Engineering Aspects
j o u r n al ho m e p age :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
Emulsionsstabilizedwithorganicsolid particles
FaizaLaredj-Bourezga, YvesChevaliera,∗, OlivierBoyronb, Marie-AlexandrineBolzingera
aUniversityofLyon,Laboratoired’AutomatiqueetdeGéniedeProcédés(LAGEP),CNRSUMR5007,UniversitéClaudeBernardLyon1,43Bd11Novembre,69622Villeurbanne,France
bUniversityofLyon,LaboratoiredeChimie,Catalyse,PolymèresetProcédés(C2P2),CNRSUMR5265,ÉquipeChimieetProcédésdePolymérisation(LCPP),UniversitéClaudeBernard Lyon1,43Bd11Novembre,69622Villeurbanne,France
a r t i c l e i n f o
Articlehistory:
Received1November2011 Receivedinrevisedform 21December2011 Accepted22December2011 Available online 30 December 2011 Keywords:
Pickeringemulsions Blockcopolymer Micelles
a b s t r a c t
Biodegradableandbiocompatibleo/wemulsionswerepreparedusingtriglycerideoilandsolidorganic particlesmadeofblockcopolymernanoparticlesasstabilizers(Pickeringemulsions).Inordertoreach highconcentrationofinternalphase,ratherconcentrateddispersionsofnanoparticleswererequired. Nanoparticlesofpoly(caprolactone)-block-poly(ethyleneoxide)(PCL-b-PEO)diblockcopolymerwere obtainedusingthe“nanoprecipitation”processrelyingofthespontaneousemulsificationuponsolvent shifting.Theclassical“nanoprecipitation”processwasimprovedsoastoaffordmoreconcentrated sus-pensionsofnanoparticles,andthenanoparticleswerecharacterizedbymeansofdynamiclightscattering and1HNMRspectroscopy.TheprocessallowedthepreparationofaqueousdispersionsofPCL-b-PEO nanoparticleswith35–50nmdiameteratconcentrationsover5wt.%.InD2O,thePCLblocksformeda centralhydrophobiccoreofreducedmobility,whilethePEOblocksformedahydrophiliccoronalayer swollenbywater.O/wemulsionsofmediumchaintriglyceridesweresuccessfullypreparedusingthe suspensionsofPCL-b-PEOnanoparticlesasstabilizers.Typicaldropletsizeswerebetween2mand 15m.Theemulsionsshowedgreatstabilityuponstorageandtheirparticlesizedistributionsdidnot showexcessnanoparticlespresentintheaqueousphaseassubmicronnanoparticles,evenwhenlarge amountsofnanoparticleswithrespecttooilwereused.Themeandropletdiameterofemulsionswas controlledbythemassratioM(oil)/M(nanoparticles).SANSandTEMexperimentsperformedon PCL-b-PEOnanoparticlesandmicelle-stabilizedemulsionsdisclosedarearrangementofthenanoparticlesat theoil/waterinterfaceduetotheliquidstateofthemicellecoreofPCL.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Pickeringemulsionsare stabilizedbysolid particlesin place of surfactants[1]. Their“surfactant-free”charactermakesthem attractiveforcosmeticandpharmaceuticalapplicationwhere sur-factantsoftenshowadverseeffects(irritancy,hemolyticbehavior, etc.)[2].Towardssuchdomains,biocompatibleandbiodegradable Pickeringemulsionswouldbeanobviousbenefit.Theycanbemade upfromtheoilsusedinpharmaceuticalapplications,andorganic solidparticlesmadefrombiodegradablematerials.Sincesolid sta-bilizingparticlesarenecessarilysmallerthanemulsiondroplets, solidparticlesofnanometricsizewereselectedsoastoallowthe fabricationofPickeringemulsionsoverawidedropletsizerange. Therearetwoissuestobeovercomeinordertoreachsuchgoal:(i) thechoiceoforganicnanoparticlesthatarepartiallywetbywater andoilinordertoensuretheiranchoringtotheoil/waterinterface; and(ii)thepreparationofsuspensions ofsolidparticlesofhigh enoughconcentrationinordertoallowfullcoverageofthedroplet
∗ Correspondingauthor.Tel.:+33472431877;fax:+33472431682. E-mailaddress:chevalier@lagep.univ-lyon1.fr(Y.Chevalier).
surface,even for concentratedemulsionsof smalldroplets that havealargeinterfacialarea.Thepurposeofthepresentresearch isthepreparationofsuchemulsionsstabilizedbyblockcopolymer nanoparticles.Thiscanbeachievedifthetwoissuesquotedabove receivesatisfactoryanswer.
