Vinyl alcohol to alginic acid molar ratio

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ContentslistsavailableatScienceDirect

International

Journal

of

Biological

Macromolecules

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / i j b i o m a c

In

vitro

hemocompatibility

of

PVA-alginate

ester

as

a

candidate

for

hemodialysis

membrane

Choirul

Amri

∗

_,

_Mudasir

_Mudasir,

_Dwi

_Siswanta,

_Roto

DepartmentofChemistry,FacultyofMathematicsandNaturalSciences,UniversitasGadjahMada,SekipUtara,Yogyakarta55281,Indonesia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3March2015

Receivedinrevisedform18May2015

Accepted7October2015

Availableonline13October2015

Keywords:

Invitrohemocompatibility

PVA-alginateester

Hemodialysismembrane

a

b

s

t

r

a

c

t

Alginatebasedbiopolymerwithimprovedphysicalandchemicalpropertiesafteresterificationusing polyvinylalcohol(PVA)hasbeenstudiedforpossibleapplicationasahemodialysismembrane.The alginicacidtovinylalcoholmolarratiowaspredeterminedat0,0.1,0.5and1.Mechanicalstrength, hydrophilicityandCa2+_adsorption_of_the_membrane_before_and_after_modification_were_evaluated._The

obtainedPVA-alginate(PVA-Alg)estermembranewasalsoconfirmedusingFTIRandSEM.Itshows thatthePVA-Algmembranetensilestrengthishigherthanthatofnativealginate.Thewatercontact angleofthemembranewasfoundtobearound33–50◦_._The_Ca2+_adsorption_capacity_tends_to_decrease

withtheincreaseinmolarratio.Furthermore,themodifiedPVA-Algestermembraneachievesbetter proteinadsorptionandplateletadhesionthantheunmodifiedone.Italsoexhibitsadialysis perfor-manceof47.1–50.0%forclearanceofureaand42.2–44.6%forclearanceofcreatinine,respectively.Itis expectedthatthisPVA-Algestermaychallengecelluloseacetateforpotentialapplicationashemodialysis membranes.

1. Introduction

Hemodialysisisanessentialmedicaltreatmenttoremovetoxic

compounds in the blood serum of a patient with severe renal

disease.Themaincomponentofa hemodialysisinstrumentis a

semipermeablemembranethatallowsselectivetransportoflow

molecularweightsolutespresentinthebloodserumi.e.ureaand

creatinine[1,2].Celluloseacetateis oneofthefirst generations

of natural biocompatiblepolymers usedto prepare

hemodialy-sismembranes [3,4].Another natural polymer being evaluated

as a membrane material and expected to compete with

cellu-loseacetateisalginate.Thisnaturalpolymerhasattractedmany

researchers in this field due to its high biocompatibility and

lowtoxicity.It alsohasotherreasonablecharacteristicssuchas

biodegradability,having a rigid molecularchain, flexibility and

abilityto formfilm. Thispolymer canform aninsolublegel in

water, which is important for membrane preparation. Alginate

alsopossessesbothhydroxylandcarboxylgroupsinitsstructure

thatcanpossiblyfacilitatefurtherstructuremodification[5–12].

Basedonthosequalities,alginatecanbeconsideredasthefutureof

∗Correspondingauthor.

E-mailaddress:chamri@hotmail.com(C.Amri).

hemodialysismembranependingsignificantimprovementofboth

physicalandchemicalproperties.

A hemodialysis membrane usually has good mechanical

strength,permeabilityforwaterandsolutesaswellas

hemocom-patibility[13].Sincealginatehasbothhydroxylandcarboxylate

groupsinthestructure,itmaybeabletoformchannelsfortoxic

uremicofureaandcreatininethrougha hydrogenbond.

Unfor-tunately,thealginatemembraneisnormallyfoundinawetstate

becausethepresenceofhydroxylandcarboxylgroupsinthe

struc-turecanattractexcessivewatermoleculesthateventuallylowerits

mechanicalstrength.Therefore,themechanicalstrengthofalginate

membranemustbeimproved.Onewaytoimproveitsmechanical

strengthisbychemicalmodificationwithamuchstrongerpolymer

backbonewithoutlossofbiocompatibility.

