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Colloids and Surfaces A: Physicochemical and Engineering Aspects
j ou rn a l h om ep 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 a
pH-sensitive multilayers based on maleic acid terpolymers with weak and strong acid moieties
Dana Mihaela Suflet, Irina Mihaela Pelin, Daniel Timpu, Irina Popescu
∗“PetruPoni”InstituteofMacromolecularChemistryofRomanianAcademy,AleeaGrigoreGhicaVoda41A,700487Iasi,Romania
h i g h l i g h t s
•A maleicacidterpolymer wassyn- thesizedandusedinlayerbylayer deposition.
•Theratiobetweenweakandstrong acidgroupsinfluencedthemultilayer properties.
•The assembling pH and the poly- cationnaturealsoinfluencedthefilm thickness.
•Theloadingofacationicdyeintothe filmswaspH-sensitive.
•ThedyereleaseprofileswerealsopH- dependent.
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Articlehistory:
Received22March2013
Receivedinrevisedform10June2013 Accepted11June2013
Available online xxx
Keywords:
Polyelectrolytemultilayers Maleicacidcopolymers pH-sensitivematerials Dyeabsorption Controlledrelease
a b s t r a c t
Multilayer films were assembled from a maleic acid terpolymer, poly[(maleic acid-alt-styrene)- co-2-acrylamido-2-methyl-1-propansulfonic acid],deposited in alternation with weakpolycations:
poly(allylaminehydrochloride)orchitosan.Thestrongacidgroupsoftheterpolymerassuredthefilms stabilitybyelectrostaticlinkages,andtheweakcarboxylicgroupsfromthemaleicacidassuredthepH- responsivenesstoexternalpHchanges.Inordertostudytheinfluenceoftheratiobetweentheweakand thestrongacidgroupsofthepolyaniononthemultilayerproperties,twomaleicacidterpolymerswere synthesizedandcharacterizedbypotentiometrictitration,FTIRand1HNMRspectroscopy.Theinfluence oftheassemblingpHandofthenatureofthepolycationonthemultilayerpropertieswasinvestigated.
ThepH-sensitivityoftheobtainedfilmswasdemonstratedbyloadingandreleasestudiesofamodel cationicdye:Rhodamine6G.Theamountofthecationicdyeincorporatedintothefilmsincreasedwith theincreaseofthepHofthepost-treatmentsolution,butwasalsoverymuchinfluencedbytheassem- blingconditions.Ahighratiobetweentheweakandthestrongacidgroupsofthemaleicterpolymerand anacidicpHdepositionwereneededtoassureahighloadingcapacityofthemultilayers.Thedyerelease profileswerealsopH-dependentduetothevariationoftheanionicchargesofthemaleicacidterpolymer fromthefilms.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
The auto-assembling of polyelectrolytes by the alternating depositionofpolyanionsandpolycationsfromtheaqueoussolu- tion ondifferentsupports is a simple and ecologically friendly
∗Correspondingauthor.Tel.:+40232217454;fax:+40232211299.
E-mailaddress:[email protected](I.Popescu).
methodfor coating and even for the obtainingof newmateri- als[1].Thecomposition,thechemicalfunctionality,thethickness, themorphology,andthemechanicalpropertiesofthemultilayer filmscanbecontrolledbytheproperchoiceofthepolyelectrolyte structure and of thedeposition conditions (pH, ionic strength, temperature,possiblechemicalcross-linking)[2,3].Thefilmsfab- ricatedbythelayer-by-layer(LbL)depositiontechniquehavebeen proposedforvariousbiomedicalapplications:incontrolleddrug release,ascoatingsofimplantablematerials,intissueengineering, 0927-7757/$–seefrontmatter© 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfa.2013.06.022
Table1
Compositionandmolecularweightoftheterpolymers.
Sample Monomerfeedratio[MAn-St]:[AMPS] Terpolymercomposition MwdeterminedbyGPC
[MAc-St]:[AMPS]fromS% [MAc-St]:[AMPS]frompotentiometrictitrations
TMSA1 1:1.50 1:1.2 1:1.3 52,000
TMSA2 1:0.43 1:0.4 1:0.4 57,000
in theconstruction of biosensors [4,5], and others. In thecon- trolledreleasedapplications,themultilayershavetoberesponsive tovariousexternalstimulisuchas:pH,ionicstrength,temperature, enzymes,light,andelectricormagneticfield[6].