Solid particles can spontaneously adsorb at fluid interfaces formingeither a densemonolayer of particles,or a thicklayer of aggregated solid particles that behavesas a rigid stabilizing layeractingagainstcoalescence[3,4].Manytypesofsolidparticles (hydrophilicsilica,hydrophobicsilica,clay,bariumsulfate,calcium carbonate,polystyrene,spores,etc.)[1,5–12]wereusedtostabilize emulsions.
Biodegradablenanoparticleswoulddecreasetheriskof toxic-ityalreadyobservedwithalotofcommonchemicalsurfactants and inorganic nanoparticles, and they are expected to create a barrier to diffusion that allows a controlled release of drug substances incorporated either in the oily layer or inside the polymeric nanoparticles. Poly(caprolactone)-block-poly(ethylene oxide)(PCL-b-PEO)copolymershaveraisedmuchinterestbecause theyarebiocompatibleandpartlybiodegradable[13–16].ThePCL blockismadeofbiodegradablepolyester,andthePEOblockisa water-solublepolymeroflowmolarmassthatis bioresorbable.
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sionstopharmaceuticalorcosmeticdomains.
Thisworkfirstlydealswiththepreparationofparticlesof non-ionicamphiphilicdiblockcopolymersthatcontainahydrophilic poly(ethylene oxide) (PEO) block and a hydrophobic poly( -caprolactone)(PCL) usinga modifiednanoprecipitation process. In a secondtime, those nanoparticlesareusedto stabilizeo/w emulsions made with medium chain triglyceride (MCT) as oil. Suchparticlesareoftencalled“blockcopolymermicelles”although theyarebetterparticlesthanclassicalmicelles. Indeedclassical micelles formed with water-soluble surfactants form sponta-neously,andareat equilibriumwitha residualsolublefraction in water. Blockcopolymer micelles do not form spontaneously and the residual concentration of block copolymer in solution is vanishinglow. In theliterature,thepreparation processes of block copolymer micelles consist in either dialysis of organic solution of block copolymer or the present nanoprecipitation method.Theterm“Blockcopolymernanoparticles”willbeused throughoutthewholepaper.Blockcopolymernanoparticleswere characterizedfortheirsizeandinternalstructureusingdynamic lightscattering,transmissionelectronmicroscopy,and 1HNMR measurements.Oil-in-wateremulsionswerepreparedusinga con-ventionalmechanicalshearingprocess.Lastly,small-angleneutron scattering (SANS) experiments were used for investigating the structureoftheinterfaciallayerattheoil/waterinterfacewhere thepresenceofmicelleswasexpected.
2. Experimental
2.1. Materials
Epsilon-caprolactone(-CL)(Sigma–Aldrich) waspurified by vacuum distillation over calcium hydride (CaH2, Acros Organ-ics). Poly(ethylene glycol) monomethyl ether (mPEG) with of molarmass5000gmol−1(Sigma–Aldrich)wasdriedbyazeotropic distillationoftoluene (anhydrous toluene, Sigma–Aldrich). Ace-tone (Laurylab), dichloromethane (Acros Organics), stannous 2-ethylhexanoate (Sn(Oct)2, Sigma–Aldrich), deuterated water (D2O) and dodecane-d26 (Eurisotop, Saclay, France) were used asreceived.Deionizedwaterof18Mcm−1resistivitywasused throughoutthework.
