hydrogels Surfactant ion effects and metal on the mechanical properties ofalginate International Journal of Biological Macromolecules

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Surfactant

and

metal

ion

effects

on

the

mechanical

properties

of

alginate

hydrogels

Hakan

Kaygusuz

a,b

_,

_Güls¸

_en

_Akın

_Evingür

c

_,

_Önder

_Pekcan

d

_,

_Regine

_von

_Klitzing

b

_,

F. Bedia

Erim

a,∗

a_Istanbul_Technical_University,_Faculty_of_Science_and_Letters,_Maslak₃₄₄₆₉_Istanbul,_Turkey

b_{Stranski-Laboratorium}_für_{Physikalische}_und_Theoretische_Chemie,_Institut_für_Chemie,_Technische_Universität_Berlin,_Strasse_des_17._Juni_124,₁₀₆₂₃

Berlin,Germany

c_Piri_Reis_University,_Faculty_of_Science_and_Letters,_Tuzla₃₄₉₄₀_Istanbul,_Turkey d_Kadir_Has_University,_Faculty_of_Science_and_Letters,_Cibali,₃₄₀₈₃_Istanbul,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received25March2016

Receivedinrevisedform4June2016 Accepted1July2016

Availableonline2July2016 Keywords:

Alginate Surfactant

Mechanicalproperties

a

b

s

t

r

a

c

t

Thispaperaddressesthecontrolledvariationofthemechanicalpropertiesofalginategelbeadsby

chang-ingthealginateconcentrationorbyaddingdifferentsurfactantsorcross-linkingcations.Alginatebeads

containingnonionicBrij35oranionicsodiumdodecylsulfate(SDS)surfactantswerepreparedwithtwo

differenttypesofcations(Ca2+_,_Ba2+₎_as_{crosslinkers.}_Compression_measurements_were_performed_to

investigatetheeffectofthesurfactantandcationtypesandtheirconcentrationsontheYoung’smodulus

ofalginatebeads.TheYoung’smoduluswasdeterminedbyusingHertztheory.Foralltypesofalginate

gelbeadstheYoung’smodulusshowedanincreasingvalueforincreasingalginatecontents.Addition

oftheanionicsurfactantSDSincreasestheYoung’smodulusofthealginatebeadswhiletheadditionof

non-ionicsurfactantBrij35leadstoadecreaseinYoung’smodulus.Thisoppositebehaviorisrelatedto

thecontraryeffectofbothsurfactantsonthechargeofthealginatebeads.WhenBa2+_ions_were_used_as

crosslinkercation,theYoung’smodulusofthebeadswiththesurfactantSDSwasfoundtobe

approxi-matelytwotimeshigherthanthemodulusofbeadswiththesurfactantBrij35.Anionspeciﬁceffectwas

foundforthecrosslinkingabilityofdivalentcations.

1. Introduction

Alginateisamaterialofinterestduetoitsuniqueanduseful properties.Beingextractedfrommarinebrownalgae,alginatesare non-toxicand ediblepolysaccharides.Thispolymerisa copoly-mer of 1–4 linked ␤-d-mannuronate (M) and ␣-l-guluronate (G)homopolymericblocks.Thispolyelectrolyteformscrosslinked hydrogelswithdivalentcations,andthishydrogelstructureisused inmanyapplications.Applicationsofcrosslinkedalginateshavea widerange,includingcontrolledrelease,drugdelivery formula-tionsandwasteremovalagents[1–5].

Addition of various dopants to alginate formulations may increasethechemicalandmechanicalstabilityofthegels.Oneof thecandidatesfordopantsissurfactants.Previously,theeffectof cationicsurfactantcetyltrimethylammoniumbromide(CTAB)on viscosityand theeffectof SDSonaggregationof alginate

solu-∗ Correspondingauthor.

E-mailaddress:erim@itu.edu.tr(F.B.Erim).

tionwerestudiedbyYangetal.[6,7].Rheologicalandturbidity measurementswerecarriedoutinaqueousmixturesof hydropho-bically modiﬁed alginates with cationic, anionic and nonionic surfactantswerealsoreportedbefore[8].

