14].Chemicalcompositionandmorphologyofa substrateareimportantparametersaffectingtheadsorptionof pro-teinspresentonthecellsurfaceandinitialcelladhesion[15–17].

Whenbloodcontactswithasubstrate,theadhesionproperties ofnormalbloodcells(i.e.leukocytes,platelets)changedepending onthesurfacepropertiesofthesubstrate[18,19].Thesubstrateis quicklycoveredwithproteinspresentintheserum.Differences intopography, chemical composition,charge and wettabilityin thesubstratesurfaceresultinchangesintype,conformationand

∗ Correspondingauthor.Tel.:+902626052114;fax:+902626052105. E-mailaddresses:yerbil@gyte.edu.tr,yerbil19@yahoo.co.uk(H.Y.Erbil).

amountoftheadsorbedproteins.Proteinadsorptionisadynamic processandproteinsdonotstayonthesurfacepermanently,and overtimethehigher-afﬁnityproteinsreplacethepreviously pre-adsorbedlower-afﬁnityproteins[20].Proteinadsorptionincreases withtheincreaseinhydrophobicityofthesubstratesincewater moleculesintheseruminteractweaklywiththesurfaceenabling moreproteinstoadsorb.Ontheotherhand,proteinadsorption alsoincreaseswiththeincreaseinthesurfaceroughnessdueto theincreaseofthetotalsurfacearea.Attheend,compositionand conformationofproteinsaredifferentonthesurfacedependingon thehydrophobicityandroughnessofthesurface[20].These varia-tionsinproteinsarerecognizedbydistinctcelladhesionmolecules; hence,celladhesionishighlydependentoncelltype.Everycell hasadifferentcombinationofadhesionmoleculesexpressedon theirsurface.Forexample,leukocytesarewellendowedwith integ-rinadhesionreceptors[21].Moreover,cancercellsareknownto altertheiradhesionmoleculesinordertoinvadeandmetastasize [22–25].Therefore,itwillbepossibletoallowadesiredcelltype toattachtoasubstratebychangingsurfacetopography, composi-tion,andwettability.Ouraimistodesignapolymersurfacewhich allowsL1210adhesionandinhibithealthyleukocyteattachment. Thisselectivityisexpectedtobearesultofthefactthatsurface receptorsofhealthyleukocytesandL1210cancercellsmightbe different.

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leukemiacellstosolidshavebeenexaminedpreviouslyanditwas foundthattheydonotadheretoglasswhereastheyeasilybindto embryonicﬁbroblastmonolayers[26].Itwasalsodeterminedthat adhesionofL1210cellsincreasedlinearlywiththeincreaseofthe sulfogroup( SO3H)concentrationonsulfonatedPSsurfaces[27].

Adhesionproperties ofhumanleukocytestosurfaceswerealso investigatedpreviouslyanditwasfoundthatthenumberof leuko-cyteswhichadheredtothepolymericsurfacesmodifiedbysurface sulfonationoradditionofotherfunctionalgroupswashigherthan thenon-modifiedsurfaces[28].Theactivationofadherent leuko-cyteswasfoundtobedependentonsurfacetopography,pattern geometryandsurfacechemistryof thesubstrate[18].Davidson etal.showedthatinteractionofthenormalcellswiththe topo-graphicallypatternedsurfaceswasdifferentfromthatofthecancer cellsandplasmamembranesofthenormalcellswerenoteasily deformedwhenthecontactareabetweensurfaceandcellswas increased[4].Yanetal.suggestedthatcellbindinglocationsand propertiescanbe controlledbydesigning specificmicropattern sizes[29].Thesefindingsshowthatmicropatternedsurfacesmay beusedinselectiveadsorptionofdifferentcelltypes.

Polymericmaterialsarewidelyusedasbiomedicalmaterials. Surfacepatterningofpolymershasbeenperformedbyapplying severalapproachessuchaslithography,UVlightsensible photore-sist,anduseofspeciallysynthesizedpolymers[30].Mostofthese patterningmethodsaretimeconsumingandexpensive;however, Erbiletal.havedemonstratedthatacheapcommercialpolymer suchaspolypropylene canbeconverted toa superhydrophobic surfacebyusingsolvent–nonsolventphaseseparationprocessand thismethodcanalsobeappliedtolargeareasurfacesand three-dimensionalmaterials[31].Polymerblendswhicharethephysical mixturesoftheindependentpolymerscanpreferablybeusedfor thispurposebecauseblendingmayprovideoptimizationofthe desiredpropertiesof theﬁnalsurface [32].Solventevaporation rate,phaseseparation,solventdiffusion,andSFEofthepolymers have substantialeffects on surface morphologyof the polymer blends[31,33–35].Micropatternedsurfacescanalsobeprepared usingpolymerblends[36,37].Kajiyamaandco-workersobserved that PMMA formed cylindrical micropatterns with controllable diametersbycreatingcrestsonthePSstructuresusingspin-coating withPMMA/PSblends[38].Overneyetal.obtainedmicropatterned surfacesbyperformingannealingprocessthroughformationofPS monolayersbyspin-coating onpoly-ethylene-co-propylene ﬁlm [39].