2.2. Methods
2.2.1. SynthesisofthePCL-b-PEOdiblockcopolymer
mPEG wasdried byazeotropic distillation of its solution in anhydroustolueneunderdrynitrogenatmosphere.ThePCL-b-PEO diblock copolymer was synthesized by ring-opening polymer-ization of -CL with mPEG as a macroinitiator and stannous 2-ethylhexanoateasacatalyst[14,16–19].Adeterminedamount of-CL,mPEG,andSn(Oct)2(0.1mol%of-CL)wereweightedinto aroundedthree-neckedglassflaskequippedwithamagnetic stir-ringbar.Thereactorwasclosedunderdrynitrogenandheatedin anoilbathat130◦Cfor12h.Thereactionmixturewascooleddown toroomtemperature,andtheblockcopolymerwasprecipitatedin anexcessofcolddiethylether.Thecopolymerrecoveredby filtra-tionwaspurifiedbyprecipitationofitssolutionindichloromethane intoanexcessofcolddiethylether.Finallythemixturewasfiltered anddriedatroomtemperatureundervacuumfor24h.Themean degreeofpolymerizationofthePCLblockwasdeterminedby1H NMRspectra(Fig.1)andsizeexclusionchromatography(SEC)as showninTable1.Attheend,theblockcopolymerchemicalformula isPCL65-b-PEO113.Thisblockcopolymerwasnotsolubleinwater [14].
Fig.1. 1HNMRspectrumofPCL-b-PEOinCDCl3andtheassignmentofthelines.
2.2.2. Preparationofaqueousdispersionsofblockcopolymer nanoparticles
Nanoparticleswerepreparedusingthespontaneous emulsifi-cationprocess[20].Thismethodwasmodifiedinordertoincrease theconcentrationofsolidparticlesbeyondtheusuallimitsofthe nanoprecipitationprocess.Asatypicalrecipe,0.75mgof PCL-b-PEOwasfirstdissolvedin50mLofacetoneandthenintroduced dropwisein15mLofwaterundergentlestirring.Theacetonewas evaporatedunderreducedpressureinasecondstage.
2.2.3. Preparationofemulsions
TheMCToilandaqueoussuspensionofPCL-b-PEOmicelleswere mixedtogetherwithanUltraTurrax® T25deviceequippedwith aS25N-18Gshaft (IKA,Germany)rotatingat22,000rpmduring 5min(coldprocess)andano/wemulsionwasobtained.
2.2.4. NMRanalyses
1HNMRspectraofPCL-b-PEOdiblockcopolymerwererecorded byusingaBruckerDRX300spectrometeroperatingat300MHz. Eitherdeuteratedchloroform(CDCl3)ordeuteratedwater(D2O) wereusedassolvents.Chemicalshiftsweremeasuredinppmfrom tetramethylsilane.
2.2.5. Sizeexclusionchromatography
Sizeexclusionchromatography(SEC)measurementswere per-formedwithaViscotekTDAmaxsystemfromMalvernInstruments thatconsistsofanintegratedsolventandsampledeliverymodule (GPCmax)andaTetraDetectorArrayincludingaright(90◦)and low(7◦)anglelightscatteringdetector(RALS/LALS),a4-capillary differentialviscometer,adifferentialrefractiveindexdetector,and aDiodeArrayUVDetector.THFwasusedasthemobilephaseata flowrateof1mLmin−1;toluenehasservedasaflowratemarker. Allpolymerswereinjectedafterfiltrationthrougha0.45m pore-sizemembrane.TheseparationwascarriedoutonthreePolymer Laboratories columns[3× PLgel 5mMixed C (300×7.5mm)] andaguardcolumn(PLgel5m).Columnsanddetectorswere maintainedat40◦C.TheOmniSEC4.6softwarewasusedfordata acquisitionanddataanalysis.Theabsolutemolarmasswas calcu-latedusingtripledetectionfromcombinedLSandRIsignals.The averagerefractiveindexincrement(dn/dC)wasmeasuredwiththe onlinerefractometerinjectingpolymersolutionsatdifferent con-centrations.Then,thedn/dCcanbecalculatedbyplottingtheRI area(integratedfromtheRIsignal)versusinjectedconcentration
PCL-b-PEOdiblockcopolymercharacteristicsasdeterminedby HNMRandSECmeasurements.