Strongpolyelectrolytes that bear completely ionized groups have a charge density constant over a broad pHrange. In the caseoftheirassembling, thecharge density,thepolymerstruc- tureandthe molarmassare theparametersthat influencethe multilayersproperties [7,8]. Weakpolyelectrolytes, instead,are notcompletelyionized,theirchargedensitybeinginfluencedby thepH.The properties of themultilayers obtained usingweak polyelectrolytescanbechangedbyvaryingthepHsolutiondur- ingtheassemblyprocess[9,10].Moreover,theexposureofthose filmsto solutions withdifferent pH values leads to thevaria- tionoftheswellingdegree,thickness,surfaceroughness,andof themicro/nano-porosity[11–14].Thesepropertiesaffecttheload- ing/releaseinto/fromthefilmsofsmallmoleculessuchasdrugsor dyes[15–19].Whencolloidalparticlesarecoveredwithmultilay- ersfromweakpolyelectrolytesandthetemplatesaresubsequently removed,pH-sensitive microcapsules are obtained[20,21]hav- ingpotentialapplicationincontrolleddelivery[22,23].Multilayers from weak polyelectrolytes are not stable in swollen state at limitingpHconditions(whenoneofthepolyelectrolytesislessthan 10%charged),butwhenoneofthepartnersismorehydrophobic orwhenoneofthemcontainsstrongacid/basicgroupsbesidethe weakones,thestabilityofthelayersincreases[20,21,24–26].
Maleic acid copolymers, synthesized by radical polymeriza- tionofmaleicanhydrideanddifferentcomonomersfollowedby hydrolysisareweakanionicpolyelectrolyteswell-knownfortheir dissociationintwosteps[27,28].Theaciddissociationconstants for the two neighbouring carboxylic groups (pK01∼3–4, and pK02∼5.5–10) are influenced bythe comonomer structure and bythepresenceofthesupportingelectrolytes[29].Layer-by-layer depositionswithmaleicacidcopolymersanddifferentpolycations havebeenstudied[25,26,30] andhave even beenproposedfor applicationinnanofiltration[31]orinlowhumiditydetection[32].
Copolymersofmaleicacidwith4-styrenesulfonicacidhavebeen usedtoassurebothstabilityandtuneablepropertiesofthelayers [25,26,31,32].
Chitosan (CS) is the only pseudo-natural polycation, being obtainedfromchitinbypartialdeacetylation.Thislinearpolysac- charidewithfree–NH2 groups attheC-2positionfrom(1–4) d-glucosamineunits(usuallypartlyN-acetylatedinchitosan)has a protonation constant around 6.5, depending on molar mass, deacetylationdegreeandmeasurementconditions[33].Thelack oftoxicity,thegoodbiocompatibility,theantibacterialactivity,and thewound-dressingpropertyofchitosanhaveledtothedevelop- mentofitsapplicationsinfood,pharmaceutical,andbiomedical fields[34].
In this work we synthesizedand characterizedpoly[(maleic acid-alt-styrene)-co-2-acrylamido-2-methyl-1-propansulfonic acid](TMSA),aterpolymerwithweakandstrongacidgroupswith acertainhydrophobiccharacterduetothestyrenecomonomer.
Twocompositionsoftheterpolymerwereusedinordertostudy theinfluenceoftheratiobetweenthestrongandtheweakacid groupsonthemultilayersproperties.Poly(allylaminehydrochlo- ride)andchitosanhydrochloridewereusedasweakpolycations intheconstruction oflayer-by-layer films.The influenceofthe
assemblingpHonthemultilayerpropertieswasalsoinvestigated.
The strong charged groups of the terpolymer were expected toformelectrostatic linkagesand toenhancethefilm stability, while the weak carboxylic groups were expected to assure a pH-responsiveness. After multilayer deposition, the filmswere treatedbyimmersionintosolutions withhigherpHinorderto produceanexcess ofnegativecharge withinthefilm toassure theabsorptionofcationicmolecules,suchasRhodamine6G.The release of the dye in neutral and acidic environment wasalso studied.
2. Experimental 2.1. Materials
Maleicanhydride(MAn)(AcrosOrganics,Germany)wasrecrys- tallizedfromchloroform,styrene(St) (Sigma-Aldrich,Germany) was purified by vacuum distillation, while 2-acrylamido-2- methylpropanesulfonic acid (AMPS)(Fluka, Germany) and 2,2- azobis(isobutyronitrile)(AIBN)wererecrystallizedfrommethanol.