TheeffectofdifferentcrosslinkingcationsonYoung’s modu-lusvaluesofalginatebeads[9],theeffectofcompressionspeed [10],theeffectsofM/Gratioofalginateandofthecrosslinking cationtype[11]onmechanicalbehaviorhavebeenreportedbefore. Recentlytheeffectsofthecrosslinkingionandpolyaminoacid coat-ingonthemechanicalpropertiesofalginatebeadswerereported [12].Besidesbeads,theeffectofthecrosslinkingiononthe mechan-icalpropertiesofdisc-shapedalginatesarealsoreported,suchas therecentstudybyKaklamanietal.[13].

Theimportanceofthealginatematerialsinbiomedical appli-cations such as drug release studies and scaffolds for tissue engineering requires mechanical strength of thesegels. Surfac-tantsplayanimportantrolefortheuptakeandreleaseofdrugs. Accordingtoourknowledge,sofartheeffectofsurfactantonthe mechanicalpropertiesofalginategelbeadshasn’tbeenstudied. Thispaperreportsfortheﬁrsttime,theeffectofsurfactant incor-http://dx.doi.org/10.1016/j.ijbiomac.2016.07.004

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Fig.1.Schematicrepresentationoftheunaxialcompressionmeasurement.

poration intoalginate gelsonthe Young’s modulusof alginate beads.Twodifferenttypesofsurfactants (nonionic:Brij35 and anionic:sodiumdodecylsulfate)wereused.Surfactantadded algi-nateswerecrosslinkedbycalciumorbariumions.Theeffectof crosslinkingionsonYoung’smoduluswasalsostudied.

2. Materialsandmethods

Alginicacidsodium salt (viscosity of 2% solution∼250 cps) wasfromSigma-Aldrich.Thisalginateisextractedfrom Macrocys-tispyriferaandhasaM/Gratioaround1.6[14].Calciumchloride dihydratewaspurchasedfromJ.T.Baker.Brij®35,sodiumdodecyl sulfate(SDS)andbariumchloridedihydratewereobtainedfrom Merck.Allreagentswereusedwithoutfurtherpuriﬁcation.The criticalmicelleconcentration(cmc)ofBrij®35isabout0.09mmol/L andabout8mmol/LforSDS.Inthepresentstudybothsurfactants wereusedwellabovetheirrespectivecriticalmicelle concentra-tions(cmc).

Accuratelyweighedalginatewasdissolvedindeionizedwater andnecessarilyamountsofsurfactantswereaddedintothealginate solutions.Thesolutionswerestirredcarefullyinordertoprevent bubbleformation.ThepHofthe1%alginatesolutionwasaround 6.7.AdditionofSDSintoalginatedidnotchangethepH signiﬁ-cantly.Ontheotherhand,incorporationofBrij35 intoalginate solutions decreased thepH slightly toaround 6.45. After com-pletedissolution,themixtureofalginateandsurfactantwereadded dropwisetothegellingsolution(e.g.CaCl2orBaCl2)usingasyringe

of 0.8cm inner diameter.The concentrationof thecrosslinking solutionswasselectedas3%ofCaCl2orBaCl2(w/v)inthe

exper-imentsdealing withthe effect of surfactants. For the effect of crosslinkerionexperiments,theconcentrationswere2,3and5%of CaCl2orBaCl2(w/v).Formedbeadsweretransferredintostorage

vesselsandkeptinthegellingmediafor12hatroomtemperature inordertocompletegelation.

CompressionmeasurementswerecarriedoutusinganInstron 3345testingmachineattachedwitha10Nforcetransducer.The diameterofeachbeadwasmeasuredusingadigitalcaliperandall ofthemeasurementswereconductedatleastintriplicate.Asingle beadwasplacedontoaplatform,asshowninFig.1.Aprobewith aﬂatendwasusedtocompressthebead.Compression measure-mentswereperformedataspeedof0.5mm/minandupto40% deformationratioat25◦C.

Inordertoclarifythestatisticalsigniﬁcanceoftheresults,single factoranalysisofvariance(ANOVA)testswereconductedforeach dataset.Thelevelofstatisticalsigniﬁcancewasassumedas0.05 andstatisticalcalculationswasdoneusingRstatisticalsoftwarev. 3.02[15].