Whitesides and co-workers were the first to use micro-patterned surfaces in the biomedical field in 1997 [12] and determinedthathumancellsdieonsolidswithparticularpatterns whereastheyremainaliveonsolidswithdifferentpatternsand concludedthatcellsizeandshapeareimportantfactorsforthis cellularbehavior[2,12].Theinteractionsbetweenmicrostructured surfacesand cellshave beenintensivelyinvestigated inthelast decadesanditwasfoundthatcellsstronglyrespondtotopographic changesonsurfaces[40–42]. Theamountand/ororientation of theinitialproteinadsorptionontopographicallyreconstructed sur-facesoccurdifferentlyfromtheflatsurfaces[41].

In this study, we prepared ﬂat, rough and micropatterned surfaces on round glass slides using commercial polymers such as polystyrene (PS), high density polyethylene (HDPE), polyethylene-polypropylene copolymer (PPPE), ethylene-vinyl acetatecopolymer(EVA),andpolyvinylalcohol(PVOH)andalso polymerblends(PS/HDPE,PS/PPPE)bydipcoating method.We determined thespeciﬁc surfaces where selective adsorption of L1210leukemiacellsoccursbuthumanleukocytesdonotadsorb. Thesesurfacesmaybeused duringblood exchangetransfusion ofleukemia patientstoremove thecancerouscells (likeL1210 leukemia cell) out of blood. The effectiveness of the parame-ters such as surface roughness, surface chemical groups and

wettabilityontosurface/cellselectiveadsorptionwasalsostudied. Smoothpolymersurfaceswerepreparedtoobservetheeffectof roughness. It was found that selective adsorption performance changeddependingonthediametersofshortcylindricalislands and separation distance between the islands on the polymer surfacesespecially when PS/PPPE polymer blend surfaceswere usedwhereislanddiameterscanbeeasilycontrolled.

2. Materialsandmethods

2.1. Materials

PVOH (Merck, Mw=160,000), PS (Sigma–Aldrich,

Mw=350,000), HDPE (Basell Inc., HOSTALEN-GM8255,

Mw>1,000,000), PPPE copolymer containing 12% PE content

byweight(DowChemicalCo.,VERSIFY2300),andEVAcopolymer containing12%vinylacetatecontentbyweight(Dupont,ELVAX 660) wereused as received. These polymerswere dissolvedin THF (Merck), xylene and toluene solvents (Tekkim, Turkey) to prepare polymer solutions. Ethanol (EtOH, Merck) was used as non-solvent. Ultrapure water, diodomethane, formamide, ␣-bromonaphthalene,andethyleneglycol(allfromMerck)were usedascontactangledropliquids.Roundglassslides(Thermo Sci-entiﬁc)withdiametersof13and15mmwereusedassubstrates. Allbiologicalmaterialsusedinthisstudyareanalyticalgradeand werepurchasedfromInvitrogenandSigma–Aldrich.

2.2. Preparationofhomo-andcopolymerﬁlms

PVOH wasdissolvedinwater, PSand EVA intoluene, HDPE in xylene, PPPE in THFsolvents at a constant concentration of 10mg/mL.EtOHwasaddedtoPSandPPPEpolymersolutions pre-paredin THF solventby 10% (v/v).The withdrawal rate ofthe mechanicaldipperwasvariedbetween320–764mm/minatroom temperatureforPVOHandPSpolymers,at60◦CforPPPEandEVA, andat115◦CforHDPE.Cleanroundglassslideswaskeptinthe polymersolutionfor1mintoreachthermalequilibrium,andthen withdrawnwithaconstantspeed.Polymerﬁlmswerekeptina des-iccatorfor3–4handcompletelydriedinavacuumovenat40◦C overnight.PVOHﬁlmwasdriedundervacuumovernight,thenwas keptinglutaraldehydesolutionfor2handthenwashedusing dis-tilledwateranddriedundervacuumat50◦Covernighttoprepare cross-linkedPVOHsurfaces.Surfacesulfonationwascarriedout bysubmergingPSsamplesinsulfuricacid(60vol.%)for24h. Sul-fonatedPSsurfaces(PS-sulfo)wererinsedindeionizedwater,and air-dried.