Drieddiethylether(Riedel-deHaën,Germany),chloroform,Rho- damine6G(Fluka,Germany),lithiumhydroxide(Merk,Germany), N,N-dimethylformamide,sodium chloride and calcium chloride dehydrate(Sigma-Aldrich,Germany)wereusedasreceived.Dou- bledistilledwaterwasusedinalltheexperiments.
Branched poly(ethyleneimine) (Mw=720kDa) solution (50%) inwaterandpoly(allylaminehydrochloride)(PAH)(Mw=15kDa) werepurchasedfromSigma–Aldrich.Chitosan,kindlyprovidedby YaizuSuisankagakuInd.,Japan,hadadeacetylationdegreeof82.5%, determinedby1HNMR[35]andbypotentiometrictitrationwith NaOHofanaqueoussolutionofchitosanwithaknownexcessof HCl.Itsmolarmass,determinedbyviscometricmeasurementsin 0.3Maceticacid/0.2Msodiumacetate(1:1,v:v)solutionat25◦C usingtheequation[Á]=1.38×10−4Mv0.85[36]was320kDa.Itwas solubilizedinwaterwithastoichiometricamountofHClandpuri- fiedbydiafiltration.Thehydrochloridesaltofchitosan (CS)was recoveredbyfreeze-drying.
2.2. Terpolymersynthesis
According to the literature [37], the radical polymeriza- tion of MAn, St and AMPS involves the well-known charge- transfer complex formed between MAn and St that inter- act by hydrogen-bonding with AMPS, leading to the forma- tion of poly[(maleic anhydride-alt-styrene)-co-2-acrylamido-2- methylpropanesulfonicacid].Twoterpolymersweresynthesized usinganinitialmolarratiobetween[MAn-St]:[AMPS] of1:0.43 and 1:1.5, as presented in Table 1. The total concentration of the monomers in N,N-dimethylformamide was 4.5M, and AIBN was used as radical initiator (0.01mol AIBN: mol of monomers). The reaction proceeded for 24h at 80◦C. The obtainedpoly[(maleicanhydride-alt-styrene)-co-2-acrylamido-2- methylpropanesulfonicacid]insolutionwasprecipitatedindiethyl ether:chloroformmixture(1:1,v/v)anddriedatreducedpressure and40◦Cfor48h.
TMSA1andTMSA2terpolymerswereobtainedbythehydrolysis ofthecorrespondingmaleicanhydrideterpolymersat40◦C.The
resultedaqueoussolutionwasthenpurifiedbydiafiltrationand thepolyelectrolytewasrecoveredbyfreeze-drying.
2.3. Preparationofpolyelectrolytemultilayers
Quartzslides(Heraeus,Germany),glassslides(J.MelvinFreed Brand, Sigma–Aldrich, Germany) or silicon wafers (Siltronix, France) were first cleaned with Piranha solution (70:30 sulfu- ricacid:30%hydrogenperoxide)for1h,rinsedwithwater,then the substrates were cleaned by sonication at 70◦C in a 1:1:1 mixtureofammoniumhydroxide(25%solution):hydrogenper- oxide (30%solution): distilled water. This procedure generates negativechargesonthesurface.Thesubstrateswerekeptunder distilled water in the refrigerator overnight before the multi- layerdeposition.Allthesubstrateswerefirstdippedfor15min inpoly(ethyleneimine)solution(2g/L,pH=5)toobtainahighly chargedlayeronthesurfacethatactedasananchoringnetwork fortheconsecutivelayersformation[38].
Aqueouspolyelectrolyte depositionsolutions were prepared withaconcentrationof2g/Lin0.1MNaCl.Aqueoussolutionsof 1MHClor1MNaOHwereusedtoadjustthepHofthepolyelec- trolytesolutionsatdesiredvalues.Bothpolyanionandpolycation solutionswereadjustedtothesamepH(either2.5or5.5).The substrateswerethendippedinthreevesselswithdoubledistilled waterfor1min.Thealternantdepositionofanionicandcationic polyelectrolyteswascarriedoutfor15mineach.Thewashingstep was repeatedafter each deposition toremove the unabsorbed polyelectrolyte and toprevent theformation of polyelectrolyte complexes in solution. The substrates were dried betweenthe depositionstepswhentheUVabsorptionspectraweremeasured inordertomonitorthegrowthofthemultilayers;otherwise,no dryingstepwasneeded.Takingintoaccountthefactthatthebind- ingofaprobetothepolyelectrolytemultilayersrequiresthatthe outermostlayerbeoppositelychargedtotheprobe[39],thefinal depositedlayerwasTMSA.