3. Results

Theeffect ofcrosslinkercation, alginateand surfactant con-centrationson beadsize is shown onTable 1. Bariumalginate

Fig.2. TheForceF(N)and(H/2)3/2_curves_of_4%_(w/v)_alginate_beads_crosslinked

with3%(w/v)BaCl2containing(a)0,5,111and333timescmcBrij35and(b),0,

3.1,12.5,25and50timescmcSDS.Thearrowsindicatethedirectionofincreasing surfactantconcentration.

beadshavebiggerdiameterthancalciumalginatebeads.Thesizes increasedwithincreasingalginateconcentrations forall formu-lations.Incorporatingbothtypesofsurfactantsintoformulations decreasesthesizesofthebeadsinitially,thenthesizesincrease withincreasingsurfactantconcentrations.For333cmcofBrij35 (i.e.themolarconcentrationofBrij35has333timesofcmcofBrij 35)and50cmcofSDS,thesizesbecamealmostequalto formula-tionswithoutanysurfactantforeachcrosslinkerconcentration.

Theforce(F)versusdisplacement(H)datawasgeneratedfrom thecompressionmeasurements.HertzTheory[16] wasusedto determinetheYoung’smodulus,asshownbelow:

F=4R₃0.5 E 1−

v

2

_H 2

3/2 (1) where Ris theradiusofa bead,EistheYoung’smodulus,His thedisplacement,and

isthePoisson’sratio.First,theforce(F) wasplottedagainstthedisplacement(H)3/2_._The_Poisson_ratio_was

takenas0.5for0.5mm/mincompressionspeedapplied.Thisvalue iscompatiblewithliteraturevalues[10,17].Intheliterature,for the compression speed range between 0.075mm/min [17] and 60mm/min,[10]thePoissonratiowasselectedas0.5.TheYoung’s moduluswasthendeterminedfromtheslopeoflinearregionusing theleastsquareregressionoftheplotofFversus(H/2)3/2_.

Twoexamplesofforceversus(H)3/2_curves_for_4%_(w/v)

algi-natebeadscrosslinkedwith3%(w/v)BaCl2areshowninFig.2(a)

and(b).Fig.2(a)correspondstothe4%(w/v)alginatebeads con-taining0,55,111and333timescmcofBrij35.Fig.2(b)showsthe curvesof4%(w/v)alginatebeadscontaining0,3.1,12.5,25and 50timescmcofSDS.FromcomparingFig.2(a)withFig.2(b),the largerconcentrationsofSDSsurfactantrequiredlargerforce val-uestoproduceagivendegreeofdeformation.Ontheotherhand, thenonionicsurfactantBrij35showstheoppositeeffect.Thesame trendwasobservedforcalciumalginatebeads,withsmallerslope values.Foreachformulation,theYoung’smodulusofatleastthree differentbeadswerecalculatedfromthelinearregionoftheforce versus(H)3/2_curves.

StatisticalsigniﬁcanceofYoung’smodulusvaluesamong chang-ingsurfactantconcentrationsateachalginateconcentrationwas investigated usingonewayANOVAtests atp=0.05. Except the 1%and2%(w/v)alginatebeadscrosslinkedwithbarium,allseries resultedinp<0.05.Thus,incorporationofBrij35showsnoeffect

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SDS

3.1cmc 2.6±0.3 2.7±0.1 3.1±0.1 3.2±0.6 3.1±0.2 3.3±0.1 3.2±0.2

12.5cmc 3.0±0.2 3.2±0.1 3.1±0.1 3.2±0.1 3.1±0.2 3.4±0.1 3.8±0.2

25cmc 3.1±0.5 3.4±0.1 3.2±0.1 3.8±0.3 3.3±0.1 3.7±0.2 3.6±0.1

50cmc 3.2±0.2 3.5±0.2 3.2±0.1 4.0±0.2 3.4±0.3 3.8±0.1 4.1±0.5

Fig.3.Effectof(a)Brij35and(b)SDSconcentrationsontheYoung’smodulusof thealginatebeadswithvariousalginateconcentrations.Crosslinkingsolution:3% (w/v)CaCl2.Errorbarsindicatestandarderrorsofthemean.

onbariumalginatebeadsatanalginateconcentrationupto2%and

otherresultsarestatisticallysigniﬁcant.