2.3. PreparationofPS/HDPEandPS/PPPEblendﬁlms

PS,HDPEandPPPEpolymerstocksolutionswerepreparedin xyleneandTHFsolventsat10mg/mLconcentrationat10◦Cbelow theboilingpointsofthesolvents.PS/HDPEandPS/PPPEblend solu-tionswerepreparedby mixingthedifferentcompositionsfor a finalconcentrationof10mg/mLandstirredmechanicallyfor2–3h at60◦CtoreachequilibriumwhenTHF/xylenemixturewasused and115◦C whenonly xylenewasused.EtOHwasadded drop-wiseintotheblendsolution.Dipcoatingoftheroundglassslides byblendpolymerswascarriedoutwithamechanical dipperat a removalspeed of320–784mm/min.Coatedpolymer filmson glassslides werekeptin a desiccatorfor 3–4handcompletely dried in a vacuum oven at 40◦C overnight.Thicknesses of the coatings were between 0.5 and 2.0␮m. Surface topography of roughpolymerblendswasexaminedby3Dprofilometry(Nikon, Eclipse-LV100DMicroscope)andenvironmentalscanningelectron microscopy(ESEM,Quanta200FEG).

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2.4. Contactanglemeasurement

Staticcontactanglesunderairweremeasuredbyusinga KSV-CAM200contactanglemeter.Equilibriumcontactanglevalues(e)

weredeterminedaftertheneedlewasremovedfroma5␮Ldroplet formedonthesolidsurface.Inaddition,wemeasuredboth advanc-ing(adv)andreceding(rec)watercontactangles(WCA)onthe

samplesurfacesbyincreasingthevolumeofthedropletsfrom3to 8_␮Landdecreasingfrom8to4_␮Lrespectivelythroughtheneedle byusingtheautomaticdispenserwhiletheneedlewaskeptwithin theliquiddroplet.rec werealsomeasuredbydropevaporation

methodforbetterprecision[43,44]. 2.5. Cellcultureandmaintenance

L1210(ATCC®_CCL-219)_and_human_primary_leukocytes_were

usedforinvitrostudies.Humanprimaryleukocyteswereisolated fromwholeblood withstandard separationby densitymethod usingHistopaque(Sigma).L1210cellsweremaintainedinhigh glu-coseDMEM,4mMl-glutamine,1%Penicillin–Streptomycin,10% horseseruminstandardcellcultureconditions(37◦C,5%CO2,and

95%humidity).HumanprimaryleukocytesweremaintainedinF12 mediumsupplementedwith2mMl-glutamine,10%calfserum, and1%Penicillin/Streptomycininstandardcellcultureconditions. 2.6. AdhesionofL1210cellsandleukocytesonpolymersurfaces

PolymercoatedsurfacesweresterilizedunderUVlightbefore cellcultureexperiments.Priortocelladhesionexperiments,cells wereincubatedinserumfreemediacontaining4mg/mLBSAand 50␮g/mLcyclohexamidefor1hinstandardcellcultureconditions. Polymercoatedsurfacesandbaresurfacesthatwereusedascontrol wereplacedin24-wellplatesandcellswereseededonsurfacesat densitiesof15,000cells/cm2_for_leukocytes_and_45,000_cells/cm2

forL1210cellsinserum-freemedia.Cellswereincubatedfor2h beforetheywerewashedwithHBSS(Hank’sBalancedSalt Solu-tion).Attachedcellswerestainedwith1␮MCalceinAMfor30min. Randomphotosweretakenfromeachwellandcellswerecounted usingImageJ.

2.7. CellularmorphologystudiesofL1210cellsonpolymer surfaces

In order to visualize the actin cytoskeleton of L1210 cells on polymer coated surfaces, cells were seeded at a density of 25,000cells/cm2_. _After ₄₈_h _of _incubation, _they _were _ﬁxed

with3.7%formaldehyde,permeabilizedwith0.25%Triton-Xand stainedwithTRITCconjugatedphalloidin.Thecellswere visual-izedunderanuprightmicroscope(ZeissAxioScope).Toanalyze themorphologiesof L1210cells onsubstrates,cells were ﬁxed in2%gluteraldehydesolutionanddehydratedinincreasing alco-hol concentrations, dried with critical point-dryer (Tourismis Autosamdri®_-815B)_and_imaged_with_a_scanning_electron

micro-scope(FEIQuanta200FEG).

3. Resultsanddiscussion

Codes of thesurfaces prepared using PVOH, PS, sulfonated-PS,HDPEhomopolymers,PPPEandEVAcopolymersandPS/PPPE, PS/HDPEpolymerblendsareshowninTables1–3.