2.4. Loadingandreleasestudies
Theaqueoussolutionof0.005mg/mLRhodamine6G(R6G)was preparedandadjustedtodifferentpHsusingNaOHorHClsolu- tion.Thepolyelectrolytemultilayersontheglasssubstratewere exposedtothedyesolutionsofdifferentpHvaluesfor24htoallow thereachingoftheequilibrium.Thefilmswererinsedwithwater toremove theexcessdyeandthendriedfor24hatroomtem- perature.TheUV–visspectraofthefilmswerethencollectedand theabsorbanceat538nmwasusedtomonitortheamountofR6G withinthefilms.Thedecreaseofthesolutionabsorbanceafterthe filmloadingcouldnotbemeasuredinallthecases.Thatiswhythe dyeloadinginfilm(mg/cm2)couldbecalculatedonlyforthefilms thatabsorbedahighamountofR6G.Apreviouslymadecalibration curvewasused.
Fortheinvitroreleasestudy,theR6G-loadedLbLfilm-coated glassslidewasimmersedin3LwaterwiththedesiredpHunder gentilestirring.Atpredeterminedtimeintervalstheglassslidewas driedandtheUV–visspectrumwasrecorded.
2.5. Characterizationmethods
FTIRspectrawererecordedonKBrpelletsusinga Vertex70 Bruker spectrometer and 1H NMR spectra in deuterium oxide wereobtainedusingaBrukerAvanceDRX400MHzspectrome- ter.The sulfur contentofthe polymerswasdeterminedbythe Schönigermethod.Potentiometrictitrationsofthemaleicacidter- polymeraqueoussolution(0.67g/L)weremadewithanall-purpose Metrohm716DMSTitrinoapparatusequippedwithadosingunit andacombinedglasselectrode forthemeasurementofthepH.
0.0 0.8 1.6
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0 2 4 6 8 10
12 without salt, pH with 0.005 M CaCl2, pH
dpH
V, mL base
pH
(a)
without salt, dpH with 0.005 M CaCl2, dpH
0.0 0.8 1.6
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0 2 4 6 8 10
12 witwithout salt,h 0.005 M C pHaCl2, pH
(b)
V, mL base
pH
without salt, dpH with 0.005 M CaCl2, dpH
dpH
Fig.1.PotentiometrictitrationofTMSA1(a)andTMSA2(b)terpolymersinaqueous solutionwith0.1NLiOH,intheabsenceandinthepresenceofCaCl2.
0.1NLiOHsolutionwasusedastitrationagentandCaCl2wasused asadditionallyaddedelectrolyte.
The average-molecular weight of the terpolymers was measured by gel permeation chromatography (GPC) using a Waters GPC apparatuswithShodexcolumn, Refractionand UV
500 700 900 1100 1300 1500 1700 1900 2100
-1 TMSA1 TMSA2
1720 1650 1556 1039 703765
500 700 900 1100 1300 1500 1700 1900 2100
Wavenumber, cm-1
Transmitance
TMSA1 TMSA2
1720 1650 1556 1039 703765
Fig. 2. FTIR spectra of TMSA terpolymers with different ratio between the monomers.
Fig.3. 1HNMRspectrainD2OofTMSA1(a)andTMSA2(b).
Photodiodearraydetectors.DMF/0.1molNaNO3wasusedassol- ventandeluent,andthecalibrationwasmadeusingpolystyrene standards.
UV-VisspectrawerecollectedwithaSPECORD200AnalytikJena spectrometer.Thetopographyofthemultilayerfilmswasstudied usingaSPM-ScanningProbeMicroscope,SOLVERPRO-Minstru- ment(NT-MTDCo.Zelenograd,Moscow,Russia),theimagesbeing takeninairatroomtemperature.ANSG10/AuSilicontipwitha 10nmradiusofcurvatureand255kHzoscillationmeanfrequency was used. The apparatus was operated in semi-contact mode, 256×256scanpointsize,withascanvelocityof6–20m/s,atdif- ferentscanareas(foursquarewiththesideof10,5,2,1and0.5m).
Theroot-mean-square roughness(RMS)andaverageheight(ha) were calculated for different scanning areas: 0.5m×0.5m, 1m×1m, 2m×2m,5m×5m,and 10m×10mto provideabroadroughnessanalysisofthefilms.Theexperimen- taldatawereprocessedusingthesoftNOVA1443andIA-9Image Analysis3.5fromNT-MDTRussia(SPMproducer).