Mwa(gmol−1) MNMRb(gmol−1) MSECc(gmol−1) PId me dn/dCf(cm3g−1)
12,410 12,992 11,180 1.12 65 0.068
aTheoreticalmolarmassascalculatedaccordingtothe-CL/MPEGfeedratioofthereactor.
bThemolarmassascalculatedfromtheratioofintegralsofthe1HNMRresonancepeaksofthePCLblockat4.07ppmandofthePEOblockat3.65ppm.
c NumberaveragemolarmassasmeasuredbySECanalysiswithcombinedLSandRIdetection.
dPolydispersityindex(PI)asmeasuredbytheSECanalysis.
ePolymerizationdegreeofthePCLblock(m)ascalculatedfromthe1HNMRspectrum.
f AscalculatedbySECanalysis.
directlyintheOmniSECsoftware.BecauseRIareaisassumedtobe proportionaltotherefractiveindex,theslopegivesdn/dC. 2.2.6. Particlesize
Size measurements of nanoparticles were performed by dynamiclightscatteringat25◦CusingaMalvern®NanoZS instru-mentoperatingat633nmwavelengthand173◦scatteringangle. Therefractiveindicesofwaterandblockcopolymerweretaken as 1.33 and 1.45 respectively, and the viscosity of water was 0.8872mPas. The size distributions obtained by the CONTIN methoddisplayedasinglepopulation,whichmadesensetothe meandiameterandwidthofthesizedistribution.Particle sizes wereexpressedasthez-averagediameterandthepolydispersity index(PdI)givenbythecumulantmethod.Formeasurementsin water/acetonemixtures,therefractiveindicesandtheviscosities ofthemixedsolventsweremeasuredusinganAtagoPAL-1 refrac-tometerandaUbbelhodecapillaryviscometerrespectively.
Emulsionsdroplet sizesweremeasuredbysmallangle light scatteringusingaMasterSizer®2000granulometer(Malvern,UK). Therefractiveindicesofwater,MCTanddodecanewere1.33,1.45 and1.42respectively.
2.2.7. Transmissionelectronmicroscopy(TEM)
NanoparticlessuspensionsandMCTinwateremulsions stabi-lizedusingmicelleswereimagedattheCentreTechnologiquedes Microstructures(CT)technicalplatformoftheUniversityofLyon [21]usingatransmissionelectronmicroscopePhilipsCM120 work-ingat80kVacceleration.Theparticlessuspensionsandemulsions wereplacedonformvar/carbon-coatedcopperTEMgridsanddried underatmosphericconditionsbeforetheirobservation.
2.2.8. Small-angleneutronscatteringexperiments(SANS)
SANS measurements were performed at 25◦C on the D22 spectrometer [22] at theInstitut Laue-Langevin (ILL, Grenoble, France).Thesampleswerecontainedinquartzcuvettesof1mm optical path, giving ∼80% transmission that ensures favorable scattering conditions (negligible multiple scattering). Scattered intensity wascollected on a 2D-detector attwo configurations withsample-to-detectordistancesof2mand14mrespectively; it wasradially averaged,giving thestandard scattering pattern ofisotropicsamplesasafunctionofthemodulusofthe scatter-ingvectorq=(4/)sin(/2)(momentumtransfer)intherange 4×10−3 ˚A−1<q<0.5 ˚A−1.Data wasprocessedaccordingto
stan-dardproceduresavailableattheILLassoftwarepackages[23]. Three sampleswere measured: A suspension of micelles of PCL-b-PEOblockcopolymerat3%(w/v)inD2O,aclassicalanda Pickeringo/wemulsionofdodecaneinD2Ostabilizedusingthe PCL-b-PEOblockcopolymer.Theoilphasewasamixtureof95vol% ofdodecane-d26and5vol%ofdodecaneofnaturalisotopic abun-dance; thescatteringlengthdensity themixturewasmatching thatofD2O.The“reference”emulsionwaspreparedbya conven-tionalemulsificationof33%oilphasemadeofa50/50(v/v)mixture ofdodecaneand dichloromethanecontaining 1.5%(w/v)of dis-solvedPCL-b-PEOinto67%ofwaterasaqueousphase.Evaporation
ofdichloromethanewasperformedaftertheemulsificationstep. Thefinalemulsionhadanoil contentof20vol%.The dodecane-in-wateremulsionstabilizedusingPCL-b-PEOnanoparticleswas preparedwithequalvolumesofdodecaneandaqueoussuspension ofPCL-b-PEOnanoparticles(5%,w/v).