3.1. Surfacecharacterizationofﬂatpolymerﬁlmsandadhesionof L1210cellsandleukocytesonthesesurfaces

PVOH,PSandPS-sulfohomopolymer surfaceswerefoundto benearlyﬂatafterexaminingtheirSEMimageswherenovisible

protrusionscanbeseenevenat40,000×magniﬁcation(Fig.1a). PVOHandPS-sulfosurfaceswerefoundtobehydrophilic,whereas PSsurfacewashydrophobicasseeninWCAresults(Table1). Con-tactanglehysteresis(CAH)resultswerevariedbetween12–32±1◦. SurfacesulfonationofPSresultedinnearly2-foldincreaseinCAH indicatingahighincreaseinthechemicalheterogeneityonPS-sulfo surface.SFEvalueswerecalculatedusingevaluesbyapplyingvan

Oss–Chaudhury–Goodmethod[44,45]andwerefoundtorange between34.8and52.5mJ/m2_.

AdhesionofL1210leukemiacellsandhealthyleukocytesonto flathomopolymersurfacesweretestedindependentlyanditwas foundthatrelativeadhesionofboth L1210cells andleukocytes onto hydrophilic PVOH and PS-sulfo surfaces was high while theiradhesionontohydrophobicPSsurface waslow (Fig.S1in the Supplementary Data). The increase of adhesion of healthy leukocyteswiththedecreaseofsurfacehydrophobicityisin agree-ment with the literature [6,7,28,46]. Our results were also in agreementwith a previousstudy where adhesionperformance ofL1210cells tothesulfonatedPSsurfacewashigherthanthat of non-sulfonated ones [27]. Regarding to cell selectivity, only PVOHsurfaceshowedselectivitywhereadhesionofhealthy leuko-cyteswashigherthantheadhesionofL1210cells.However,this wasoppositetoourexpectationswhere wetargetedtoprepare thesurfaces onwhich L1210 cells bindmore, whereas healthy leukocytes perform less adhesion. Very low positive selectiv-ity fitting our purpose was obtained only on the flat PS-sulfo surface.

3.2. Surfacecharacterizationofroughpolymerﬁlmsand adhesionofL1210cellsandleukocytesonthesesurfaces

SEMimagesoftheroughpolymersurfaces(HDPE,PPPEand EVA)weregiveninFig.1a.Thepresenceoflargespheruliteswas observed onHDPE coatingwithdiameters of 10–20_␮m,where somenanoﬁbrillarstructureshavingadimensionoflessthan1␮m wereseenatthecenter(Fig.1a).PPPEhadalowsurface rough-nesswithoutanyspheruliteformation.EVAsamplewasﬂatand smallprotrusionsof1–3␮mindiameterweredispersedacrossthe wholesurface.Theorder ofthesurfaceroughness(Rrms)of the

sampleswasHDPE>PPPE>EVA(Table1),which wasconsistent withourpreviouspublication[47]althoughthesurface prepara-tionmethodsarenotthesame.RrmsofEVA,HDPEandPPPEwere

generallysmall.HDPEandPPPEsurfaceswerehydrophobicand EVAwashydrophilicandthehighertheroughness,thehigherthe CAHresultswereobtained(Table1).SFEvaluesofHDPE,PPPEand EVAvariedinasmallrangebetween30.8and34.8mJ/m2₍_±12%

fromthemean).

Adhesionofbothcelltypesontoroughsurfaceswasfoundto berelativelyhigherthanthatontoﬂatsurfaces(Fig.S1).Adhesion ofbothleukocyteandL1210cellswasfoundtobehighestonthe PPPEsurface.SincePPPEandHDPEhadverycloseWCA,CAHand SFEvalues,this differenceincelladhesionperformancemaybe duetotheroughnessvariationorthepresenceofdifferentpattern structuresonPPPEsurfaceratherthanitshydrophobicity(Table1). Positiveadsorptionselectivitywasobservedonalloftherough sur-faceswhereadhesionperformanceofL1210cellsonHDPEandPPPE polymersurfaceswasnearlytwicetheleukocytes.Chemically het-erogeneousEVAandPPPEcopolymersurfacesshowedthehighest celladhesionperformanceinthisgroup.Thisfactrevealsthatrough andchemicallyheterogeneoussurfacescausehighercelladhesion inaccordancewithsomepreviouspublications[1–3,12,13,48].In summary,weobtainedpositiveselectiveadsorptiononroughand chemicallyheterogeneoussurfaceswhencommercialpolymersare usedhowevertheselectivityratioofL1210/leukocytewas insufﬁ-cient.Then,wedecidedtoprepareheterogeneoussurfaceshaving

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Table1

Polymertype,watercontactangle,surfacefreeenergyandRrmsroughnessresultsonﬂatandroughpolymersurfaces.