Fig.3(a)and(b) showtheeffect ofsurfactanttype and sur-factantconcentrationontheYoung’smodulusofthebeadswith varyingconcentrationsofalginatesolutionsbycrosslinkingwith 3%CaCl2ions.ThemostpronouncedeffectontheYoung’smodulus

isgivenbythealginateconcentration.Byincreasingthealginate concentrationfrom1to4%(w/v)theYoung’smodulusincrease byfactor5–6fromabout50kPatoalmost300kPa.Asseenfrom Fig.3(a),theYoung’smodulusofalginatebeadsdecreaseswithBrij 35concentration.Thisdecreaseismoreapparentforhigher algi-nateconcentrations.Ontheotherhand,thevalueoftheYoung’s modulusofalginatebeadsincreaseswithSDSconcentrationsfor beadscontainingdifferentalginateconcentrations.Itisimportant tonotethattheYoung’smodulusforcalciumcrosslinkedpure algi-natebeadsareintherangeof60–300kPa,whicharecomparable withtheresultsbyKaklamanietal.reportedfor2.5–5%alginateand 1–5MCa2+_although_they_manufactured_disc-shaped_pure_alginate

hydrogels[13].

TheeffectofBrij35andSDSconcentrationsontheYoung’s mod-ulusofalginatebeadscrosslinkedwith3%(w/v)BaCl2ionsisshown

inFig.4.WhiletheYoung’smoduliofthebeadsslightlydecrease withBrij35concentration,aconsiderableincreaseintheYoung modulusisobservedwithincreasingSDSconcentration.From com-paringFig.3(b)andFig.4(b),theSDSeffectontheelasticmodules ofalginatebeadsisstrongerforBa-Alginatebeadscomparedto Ca-Alginatebeads.TheYoung’smodulusofbariumalginatebeads

Fig.4.Effectof(a)Brij35and(b)SDSconcentrationsontheYoung’smodulusof thealginatebeadswithvariousalginateconcentrations.Crosslinkingion:3%(w/v) BaCl2.Errorbarsindicatestandarderrorsofthemean.

containing50cmcSDSwasapproximatelytwotimeshigherwhen comparedwiththemodulusofpurebeads.Itshouldbenotedthat therewasnosphericalbariumbeadformationfortheformulations containingSDSand1%(w/v)BaCl2.Theformedgelswereﬂatand

thereforearenotincludedinthemeasurements.

Inordertounderstandtheeffectofcationconcentrationonthe beadscontainingtheanionicsurfactantSDS,thealginateandSDS concentrationswerekeptconstant(2%and50cmc,respectively), whereastheconcentrationsofbothCaCl2 andBaCl2werevaried

from1to5%(w/v).ThecationeffectonYoung’smodulusofthe beadsisgiveninFig.5.AsseenfromFig.5,theYoung’smodulusof alginatebeadscrosslinkedwithCa2+_ions_decreases_slightly_with

increasingcalciumionconcentration,whereastheYoung modu-lusofbariumalginatebeadsincreasesdramaticallywithincreasing bariumionconcentration.BariumalginategelshavelargerYoung’s modulusthancalciumalginateonesandthisorderisthesamefor formulationscontainingSDS.

4. Discussion

4.1. Effectofalginateconcentration

ThestrongesteffectontheYoung’smodulusiscausedbythe changeinalginateconcentration.Theincreaseinalginatefrom1to 4%leadstoanincreaseinYoung’smodulusbyafactorofabout5–6. Theincreaseinalginateleadstoadensiﬁcationofthebeads’

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mate-Fig.5.EffectofcationcrosslinkerconcentrationontheYoung’smodulusof2%(w/v) alginatebeadscontaining50cmcSDS.Errorbarsindicatestandarderrorsofthe mean.

rial.Onehastokeepinmindthatthealginatechainsthemselves formaggregatesviahydrogenbonding.

4.2. Effectofsurfactant

Theconstituentsof thealginatecopolymer,mannuronicacid and guluronicacid,are acidicmonomers havingpKa values 3.2

and 3.6, respectively [18]. However, there is not anyliterature reportaboutthepKavalueofthecopolymer.Accordingtoour

pre-viouselectrophoreticstudywithalginatepolymer,alginatedoes notgain a noticeable electrophoreticmobilitybetweenpH=3.5 and 8.5. However, when SDS added to the medium above its cmc,alginategainsanelectrophoreticmobilitylikea negatively chargedmolecule.Thisbehaviorshowedusaninteractionbetween alginate biopolymer and SDS micelles [19]. Since the interac-tionisnotelectrostatic,itcanbetheoreticallyexpectedthatthe SDS carbon chain and the alginatecopolymer chain show this hydrophobic–hydrophobicinteraction.Inthepresentstudy,since theusedSDSconcentrationiswellabovethecmc,itcanbeassumed that half micelles are formed along the alginate chains which increasethenegativechargedensityofthealginate/SDSassociate. Thisadditionalchargeoffersmoreadsorptionsiteforthedivalent cationsandthereforeahigheramountofcross-linkersthaninthe SDS-freegelbead.Thisincreasingamountofcross-linkerpoints increasesthestiffnessandtheYoung’smodulus.SincetheSDS con-centrationsarewellabovethecmc,asalteffectofAlginateonthe formationofmicellescanbeneglected.