Inthisstudy,PVAisusedaspolymermodifier.PVAhas

excel-lent mechanical strength and biocompatibility.This polymer is

alsonon-toxic[9–11].ThePVAhydroxylgroupsareexpectedto

reactwithcarboxylgroupsofalginatetoformanesterderivative.

Themechanicalstrengthoftheproducedmembraneisthoughtto

improveduetotheesterificationreaction.Thecarboxylgroupsin

thestructureofnativealginatecaninteractwiththeaminegroups

ofproteincausingmuchmoreproteintobeadsorbed.However,

themembraneproteinadsorptionandplateletadhesionare

pre-dictedtobelowaftersuccessfulesterification[12].Therefore,the

esterification of alginateby PVA can resultin an improvement

http://dx.doi.org/10.1016/j.ijbiomac.2015.10.021

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ofhemocompatibilityasimpliedbyadecreaseinsurfaceprotein

adsorptionandplateletadhesion.

2. Experimental

2.1. PreparationandcharacterizationofPVA-alginateester membranes

Sodiumalginate(2%aqueoussolutionwithviscosityof250cps

atroomtemperature)waspurchasedfromSigmaandPVA

(CAS-No.9002-89-5withdegreeof hydrolyzation>98%)wasacquired

fromMerck.Thesechemicalswereusedwithoutfurthertreatment.

AlginatewasreactedwithPVA toformaPVA-Alg ester

deriva-tive.ThePVA-Algmembranewaspreparedaftertheesterification

reactionwascompleted.Analiquotof10.0mLofaqueoussodium

alginate(2%w/v)andPVAwithpredeterminedweight(0,7.7,38.6

and77.2mg)waspouredintoa6-cmPetridishtoformamixture

andgivevinylalcohol toalginicacida molarratioof0,0.1,0.5,

and1.0.Themixturewasstirredfor30minandwasallowedto

coolinarefrigeratorat4◦_C_for₂₄_h_to_eliminate_air_bubbles._The

alginatemixturewasdriedat80◦_C_for₈_h_before_the_addition_of

10.0mLHCl1.0M.Themixturewasincubatedfurtherat40◦_C_for

1h.Theresultingfilmwaswashedwithdistilledwateranddriedat

40◦_C_for₂₄_h._The_FTIR_spectra_were_obtained_on_a_Shimadzu_FTIR

spectrometerinwithawavenumberrangeof400–4000cm−1_.

2.2. Mechanicalstrengthmeasurement

Thefilmwascutto2-cm×11-cmsize.Themechanical

prop-ertiesofthemembraneweremeasuredusingauniversaltesting

machine.ThestressappliedismeasuredinMPawhiletherateisin

mm/min.

2.3. Hydrophilicitytest

Filmhydrophilicitywasestimatedbasedonthedataofwater

contactanglemeasurement[14].Adriedfilmwithflatsurfacewas

placedonaglassslide.Adropofwater(10.0␮L)waslaidonthetop

ofthefilmfrom1-cmabove.Thewatercontactanglewasrecorded

everyminuteforthefirst10minandevery5minafterthat.The

watercontactanglewasdeterminedbasedontheimageproduced.

2.4. MeasurementofCa2+_adsorption

Aportionof100-mgofthefilmwassoakedin10.0mLsolution

of0.025MCaCl2.ThemembraneCa2+adsorptionwascalculated

basedontheCa2+_{concentration}_in_the_solution_after_15,_30,_60,

120,and180minofsoaking.

2.5. Hemocompatibilitytest

Hemocompatibilityof themembranewasexaminedby

con-ductingatestforhemolysisratio,proteinadsorption,andplatelet

adhesion. Human whole blood (WB) with an anticoagulant of

sodiumcitratewasusedforhemolysistesting.Samplesofplatelet

richplasma(PRP)wereobtainedbyseparationofWBwith

spin-ningrateof1000rpmfor10minusinga centrifuge.Samplesof

plateletpoorplasma(PPP)wereobtainedbyseparationofWBwith

spinningrateof3000rpmfor15minusingacentrifuge.