Samplecode Polymertype Contactangle(±1◦₎ _␥TOT_(mJ/m2₎ _R_(rms)

e adv rec CAH

PVOH PVOH(100%) 46 48 38 12 34.8 N.A.

PS PS(100%) 93 96 79 17 40.6 N.A.

PS-sulfo Sulfonated-PS 55 57 23 32 52.5 N.A.

HDPE HDPE(100%) 105 114 87 27 32.9 1.07

PPPE PPPE(100%) 106 107 83 24 30.8 0.63

EVA EVA-12(100%) 85 94 81 13 34.8 0.28

PS50/HDPE50 PS/HDPE(50:50) 101 107 84 23 N.A. N.A.

Table2

Polymertype,watercontactangleandRrmsroughnessresultsonPS,PPPEhomopolymersandPS/PPPEmicro-patternedpolymerblendsurfaceshavingdifferentblend

compositionobtainedby10vol.%non-solvent(EtOH)addition.

Samplecode Polymertype Contactangle(±1◦₎ _R_(rms)

e adv rec CAH

(PPPE)EtOH-10 PPPE(100%) 106 112 89 23 N.A.

(PS10/PPPE90)EtOH-10 PS/PPPE(10:90) 106 110 85 25 0.14

(PS)EtOH-10 PS(100%) 100 101 87 13 N.A.

speciﬁcpatternstructuresusingpolymerblendsinsteadofusinga singlepolymer.

3.3. Surfacecharacterizationofmicro-patternedPS50/HDPE50 polymerblendanditsadhesionperformanceforL1210cellsand leukocytes

WeobtainedPS50/HDPE50blendsurfacewhichcontains circu-larPSislandsonitwithdiametersof5–15␮m(Fig.1a).Surface morphologyofPS50/HDPE50blendwassimilartothereportsgiven

intheliteraturewherethediametersofcircularPSpatternswere between 2 and 20␮m depending on the synthesis conditions [49,50].Ingeneral,thepolymerwithlowerMwandSFE concen-tratesonthesurfacetominimizetheinterfacialtensionbetween polymer/airinterfaceandthusHDPEusuallycoversthesurfaceof PS.WCAandCAHresultsonthePS50/HDPE50surfacewasfoundto

bebetweenPSandHDPE(Table1).

AdhesionofL1210cellswashighonPS50/HDPE50surface(Fig.

S2a)however,theadhesionofhealthyleukocytesonthisblend sur-facedecreasedapproximately2-foldwhencomparedwithHDPE surfaceandthepositiveselectivityratioofL1210cells/leukocytes increasedfrom1.8to3.5onPS50/HDPE50 (Fig.S2b).L1210cells

donotliketoadheretoPSsurfacesandtheyprefertoadhereonto HDPEregionshowever,thepresenceofcircularPSislandsincreased theadhesionofL1210cellsmorethanthatofaHDPEsurface.Then, itisassumedthatmostoftheblendsurfacewascoatedwithHDPE includingthetopofthePSislands,probablywithaverythinHDPE layeronthemandthepresenceofthesecircularHDPEpatterns

causedtheincreaseoftheadhesionofL1210cellsandthedecrease ofleukocytes.Thus,thesizeandseparationdistancebetween cir-cularPSislandsareimportantfactorsaffectingtheadhesionofboth celltypes.Thenwedecidedtosynthesizenewpatternedsurfaces havingvaryingislanddiametersandseparationdistances. 3.4. Surfacecharacterizationofmicro-patternedPS/PPPEpolymer blendsﬁlmswithvaryingblendcompositionandadhesion performanceofL1210cellsandleukocytesonthesesurfaces

Dissolutionof HDPE in an organicsolventis a difﬁcult pro-cessandwehadtousexyleneandheatuptohightemperatures topreparePS/HDPE blend solution.However,commercialPPPE copolymermaybeusedinsteadofHDPEandsincePPPEcanbe eas-ilydissolvedinTHFat60◦C.Then,wepreparedPS/PPPEpolymer blendsinTHFbyvaryingtheblendcompositionsas10:90,20:80, 30:70,50:50and70:30whereweaddedaconstant10vol.%EtOH intothesolutionforbetterphaseseparationduringdipcoating. SEMimagesofthesesurfacesweregiven(Fig.1b)anddiameters ofthecircularPSmicroislandsonPS/PPPEblendswerefoundto bebetween1and10␮mandwerelessthantheonesformedon PS/HDPEblend.

AllWCAresultsofPS/PPPEblendswereveryclosetoeachother andalsoclosetotheWCAofPPPEsurfaceobtainedby10vol.%EtOH additiontoitsTHFsolution(Table2)indicatingthatWCA’swere notaffectedbythepresenceofcircularPSislands.Thismaybe duetothecompletecoverageofcircularPSislandsbyPPPEduring ﬁlmformationandtheseblendsurfacesmaybeassumedtohavea

Table3

Polymertype,EtOHcontent,watercontactangleandRrmsroughnessresultsonmicro-patternedpolymerblendsurfaceswithaconstantPS40/PPPE60andPS20/PPPE80

compositionswheredifferentvol.%non-solvent(EtOH)wasadded.