3. Resultsanddiscussion 3.1. Terpolymercharacterization
Inordertoobtaintheratiobetweenthesulfonicandthecar- boxylicacidgroups fromtheTMSAterpolymers,potentiometric titrationinaqueoussolutionwasperformed.Maleicacidcopoly- mersgenerallypresent twodissociation stepscorrespondingto thetwoadjacentneighbouringcarboxylicacidgroups,butinpure wateronlythedissociation ofthefirstCOOHgroupisdetected.
Whenmono-monovalentsalt andespecially di-monovalent salt (CaCl2)is added,thesecondcarboxylicgroupcanbeevidenced bypotentiometrictitration[27–31].Fig.1showsthepotentiomet- rictitration curves oftheterpolymers TMSA1 andTMSA2 with different[MAc-St]:[AMPS]ratio.Thefirstderivativesofthepoten- tiometriccurvesarealsopresented.Inpurewater,thetitrationof
thesulfonicacidgroupatlowtitrantvolumescouldhardlybeseen, butthefirstcarboxylicgroupwasclearlyevidencedbytheinflec- tioninthepHcurveandbythemaximainthefirstderivativecurve.
Inthepresenceof0.005MCaCl2,twoend-pointscouldbeidenti- fiedcorrespondingtobothCOOHgroups.The[MAc]:[AMPS]ratio intheterpolymerscouldbecalculatedas(V2−V1)/[V1−(V2−V1)], whereV1andV2arethevolumeofLiOHaddeduntilthefirstand secondinflectionpointswerereached.Theobtainedvaluesforthe twoterpolymersarepresentedinTable1.
Thestructureoftheobtainedterpolymerswasconfirmedby FTIRspectra(Fig.2),wherethecharacteristicbandsofthemaleic acid (COOH groups at 1720cm−1), the styrene units (mono- substitutedbenzeneringat765and703cm−1),andtheAMPSunits (amidegroupsat1650and1556cm−1,SO3Hgroupsat1039cm−1) werepresent. Withtheincrease of [MAc-St]:[AMPS]ratiofrom TMSA1toTMSA2,thebandsintensityattributedtoMAcandto StunitsincreasedandthebandintensityattributedtoAMPSunits decreased,asshowninFig.2.
Fig.3showsthe1HNMRspectraoftheobtainedterpolymers.
Asignificantdecreaseofthesignalcentredat1.5ppm(protonsof methylgroupsfromthesidechainofAMPS)comparedwithsignal ofthearomaticprotons(7–7.4ppm)orwiththemethineprotonsof maleicacid(3.1–3.6ppm)canbeobservedwhentheAMPScontent intheterpolymerdecreases.
3.2. Polyelectrolytemultilayerfilmsformationand characterization
Multilayerthinfilmswerepreparedby alternatingassembly ofTMSAterpolymerswithPAHorwithCS.Bothpolycationscon- tainprimaryaminegroupsinthepolymericchain,buttheyhave differentchainstructureandflexibility.
Thegrowthofthelayersonquartzslidescouldbemonitored bymeansofUVspectroscopy,usingtheabsorbanceat210nm.At thiswavelengthashoulderwasobservedinthespectrumofTMSA
0 2 4 6 8 10 12 14 16 18 20 0.0
0.2 0.4 0.6
TMSA1/CS, pH = 2.5 TMSA1/CS, pH = 5.5 TMSA1/PAH, pH = 2.5 TMSA1/PAH, pH = 5.5
Absorbance (au)
Layer number (a)
0 2 4 6 8 10 12 14 16 18 20
0.0 0.2 0.4 0.6 0.8 1.0 1.2
TMSA2/CS, pH = 2.5 TMSA2/CS, pH = 5.5 TMSA2/PAH, pH= 2.5 TMSA2/PAH, pH = 5.5
Absorbance (au)
Layer number (b)
Fig.4. ThegrowthofTMSA/PAHandTMSA/CSmultilayerfilmsinvestigatedbyUV spectroscopy.Terpolymerswith[MAc-St]:[AMPS]=1:1.3(a)and1:0.4(b)wereused.