Forthehemolysisratiotest,a1×1cm2cutmembranewas

pre-paredand washed threetimes withdoublydistilled water and

0.90%NaClsolution.Themembranewassoakedin0.9%NaCl

solu-tionfor 30minat 37◦_C, _then _soaked _in _the_WB _mixture ₍₅_mL

solutionof0.9%NaCland20␮LWB)at37◦Cfor15min.Thesoaking

timevariedfrom30,45,and60min.TheWBmixtureswere

sepa-ratedusingacentrifugewithspinningrateof1500rpmfor10min.

TheUV–Visabsorbanceofthesolutionwasmeasuredat546nm.

Thehemolysisratio(HR)waslatercalculatedusingEq.(1)[15].

HR=

(AS−AN)

(AP−AN)

(1)

whereAS istheabsorbanceof samples,ANis theabsorbanceof

negativecontrol,andAPistheabsorbanceofpositivecontrol.

For protein adsorption test, a 2×2cm2 cut membrane was

immersedin1mLPPPandincubatedat37◦_C_for₁_h._It_was

sub-sequently rinsed withPBS solution and double distilled water.

The membrane was washed with 2% sodium dodecyl sulfate

(SDS)toremovetheadsorbedprotein.Theproteinconcentration

in thewashingsolutionwasdetermined bythebiuret

spectro-photometricmethod.

Forplateletadhesiontest,a2×2cm2cutmembranewasused.

AfterwashingwithPBSbuffersolution(pH7.4),themembranewas

immersedin1mLPRPat37◦_C_for₁_h._The_platelet_{concentration}

beforeandafterimmersionwasdeterminedusinga

hemocytome-ter(Neubauer,Germany).Themembranewasrinsedthreetimes

usingPBStoremovelightlyadsorbedplatelets.Anaqueoussolution

of2.5%glutaraldehydewasadded(1.0mL)intothesolutionandit

wasallowedtosettleforonenighttofixtheadsorbedplatelets.The

samplesweredehydratedstepwiseusingethanol/watersolutionof

25%,50%,75%,100%(v/v)for10mineach.Theplateletadhesionon

themembranesurfacewasobservedbySEMafterfreezedrying.

2.6. Dialysissimulation

Themembranewasfixedintothedialysisapparatusbetween

two compartments with effective diffusion area 3.14cm2_. _The

sourcecompartmentwasfilledwith30mLofPBSsolution

con-tainingureaandcreatinine.Theureaandcreatinineconcentration

inthesolutionis200mg/dLand5mg/dLrespectively.Thedialysate

compartmentwasfilledwith30mLofblankPBS(pH7.4).The

con-centrationofureainbothcompartmentsafter0,1,2,3,and4h

ofdialysistestwasdeterminedbyphenolblueenzymaticmethod

andcreatininewasdeterminedbypicricalkalimethod.Theurea

andcreatinineclearanceduringdialysistestwascalculatedusing

Eq.(2)[16].

whereSCisthesoluteclearance;C0andCtarethesolute

concen-trationsinthetestingreservoir solutionattheprescribedtime,

respectively.Thefluxofureaandcreatininewasdeterminedusing

Eq.(3)[17].

J=W

At (3)

whereJisthefluxofthesolute(mgcm−2_h−1_);_W_is_the_mass_of

thediffusesolute(mg);Aisthediffusionarea(cm2_),_and_t_is_the

diffusiontime(hour).

3. Resultsanddiscussions

3.1. Characterizationofmembranesurface

TheFTIRspectraofthePVA-AlgmembranesareshowninFig.1.

Thepeaksat1735and1250cm−1_are_observed._These_are_attributed

totheC OstretchingandC Ostretching,respectively.Thisis

con-sistentwiththeFTIRspectraofanester.Thenativealginateitself

hasapeakat1620cm−1_due_to_the_C _O_stretching_of_COOH_group.

Thesuccessoftheesterificationreactionisclearforthemembrane

withvinylalcoholtoalginicacidmolarratioof0.5orhigher.The

peakseen at3433cm−1 _is _due_to_O _H_stretching,_with_sharper

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500

% Transmittance (a.u.)

Wavenumber (cm-1₎

a

d

c

b

Fig.1.FTIRspectraofpreparedPVA-Algmembranes;nativealginate(a),PVA-Alg

withmolarratioof0.1(b),0.5(c),and1.0(d).