Samplecode Polymertype EtOH(vol.%) Contactangle(±1◦₎ _R_(rms)

e adv rec CAH

(PS40/PPPE60)EtOH-4 PS/PPPE(40:60) 4 105 110 92 18 0.22

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Fig.1. (a)SEMimagesofﬂatandroughhomopolymer,copolymerandPS50/HDPE50polymerblendﬁlms;(b)micro-patternedPS,PPPEhomopolymersandPS/PPPE

micro-patternedpolymerblendsurfaceshavingdifferentblendcompositionobtainedbytheadditionof10vol.%non-solvent(EtOH)toTHFsolution.

completePPPEupperlayerwherethesurfaceroughnessand micro-patterningwereimposedbycircularPSislands.Thisisreasonable becauseSFEofPPPEwas30.8mJ/m2_and_much_less_than_SFE_of_PS_of 40.6mJ/m2_(Table₁₎_and_PPPE_having_the_lower_SFE_may_prefer_to concentrateonPS/airinterface.Itwasfoundthatthediametersof thecircularPSislandsonthesurfacecanbecontrolledbychanging theratioofPS/PPPEblendwhile10vol.%EtOHwasaddedwhere PSdiametersincreasedfrom1to10␮mwiththeincreaseofPS componentintheblendupto50%andthendecreaseddownto 2–4␮mabove50%.

L1210celladhesionincreasedonPS/PPPEblendsurfaceswith theincrease ofthe diameterofthe circularPSislands whereas leukocytesadhesionwereclosetoeach otheronthesesurfaces regardlessof thesizeof PSislands(Fig.2a).Allof thePS/PPPE polymerblendsurfacesshowedpositiveselectivitytowardL1210 cellsinagreementwithourobjective.L1210/leukocyteselectivity ratiorangedbetween2.0and9.5ontheseblendsurfaces(Fig.2b). (PPPE)EtOH-10 surface performed thehighest positive selectivity

andL1210cellsadheredtothissurface9.5timesgreaterthanthe healthyleukocytes.Mainreasonofthishighselectivityratiowas thelessadhesionofleukocytes.Themostsuccessfulsecond sur-facewasfoundtobe(PS20/PPPE80)EtOH-10whichshoweda7.8-fold

positiveselectivity.Bothofthesesurfaceshavesmallmicroislands whichallowtheadhesionofL1210cellsbutpreventsadhesionof leukocytes.Theincreasein sizeofcircularPSislandscausedan

increase oftheadhesionofboth L1210cellsand leukocytesfor (PS50/PPPE50)EtOH-10sample.Itisobviousthatthesurface

morphol-ogyarisingfromphaseseparation hasanimportantroleonthe cell/surfaceadhesionperformance.

As given in the introduction section, when blood contacts withasubstrate,thesubstrateis quicklycoveredwithproteins present in the serum and differences in topography, chemical composition, and wettability in the substrate surface result in changesintype,conformationandamountoftheadsorbed pro-teins[20].Whensurfaceroughnessincreases,proteinadsorption alsoincreasesduetotheincreaseofthetotalsurfacearea. Com-positionandconformationofproteinsaredifferentonthesurface dependingonthehydrophobicityandroughnessofthesurface. Every cell has a different combination of adhesion molecules expressedontheirsurfaceandthevariationsinproteinsonthe sub-stratearerecognizedbydistinctcelladhesionmolecules;hence, cell adhesionis highly dependent oncell type. Moreover, can-cer cells are knownto alter their adhesionmolecules in order to invade and metastasize [22–25]. Thus, surface receptors of healthyleukocytesandL1210cancercellsmightbedifferentand onlya speciﬁccelltypeis allowedtoattachtoa substrate sur-facehavingspeciﬁcsurfacetopographyand composition.Inour work, we can only report which cell type prefers to attach to whichsurfacetypedependingonitsmorphology,roughnessand hydrophobicity.

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Fig.2. (a)L1210andleukocyterelativecelladhesion;(b)L1210/leukocytecell adhe-sionratioformicropatternedroughﬁlmspreparedfromPS/PPPEpolymerblends withdifferentblendcompositionsobtainedbytheadditionof10vol.%non-solvent (EtOH)toTHFsolution.