terpolymersduetothephenylrings,butPAHalsopresentedaslight absorptionatthiswavelength.InFig.4arepresentedtheresults oftheUVmeasurementsof TMSA/PAHandTMSA/CSmultilayer films,wheretheoddlayernumberscorrespondtomaleicterpoly- mer deposition.In almost all thecases themultilayers growth waslinear.WhentheTMSA1withahighamountofstrongacid moieties wasused (Fig.4a),the assemblingpHdidnot have a verysignificantinfluenceonthemultilayergrowthbecausethe anionicpolyelectrolytewasdepositedinanextendedconforma- tion.TheslightlyhigherUVabsorbanceforTMSA1/PAHdeposition atpH=5.5couldbeduetoPAHwhichalsohasanUVabsorbance atthesamewavelength.WhentheTMSA2with[MAc-St]:[AMPS]
ratioof1:0.4wasused(Fig.4b),theabsorbancevalueswerehigher duetothepresenceof phenylringsinahighernumber.In this case,theinfluenceofthesolutionspHonthedepositionprocess wasbetterevidenced:thedecreaseofthepHvaluesfrom5.5to2.5 ledtoanincreaseoftheabsorbedterpolymeramount.AtpH=2.5, whenonlythesulfategroupsweredissociatedandprovidedthe electrostaticbindingwiththepolycation,themaleicacid-styrene residuesformedloopsandtailsassuringthethicknessoftheter- polymerlayers.ThiseffectwasenhancedwhenCS(withhigher molarmass thanPAH) wasusedas polycation. Theincrease of
Table2
Characteristicsofsurfacetopologyof19layersofTMSA/PAHandTMSA/CS.Root- mean-square roughness and averageheight were calculated for2m×2m scanningarea.
AssemblingpH RMS(nm) ha(nm)
PAH CS PAH CS
TMSA2 2.5 9.7 54 26 213
TMSA2 5.5 3.8 7.3 6.5 24.8
TMSA1 2.5 10 4.7 19 17.4
theterpolymeramountinthefilmwasthehighestforTMSA2/CS assembled at pH=2.5, where themultilayer growthwasexpo- nential.Inthedepositionprocessitwasobservedthatthisfilm began tobecomecloudyafter8layers,suggesting ahighthick- ness.
Thesurfacetopographyof19 layersofTMSA/polycationwas investigated using AFM (Fig. 5). The influence of the assem- bling pH, of the [MAc-St]:[AMPS] ratio in the terpolymer, of the natureof the polycation on the RMSand ha values of the obtained films is shown in Table 2. Those values showed the same trendas theUV–vis data:decreased withtheincrease of theassemblingpHfrom2.5to5.5,decreased withtheincrease ofthestrongacidamountfromtheterpolymer(from TMSA2to TMSA1).
However,TMSA2/CSfilmassembledatpH=2.5hadveryhigh RMSandhavalues.Thepresenceoflargestructuresinthetopo- graphicalimageofthisfilmtogetherwiththeexponentialgrowth, suggesttheformationofpolyelectrolytecomplexnanoparticle-like structures. It is known that the films with linear growth con- sistofstratifiedlayers,whilethosewithexponentialgrowthhave intermixedlayers,resultingfromthehighmobilityof thepoly- electrolyte chainsduring film deposition[40]. In this case it is mostprobablethatTMSA2inthecoiledconformationatpH=2.5 has the highest mobility. Its migration in and out of the film wouldleadtotheformationoflargestructureswiththepolycation chains from the solution. These polyelectrolyte-like structures werelargerwhenCSwithhighmolarmasswasusedinsteadof PAH.
4. Dyeloading
Inordertoexaminetheutilityofthemultilayerfilmsbasedon maleicacidterpolymersaspotentialcarriersforsmallmolecules, R6Gwasused.Thiscationicdyewaschosenbecauseitcanelec- trostaticallyinteractwiththecarboxylicgroupsofthemaleicacid copolymersthatarenotinvolvedintheinteractionwiththepoly- cationlayers.Inaddition,thehydrophobicinteractionbetweenthe R6Gmoleculesandthestyreneunitsfromtheterpolymercanoccur.
Glassslidescoatedwith19layerswereimmersedinR6Gsolutions atdifferentpHs.
In Fig. 6 the absorption spectrum of the dye in aque- ous solution is compared with its spectrum in the polymeric films, as for example in the films formed of 19 layersofTMSA/CS.Themaximumabsorptioninaqueoussolution, observedat526nm,correspondstothemonomersR6Gmolecules, and the shoulder at 496nm corresponds to the dimmers (also called H aggregates) [41].In thepolyelectrolyte complex films (containing PAH or chitosan), the two bands were shifted to 540 and 510nm, respectively. The increase of the short-wave bandsuggeststheformationingreaternumberofH-typedimmer aggregates.Thered-shiftandthebroadeningoftheR6Gabsorption spectrainfilmcomparedwiththoseinsolutioncouldbedueto theformationofmicrocrystallineaggregatesortothefactthatthe R6Gmoleculesweresubjectedtoamorepolarenvironmentinthe
Fig.5.AFMheightimagesof19layersofTMSA/PAHandTMSA/CS;scanningarea2m×2m.
polyelectrolytemultilayers [42,43].However,theabsorbance of thefilmat540nmwasusedasameasureoftheamountofR6G loading.