Brij 35 caninteractwiththe alginatevia hydrogenbonding mediatedby theheadgroupor viaassociationbythe aliphatic chain.Thiscouldleadtoareductioninchargeofthealginatechain, sincepotentialchargesofthealginatechainarecoveredby non-ionicBrij35halfmicelles.TheassociationwithBrij35wouldreduce thedensityofadsorptionsitesforthedivalentcationsand there-forethedensityofcross-linkedalginateandtheYoung’smodulus decrease.Ontheotherhandtheassociationviatheheadgroup couldalsohydrophobizethealginatechains,whichwouldleadto anassociationbetweenhydrophobicdomainsrelatedtoanincrease inYoung’smodulus.Obviously,thisinﬂuenceisminorsincethe Young’smodulusdecreaseswithincreasingBrij35concentration. Fig.6schematizesthisdiscussion.

Fig.6.Schemeofsurfactanteffect.Calciumionsbindstonegativecenterson algi-natechain(a)AdditionofSDSincreasesthenegativechargedensityandthereforea higheramountofcrosslinkers(calciumorbariumions)localizesaroundthealginate chain.ThisincreasedamountofcrosslinkerscausesanincreaseintheYoung mod-ulusandstiffness(b)NonionicBrij35coverssomeofnegativecentersofalginate chains,leadingtoadecreaseincrosslinkingionsandconsequentlygelation.Thus theresistancetobeingdeformedelasticallydecreases(c).

4.3. Ionspeciﬁceffectsincross-linking

Sincetheionicradiusofbarium(135pm)islargerthancalcium (99pm),thehydrationshellaroundBa2+_is_smaller_and_less_ordered

thanforCa2+_.This_makes_it_easier_for_the_negative_groups_of

algi-natetointeractwithBa2+_than_to_interact_with_Ca2+_._This_leads_to

astrongercross-linkingandthereforeahigherYoung’smodulusin presenceofBa2+_than_of_Ca2+_._Of_course_an_increase_in_cross-linker

(Ba2+₎_increases_the_density_of_{cross-linking}_points_which_increases

theYoung’smodulusofalginateparticles.Itisalsoknownfrom lit-eraturethattheafﬁnityofalginatetowardsbariumisgreaterthan towardscalcium[20,21].Thisissupposedtoresultinstrongergel formationinthepresenceofbariumions.Thesameorderwasalso observedforbariumandcalciumalginatemicrobeads.[20].Itis notreallyclearwhytheYoung’smodulusdecreaseswith increas-ingCa2+_{concentration.}_Here,_we_can_only_speculate._Perhaps_the

Ca2+_acts_rather_like_a_salt_in_the_system_than_as_a_{cross-linker.}_This

meansthattheCa2+_ions_screen_the_charges_in_the_system,_which

mightreducetheYoung’smodulusoftheparticles. 5. Conclusion

ThepresentstudyshowsthattheYoung’smoduluscanbe eas-ilyvariedbymoreorderofmagnitudebychangingthealginate concentrationorthetypeorconcentrationofthesurfactantorthe cross-linkingdivalentcation.Theeffectofsurfactantonmechanical propertiesofcalciumandbariumalginatebeadswasinvestigated bycompressionmeasurementscombinedwiththeHertzTheory. The Young’s modulus of alginate beads changes withthe type ofusedsurfactantandmetalions.Brij35additiondecreasesthe Young’smoduluswhileSDSadditionincreasestheYoung’s mod-ulusofbothcalciumandbariumalginatebeads.Thisindicatesa differenttypeofassociationofSDSandBrij35tothealginatebeads duetodifferentchargesandthedifferentabilitytoform hydro-genbonding.IncaseofSDSonlyahydrophobicinteractionatlow alginatechargecantakeplace,whileBrij35 canassociatewith thealginatechainviaitheadgrouporviathealiphaticchain.The

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Acknowledgment

H.Kaygusuzacknowledges Scientiﬁcand Technical Research Council of Turkey (TÜB˙ITAK)-BIDEB 2214/B scholarship pro-gramme.

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