3.5. Effectofnon-solventaddition:surfacecharacterizationof micro-patternedPS40/PPPE60andPS20/PPPE80polymerblend

ﬁlmswithvaryingnon-solventadditionandadhesion performanceofL1210cellsandleukocytesonthesesurfaces

Additionof non-solventresultsin the variation ofPS island sizesonthePS/PPPEblendsurfaces.Wepreparedpolymerblend

Fig.4. (a)L1210andleukocyterelativecelladhesion;(b)L1210/leukocytecell adhe-sionratioformicropatternedroughﬁlmspreparedusingconstantPS40/PPPE60blend

compositionandaddingdifferentvol.%ethanol.

surfaceswithaconstantPS40/PPPE60compositionwheredifferent

amountsofEtOH(4,10,14,and18vol.%)wereaddedand WCA andRrmsresultsonthesesurfacesareshowninTable3.Allthee

andadvvalueswereveryclosetoeachotherandalsoclosetothe

valueofPPPEcopolymerasseeninthistable,whereasrecreduced

andcorrespondinglyCAHincreasedbytheadditionofEtOHwhich

Fig.3.(a)OpticalmicroscopeandSEMimagesofmicro-patternedﬁlmspreparedusingconstantPS40/PPPE60polymerblendcompositionbutaddingdifferentvol.%ethanol; (b)micropatternedﬁlmspreparedusingconstantPS20/PPPE80polymerblendcompositionbyaddingdifferentvol.%ethanol.

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Fig.5. (a)SEMimagesofL1210cells;(b)actinstainingofL1210cellsbyphalloidin.Red:actinﬁbers.(Forinterpretationofthereferencestocolorinthisﬁgurelegend,the readerisreferredtothewebversionofthisarticle.)

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increasedtheroughnessandheterogeneityofthesurfaceduring phaseseparation.DiametersofcircularPSislandswerefoundina rangeof5–18␮m(Fig.3a)andinitiallyincreasedwiththeadded EtOHamount,howeverthenstayedapproximatelyconstantafter 10vol.%.

ResultsofcelladhesionsofL1210cellsandleukocytesontothe blendsurfaceswithaconstant PS40/PPPE60composition, where

differentamountsofEtOHwasaddedwerecompared with sin-gle(PS)EtOH-10and(PPPE)EtOH-10surfaces(Fig.4).Adhesionvalues

ofL1210cellswereclosetoeachotheronthesesurfacesexcept (PS40/PPPE60)EtOH-18surface(Fig.4a).AllofthePS40/PPPE60blend

surfacesshowedpositiveselectivitytowardL1210cellswhereratio ofL1210/leukocyteranged between4.2and 6.0 onthese blend surfaces(Fig.4b).Thehighestselectiveadsorptionwasfoundon the(PS40/PPPE60)EtOH-18 surface where theseparation distances

betweencircularPSislandswereslightly higherthanother sur-faces.rec valueforPPPEwasfoundtobe83◦(Table1)andthis

valuewasveryclosetothatof(PS40/PPPE60)EtOH-18of84◦(Table3),

indicatingthatPPPEcopolymercoatedthetopofthePS microis-landsduringﬁlmformation.Again,itwasfoundthatthesizeand geometryofmicroislandshasanimportantroleonthecell/surface adhesionperformance.

TheaboveworkwasrepeatedbypreparingPS20/PPPE80blends

inordertoseetheeffectof EtOHadditionontothesizesofPS microislands.DiametersofcircularPSmicroislandsdecreaseddue totheincreaseofthenucleationratewiththeuseofEtOHand variedbetween0.5and4.0␮m(Fig.3b).ThelargestPS microis-landswereseenon(PS20/PPPE80)EtOH-14 surface. WCAandRrms

roughnessresultsonmicro-patternedpolymerblendsurfaceswith aconstantPS20/PPPE80compositionwheredifferentamountsof

EtOH(6,10,and14vol.%)wereaddedarealsoshowninTable3. SimilartotheresultspresentedaboveforPS40/PPPE60blends,e

andadvvaluesofthePS20/PPPE80blendswereveryclosetoeach

otherandalsoclosetothevalueofPPPE(Table3)whereasrecvalue

reducedconsiderablyandcorrespondinglyCAHvalueincreasedby theadditionofEtOH.

RelativecelladhesionofL1210cellsandleukocytestotheblend surfaceshavingaconstantPS20/PPPE80compositionwithdifferent

amountsof ethanol addition wasdetermined(Fig. S3). Positive selectivitytowardL1210cellswasseenonallofthePS20/PPPE80

blendsurfaces.RelativeadhesionvaluesofL1210cellsincreased linearlywiththeincreaseofEtOHaddition,howevertheadhesion of leukocytes were close to each other on these surfaces (Fig. S3a).ThistrendissimilartotheincreaseofL1210celladhesion withtheincreaseofEtOHaddition(rightsideofFig.4a).Positive selectivityratioofL1210/leukocyterangedbetween5.0and8.0on theseblend surfaces(Fig. S3b).Thehighestselectiveadsorption wasfound on (PS20/PPPE80)EtOH-14 surface. The increase of the

circularPSdiameterfrom1to3.5␮mwiththeincreaseof non-solventadditionprovidedapositiveeffectonincreasingadhesion tendencyofL1210cells.