Fig.2.SEMimagesofPVA-Algmembranesinstateofdry(A),andinstateofwet

afterdiffusionuse(B).

itssharppeak atabout3400cm−1_._The_FTIR_data_are_needed_to

ascertaintheformationofcross-linkpolymerproductofPVA-Alg

ester.Theremaininghydroxylgroupsandcarboxylgroupsfrom

respectedpolymerscouldhelptransportureaandcreatinineacross

themembrane.TheFTIRspectrasuggestthathydroxylgroupsin

PVAandcarboxylgroupsinalginatedonotentirelyformesters.

Tounderstandthemorphologyofthemembranesurface,SEM

imageswererecorded.TheSEMimagesofPVA-Algfilmsareshown

inFig.2.Itisobservedthatthesurfaceofthemembraneisflatand

hasnovisiblecracks.However,theinnerstructureofthemembrane

islessuniform.Theestimatedporesizeisaround1␮m.Afterbeing

usedinthetransportexperiment,itdoesnotshowfouling,although

ingeneralitshowsswellinganderosion.

3.2. Mechanicalstrengthofmembranes

Mechanicalstrengthofthemembraneisacriticalfactorto

con-siderfordialysisapplications.Foramembrane,tensilestrengthis

expressedinMPawhereaselongationisexpressedinpercent[18].

Theresultsoftensilestrengthandelongationmeasurementsare

0

Fig.3. TensilestrengthofPVA-Algmembranesinstateofdry(a),andwet(b).

0

Vinyl alcohol to alginic acid molar ratio a

b

Fig.4. ElongationofPVA-Algmembranesinstateofdry(a),andwet(b).

showninFigs.3and4,respectively.Themembraneofunmodified

alginatehasatensilestrengthofonly19.5MPa.Ontheotherhand,

thePVA-Algmembraneatamolarratioof0.1,0.5,and1.0each

givestensilestrengthof30.8,35.9and38.5MPa,respectively.The

presenceofPVAinthealginatefilmappearstoimproveitstensile

strengthsignificantly.Themaximumtensilestrengthwasobtained

fromPVA-Algfilmatmolarratioof1.0.

TheadditionofPVAtoalginateseemstoimprovethemembrane

elongation,indicating thatPVA hasa chemicalinteraction with

alginate.Theexplanationforthisbehavioristhatalginatereacts

withPVAtoformanesterderivative,assuggestedbyFTIRdata.

Inadrystate,theinteractionbetweenalginateandPVAcausesan

increaseinbothmembranes’tensilestrengthandelongation.The

wetPVA-Algmembranehasalowtensilestrengthbuthigh

elon-gation,whichcouldbeduetothemembrane’splasticizingeffect.

Wangetal.observedadecreaseintensilestrengthofthemembrane

preparedusingchitosan-celluloseblendsfrom55MPaforwetfilm

to35MPafordryfilm.Meanwhile,theelongationincreasesfrom

9%forwetfilmto15%fordryfilm[15].

3.3. Hydrophilicityofmembrane

Hydrophilicityofthemembranesurfacecanbeevaluatedfrom

theresultsofwatercontactanglemeasurement.Thelowwater

con-tactangleindicateshighhydrophilicityofthemembranesurface

andviceversa[19].Thedataofmembranewatercontactangle

mea-surementsareshowninFig.5.After30minofdropage,thenative

alginategiveswatercontactangleof14◦_,_which_is_low._The

PVA-Algmembraneswithvinylalcoholtoalginicacidmolarratioof0.1,

0.5,and1yieldswatercontactangleof45,50,and30,respectively.

TheadditionofPVAtoalginatecausesthemembranewatercontact

angletodecrease.ItmeansthatPVAisabletoreducehydrophilicity

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Fig.5. HydrophilicityofPVA-Algmembranes;nativealginate(a),PVA-Alginthe

Time (minute)

a

d

c

b

Fig.6.Ca2+_adsorption_by_PVA-Alg_membranes;_native_alginate_(a),_PVA-Alg_with

moleratioof0.1(b),0.5(c),and1.0(d).