TheinfluenceofthepHonthedyeloadingintotheobtainedmul- tilayersattheequilibriumisshowninFig.7.Generally,theamount ofthedyethatentersthefilmsincreaseswiththesolutionpH.The filmsformedwithPAHaspolycation(Fig.7a)wereloadedwith lowerR6Gamounts,comparedwiththefilmsobtainedusingCSas polycation(Fig.7b).Thiscanbeduetothelowerthicknessofthe filmsformedwithPAH,butalsotothewell-knowndyeadsorption capacityofCS[44].ThehydroxylgroupsofCScanformhydrogen bonds[45]thatassuretheinteractionwiththedyemoleculeseven
atbasicconditionswhenCSisuncharged[46].However,theexpo- sureofthemultilayerswithCStosolutionswithpHvalueshigher than9ledtothedecompositionofthelayers,whilethose with PAHaspolyanionweredecomposedonlywhenthepost-treatment pHwashigherthan10.ThisiswhytheabsorptionofR6Gsharply decreasedwhentheloadingwasperformedfromsolutionswithpH overthosevalues.Theexactamountofdyeloadingcouldbecalcu- latedfromthedecreaseofthedyeconcentrationinsolutiononlyfor thefilmswithhighloadingcapacity.Thus,0.002mgR6G/cm3was foundinTMSA2/PAHassembledatpH=2.5andloadedatpH=10, and 0.0076mg R6G/cm3 wasfoundforTMSA2/CSassembled at pH=2.5andloadedatpH=9.
Fig.6. NormalizedabsorptionspectraofR6Ginsolutionandinmultilayerfilms.
1 2 3 4 5 6 7 8 9 10 11
0.0 0.2 0.4 0.6 0.8 1.0 1.2 (b) 1.4
TMSA2/CS, pH=2.5 TMSA2/CS, pH=5.5 TMSA1/CS, pH=2.5
Absorbance at 540 nm, a.u.
pH
1 2 3 4 5 6 7 8 9 10 11
0.0 0.1 0.2 0.3 0.4 0.5
TMSA2/PAH, pH=2.5 TMSA2/PAH, pH=5.5 TMSA1/PAH, pH=2.5
Absorbance at 540 nm, a.u.
pH
(a)
Fig.7.Absorbanceat540nmoftheR6Gloadedinto19layersofTMSA/PAH(a)and ofTMSA/CS(b)asafunctionofthesolutionpH.TheassemblingpHwasindicated inthelabels.
ThefabricationpHandtheratiobetweenweakandstrongacid moietiesofthemaleicterpolymeralsoinfluencedthedyeloading, asshowninFig.7.WhentheassemblingpHwas2.5,bothCOOH groupsofthemaleicacidunitswereundissociatedandtheelec- trostaticinteractionsbetweenthelayerswereassuredonlybythe dissociatedsulfonicacidgroups.Whenthesefilmswereexposed tosolutionswithhigherpHs,thecarboxylicgroupsbegantodisso- ciate,leadingtotheswellingofthefilmsandtotheelectrostatic bindingof thecationicdye.Whentheassemblingsolutions pH was5.5,halfofthecarboxylicgroupswereionizedandtrapped intotheinteractionwiththepolycation.Theincreaseofthepost- treatment pHsolution above 5.5 ledto thedissociation of the secondcarboxylicgroupofthemaleicterpolymerthatcouldattract thecationicdyemolecules.WhentheTMSA1 withlow amount ofmaleicacidwasusedinthemultilayerfabrication,evenifthe assemblingpHwas2.5,thedyeloadingwaslowandwasnotinflu- encedbythepH,meaningthatahigherratiobetween[MAc-St]
and [AMPS] was required in the terpolymer to assurethe pH- sensitivity.
Themodification ofthefilmstopography aftertheexposure to basic aqueous solution (without dye) was also investi- gated. Fig. 8 presents the AFM images taken in the dry state of the films after the exposure for 24h at pH=9. The surface topography of the films indicated a certain rearrange- ment due to the dissociation of the carboxylic acid groups.