3.6. CharacterizationofL1210cellmorphologies

Uponbindingtoasubstratecellsstarttosynthesize extracellu-larmatrixproteins.Longtermadhesionbehaviorsofcells,which determinethemorphologiesandorganizationofactin cytoskele-tonsofcells,dependontheinteractionofsurfaceboundproteins andcells[51].InordertoinvestigatetheactincytoskeletonofL1210 cells,theywerestainedwithphalloidintodisplayF-actin orga-nization(Fig.5a).L1210cellsdisplayedactincytoskeletononall surfacesafter48h.SEManalyseswerealsoperformedtoinvestigate compatibilityofthesurfacestothecellsinmoredetail.These anal-ysesdemonstratedthatL1210cellsmaintainedtheirmorphology overallsurfaces(Fig.5b).Themorphologicalstudieshaveshown thatcellsgainedtheirexpectedmorphologiesattheendof48hand

maintainedtheirnaturalcytoskeletalorganization.These experi-mentsshowthatsynthesizedpolymerthinﬁlmsarebiocompatible surfacesforcellstudies.DiametersofL1210cellsusedinthisstudy wereapproximately5␮m(Fig.5a)andtheaveragediameterof aleukocyteis10␮m[52].Thus,theenhancedadhesiontendency ofleukocytesthanL1210cellsonPPPE/PSpolymerblendsurfaces withsmallmicro-patterndiametermightbecausedbythesize differencebetweenthesecelltypes.

4. Conclusions

Wepreparedpolymerblendsurfacesbydipcoatingontoglass wheremicroislandswereformedonthesurfacebyphase separa-tion.OurintentionwastoadsorbL1210leukemiacellsselectively muchmorethanthatofthehealthyhumanleukocytesonthese speciﬁcsurfaceswhichweremadeofcommercialpolymers.We determinedthesurfacepropertiesand relativeadhesion perfor-manceofthecellsonthesesurfaces.Itwasfoundthatthecontrol on thediameters of circular PS microislands on the surface is possibleespeciallywhenPS/PPPEblendsarepreparedusingTHF as solvent and EtOH as non-solvent. The top of the PS micro-islands was coated with a thin layer of PPPE having a lower SFEthan thatof PSduringﬁlmformation. Thesesurfaceswere testedfortheireffectoncellviabilitythroughMTT tests.It was foundthat all of the PS/PPPE blend surfaces showed consider-ablepositiveselectivitytoward L1210cells.The increaseinthe amountofEtOHincreasedboththediametersofPSmicroislands andrelativeadhesionofL1210cellsonthesesurfaces.However, healthyleukocytesdidnotshowthistrendandtheirrelative adhe-sionvalueswereclosetoeachother.PositiveselectivityofL1210 cells/healthyleukocytesratioapproachedto9-foldinvitro.The sur-facesshowingthehighestL1210/leukocyteselectivityratiowere (PPPE)EtOH-10,(PS20/PPPE80)EtOH-10 and (PS20/PPPE80)EtOH-14.The

diameterofcircularPSmicro-islandsonthesesuccessfulsurfaces variedbetween1.0and3.3␮m.Itwasalsoshownthatthesizes ofcircular micro-islands onthesurfacehave animportant role ontheL1210cell/surfaceadhesionperformance.Itispossiblethat theconformationandamountoftheadsorbedserumproteinson thesespeciﬁcblendsurfacesaredifferentdependingonthesizeof micro-islandsandthevariationsinproteinsonthesubstrateare recognizedbydistinctcelladhesionmoleculesdependingonthe typeofthecell.Thus,L1210leukemiacellsorhealthyleukocytes showeddifferentadsorptionbehavioronthesesurfaces.These ﬁnd-ingsmaybeusedtodevelopnewtreatmentoptionsforpeoplewith leukemiawithsuitablescale-up.

Acknowledgement

TheauthorsgratefullyacknowledgethesupportfromThe Scien-tiﬁcandTechnologicalResearchCouncilofTurkey(TUBITAK)under theproject“Synthesisand useofmicro/nano patterned chemi-callyheterogeneoussurfacesfortheselectiveadsorptionofL1210 leukemiacells”(ProjectNo:MAG-110M181).

AppendixA. Supplementarydata

Supplementarymaterial relatedto this articlecanbe found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.09.040.

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