3.4. Ca2+_adsorption

TheresultsofCa2+_adsorption_by_the_membranes_are_shown_in

Fig.6.ThemembraneCa2+_adsorption_is_expressed_in_mg_Ca2+

/100-mg of membrane. It shows that the Ca2+ _adsorption _decreases

from3.83-mg/100-mgforthePVA-Algmembraneatmolarratioof

0.1–2.66-mg/100-mgatmolarratioof0.5,andto1.9-mg/100-mgat

molarratioof1.0.Theseresultsrevealthattheincreaseinthemolar

ratiocausesadecreaseintheCa2+_adsorption._The_membranes_with

highmolarratiosdemonstratesmallerCa2+_adsorption_than_that

ofmembraneswithlow molarratios.TheCa2+_ions_form_strong

electrostaticbondswithcarboxylgroupsintheoriginalalginate

structure.Asexpected,thenativealginatemembranehasmore

carboxylgroupsthanthatofthePVA-Algmembrane.Successful

esterificationreactionisabletoreducethemembraneCa2+

adsorp-tion.Lietal.reportedthatapolyethersulfonemodifiedmembrane

hashighCa2+_adsorption_[20]_.

3.5. Hemocompatibilityofmembrane

Hemolysis ratio (HR) is an important feature of

hemocom-patibility.It isusedtodetecttheerythrocytedamagecausedby

membranematerials.Fig.7showstheHRvalueofdifferent

mem-branes.Itdemonstratesthatafter30minofcontactthePVA-Alg

membranesatmolarratioof0.1,0.5and1haveconsiderablylow

HRvaluesof0.043,0.049and0.058,respectively.

Proteinadsorptionon themembranesis one important

fac-tortoevaluatethehemocompatibilityofthemembranes[21,22].

Hydrophobicinteractionbetweenthemembranesurfaceand

pro-teincanhavegreateffectonthesurfaceproteinadsorption[20].

Inthepresentwork,themembraneadsorptionofplasmaprotein

wasstudiedinvitro.ThedataarepresentedinFig.8.The

PVA-Algestermembranesexhibitlowproteinadsorption.Thelowest

Fig.7.Thehemolysisratioofthemembranes;nativealginate(a),PVA-Algwith

molarratioof0.1(b),0.5(c),and1.0(d).

Fig.8.PlasmaproteinadsorptiononPVA-Algmembraneswithvariousmolarratios.

460

Vinyl alcohol to alginic acid molar ratio

5,9

Fig.9.PercentdegradationofplateletinPRPafterinteractingwith2×2cm2of

PVA-Algmembranesfor1h.InitialamountoftheplateletinPRPis510,000cells/␮L.

proteinadsorptionwasobservedforthefilmwithvinylalcoholto

alginicacidmolarratioof0.5.Factorsthataffecttheinteraction

betweenmembrane surfaceand proteininclude hydrophilicity,

roughness,surfacecharge,surfacefreeenergy,topological

struc-ture,solutionenvironment,andproteintype[1].Thelowprotein

adsorptionmeanslessproteinlossduringhemodialysistrial.

Theplateletadhesiononthemembranesurfaceiscloselyrelated

totheproteinadsorption[1].Fig.9showedthenumberofadhering

plateletsonthePVA-Algmembranes.After1hofcontact,the

max-imumnumberofadheringplateletstothemembraneis3.9%and

1.0%oftheinitialforthemembranewithmolarratiosof0.1and0.5,

respectively.Theinitialplateletconcentrationis510,000-cells/␮L.

Inthisexperiment,a2×2cm2 cutmembranewasapplied.Even

withalowmolarratio,themodifiedmembranesstillhavelower

plateletadhesionthanthatoftheunmodifiedones.

Bloodplasmaplateletadhesiononthemembranesurfaceand

morphologyoftheadheringplateletareusedtoevaluatethe

hemo-compatibilityof a hemodialysismembrane. TheSEM imagesof

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Fig.10.SEMimageofplateletadhesiononthesurfaceofaPVA-Algmembrane.

Dialysis time (hour) a

b c d

b

Fig.11.Performanceofureaclearance(a)andcreatinineclearance(b)ofthe

PVA-Algmembraneswithmolarratioof0(a),0.1(b),0.5(c),and1(d)inthedialysis

simulationexperimentfor1,2,3and4h.