Nevertheless, the films roughness and its variation with the scan-size were not influenced much by the pH change,show- ing that the filmswere not removed by theexposure at basic pH.
5. Thedyereleasestudy
The pH-sensitive physicochemical properties ofsome of the obtainedfilmswereusedintheloadingprocess,andtheseprop- erties were also expected to influence the dye release. Fig. 8 shows the release profiles of R6G from the LbL films that presented the highest loading capacity (deposited at pH=2.5 using the TMSA2 terpolymer and loaded at pH=9). Both films obtained with PAH (Fig. 9a) and with CS (Fig. 9b) as polyca- tions were observed to have a dye release and a release rate higherinacidicconditionsthaninneutralmedium.Thisbehaviour can be explained through the fact that at pH=3 both car- boxylic groups from maleic acid units become predominantly uncharged,and thedyeisquickly released.AtpH=7, only55%
of the dye was releasedfrom the thicker TMSA2/CSfilm after 16h.
Thereleasedataupto60%couldbeanalyzedusingthemodel proposedbyPeppasandSahlin[47]:Mt/M∞=ktn.Inthisequation Mt/M∞isthefractionofdrugreleaseattimet,kisacharacteris- ticconstantofthesystem,andnistheexponentthatdescribes the release mechanism of the polymer matrix (for thin films, n≤0.5describesFickiandiffusion,0.5<n<1indicatesanomalous diffusion,andn=1impliesCaseIItransport).Inourcase,forthe TMSA2/PAHfilm,nincreasedfrom0.21to0.42withtheincreaseof thepHfrom3to7,indicatingatransportmechanismbyBrownian motioninbothcases.FortheTMSA2/CSfilm,thenvalueis0.43at pH=3,and0.83atpH=7,meaningthatinneutralpHthetransport ofR6GthroughthethickerTMSA2/CSfilmdependedontwosimul- taneousrateprocesses:dyediffusionandswellingofthepolymeric matrix.
However, the TMSA2/CS films assembled at acidic pH can load large amounts of small dye/drug molecules at basic solution and release them by decreasing the pH of the medium. This property can be used in drug controlled release applications.
Fig.8.AFMimages(2m×2m)of19layersofTMSA/polycationpost-treatedatpH=9androot-mean-squareroughnessofthefilmsbeforeandafterpost-treatmentasa functionofAFMscan-size;thevaluesarethemeanoffour-area-independentmeasurements.
0 200 400 600 800 1000 0
20 40 60 80 100
pH = 3 pH = 7
R6G release, %
Time, min
(a)
0 200 400 600 800 1000
0 20 40 60 80 100
pH = 3 pH = 7
R6G release, %
Time, min
(b)
Fig.9. EffectofthepHonthereleaseofR6Gfrom19layersofTMSA2/PAH(a)and TMSA2/CS(b)assembledatpH=2.5andloadedwithR6GatpH=9.
6. Conclusion
Two terpolymers based on maleic acid, styrene, and 2- acrylamido-2-methyl-1-propansulfonicacidwithdifferentratios betweenweakandstrongacidmoietiesweresynthesizedandchar- acterized.NewLbLfilmswereobtainedbasedonthesecopolymers.
Thepresenceofthestrongacidgroupsintheterpolymerstructure permittedtheassemblingofthemultilayersatpH=2.5,wherethe carboxylicgroupswerenotdissociated.Wealsoconcludedthatin ordertoobtainpH-sensitivemultilayers,maleicterpolymerswith ahighratiobetweenweakandstrongacidmoietieswereneeded.
WhenthecationicpolysaccharideCSwasusedinsteadofsynthetic PAHasweakpolyanioninfilmpreparation,theobtainedfilmswere thickerand thedyeloadingcapacitywashigher.ThepHofthe depositionsolutionsalsoinfluenced thepropertiesof thefilms, thickerfilmsbeingobtainedfromacidicsolutionswhereonlythe sulfategroupsassuredthebindingsitesforcationicmolecules,the carboxylicgroupsbeingundissociated.
The loading and the release of the cationic R6G molecules into/fromthepolyelectrolytemultilayerfilmswereinfluencedby thedissociationofthecarboxylicgroupsfromthemaleicterpoly- mer:theloadingofthelowmoleculardyemoleculeswashigher withtheincreaseoftheexposurepH,butthereleasewasmore rapidinacidicmedia.
Acknowledgement
ThisworkwassupportedbyagrantoftheRomanianMinistry ofEducation,CNCS-UEFISCDI,projectnumberPN-II-RU-PD-2012- 3-0059.
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