Thrombocyteadhesiononthemembranesurfacetakesplacealong

withproteinadsorption.Therefore,thethrombocytesseemtobe

coveredbytheplasmaprotein,especiallyfibrinogen.

3.6. DialysisperformanceofPVA-Algmembrane

Clearanceofureaandcreatininewasdeterminedtoevaluatethe

dialysisperformanceoftheproposedmembranes.Theclearance

ofureaandcreatininebythePVA-Algmembranesareshownin

Fig.11.Themembraneclearanceefficiencyisevaluatedbasedon

theclearanceofuremiccompoundsi.e.ureaandcreatininefrom

thesourcecompartment.Acomparisonismadebetweenthedata

takenafter1handafter4hofdialysistrial.Itindicatesthatthe

clearanceofureaandcreatinineincreaseswithtime.

Hemodialy-sisforpatientswithrenalfailureisusuallyperformedforabout

4h.ThePVA-Algmembraneswithmolarratioof0,0.1,0.5and1.0

showtheureaclearanceof45.3,47.0,50.0,and49.3%after4hof

dialysistestwhereasthecreatinineclearanceis42.4,42.2,44.6and

Table1

FluxofureaandcreatinineacrossthePVA-Algmembranes.

Vinylalcoholtoalginicacidmolarratio Flux(mgcm−2_h−1₎

Urea Creatinine

0 2.541 0.059

0.1 2.621 0.057

0.5 2.791 0.059

1 2.782 0.060

43.7%,respectively.Ingeneral,thePVA-Algestermembranewith vinylalcoholtoalginicacidmolarratioof0.5showsthehighest clearanceofureaandcreatinine.However,theclearanceofurea andcreatininewithothermolarratioswasnotsignificantly differ-ent.ThefluxofureaandcreatinineacrossthePVA-Algmembrane inthedialysisexperimentwasalsoexamined.Thefluxofureaand creatinineisshowninTable1.

Thecelluloseacetatebasedmembraneisabletoreduceurea

and creatinine contents by 17.2 and 10.8%, respectively. Using

membranespreparedusingcelluloseacetatemodifiedwithurease,

theureaclearanceimprovedfrom45.8to53.2%withthe

creati-nineclearanceof31.2%[2].Thisfigureismuchlowerthanofthe

alginate-basedmembranepreparedinthisstudy.Thefunctional

groups present in thealginate-modified-PVA membranes could

providemolecularchannelsforuremiccompounds.The

molecu-larchannelsformedacrossthemembranecouldbebeneficialin

thedialysisprocess.Inthedialysistests,thePVA-Algmembranes

showclearanceofuremiccompoundsandfluxcomparablewith

thatofcelluloseacetatemembranes.

4. Conclusions

Insearchof abetterbiocompatiblehemodialysismembrane,

alginatehasbeencross-linked withPVAtoform a

correspond-ingester.ThePVA-Algesterbasedmembranesshowhighertensile

strengththanthatofthenativealginatemembranes.Whenthe

alginicacidtovinylalcoholmolarratiowassetto1,theresulting

PVA-Algmembranesshowthehighesttensilestrengthof38.5MPa.

Themembranehydrophobicityalsoimprovesaftermodification

usingPVAasindicatedbyanincreaseinthewatercontactangle.The

membraneCa2+_adsorption_tends_to_decrease_with_the_increase_in

molarratio.Conversely,thehemolysisratiotendstoincreasewith

theincreaseinthemolarratio.ThePVA-Algmembranesexhibit

hemocompatibilitybetterthanthatofnativealginatemembrane

assuggestedbylowproteinadsorptionandplateletadhesion.The

highest hemocompatibilitycharacteristic of PVA-Alg membrane

wasobservedatmolarratioof0.5withureaandcreatinine

clear-anceof50.0%and44.6%,respectively.Meanwhile,thefluxofurea

andcreatinineacrossthemembranewasfoundtobe2.791and

0.059mgcm−2_h−1_,_{respectively.}_In_conclusion,_this_PVA-Alg_ester

maybeconsideredasapossiblesubstituteforcelluloseacetateas

amaterialforhemodialysismembranes.

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