3. Resultsanddiscussion
3.1. Modificationofthe“spontaneousemulsification”method The“nanoprecipitation”or“spontaneousemulsification” pro-cesswasusedforthedispersionofPCL-b-PEOinwater.Theoriginal methodcalled“nanoprecipitation”iswidelyusedforthe prepara-tionofaqueoussuspensionsofpolymernanoparticles[20,24,25].In theclassicalversionofthisprocess,asolutionofpolymerinacetone ispouredintoanaqueoussolutionofsurfactant;asupersaturated solutionofpolymerresultsfromthedissolutionofacetoneinwater, andthepolymerprecipitatesasastablesuspensionofparticles of diameterintherange 100–200nm [24–27].Wateris evapo-ratedunderreducedpressureinasecondstageinordertorecover anaqueoussuspension.Suchaprocessusuallyconsistsinmixing afairlydilutesolutionoforganicmaterialsintoalargeramount ofwater.Asaconsequencethisprocessyieldsdilutesuspensions ofsuchnanoparticles[24,25].Inthecaseofpreparationof poly-mernanoparticles,attemptstoincreasethepolymerconcentration wereunsuccessful;largelumpsofsolidmaterialsformedwhenthe amountofpolymerinacetonewasincreased.Thepresentcaseis differentsincethematerialtobeemulsifiedisanamphiphilicblock copolymerthatformsself-stabilizedparticles.Aggregation,of pri-maryelementary particlesisprevented, allowingtheformation ofmuchsmallerparticlesintherange30–40nm.Itispresumed thattheformedparticlesarecorrespondingtothosegeneratedby anucleationandgrowthprocess[26,27].Alimitedcoagulationand coalescenceprocessusuallyfollowstheformationofprimary
parti-cles[28];itispreventedbythestericstabilizationofthenon-ionic
PEOblocks.Thenanoprecipitationmethodwasmodifiedby invert-ingtherelative amountsofacetoneand water,allowinghigher concentrationsofthefinalsuspensions.Asatypicalrecipe,0.75g of theblockcopolymerwasdissolvedin 50mLof acetone.The nanoprecipitationwasperformedinto15mLofwaterundergentle magneticstirring.Theorganicsolventwasthenevaporatedunder reducedpressureat40◦C.Thepathfollowedwithinthetwosteps inaternaryphasediagram(water/acetone/PCL-b-PEO)ispresented inFig.2.Asthefirststep,thestartingsolutionofPCL-b-PEOat1.5% concentrationinacetonewasmixedwithwatersoastoreacha 1.15%suspensionofPCL-b-PEOinaacetone/water77/23mixed liq-uidphase.Thesecondstepwastheevaporationofacetonewhich yielded a clearaqueous suspensionof PCL-b-PEOnanoparticles reaching5%concentration.
Sizemeasurementrevealedtheformationofnanoparticlesof 30nmdiameterasshowninFig.2.Inordertoinvestigatethe forma-tionofnanoparticles,solutionsofblockcopolymerinacetonewere combined with water and size measurements were performed bydynamiclightscattering(DLS)beforeandafterevaporationof
Fig.2. Pathsfollowedbythechemicalcompositionofthesamplesduringthetwostagesofthepreparationofmicellesusingthe“modified”nanoprecipitationprocess.The insetattheleftshowstheparticlesizedistributionmeasuredbyDLS.
acetone.Whateverthewatercontentbetween30%and100%,the micellediametersmeasuredintheacetone/watermixedsolvent werethesame; thehydrodynamicdiameters wereintherange 26–35nm(Table2).Thenanoparticlesformedduringthefirststep whenthePCL-b-PEOsolutioninacetonewasmixedwithwater.
Sinceacetoneisagood solventfor thePCLblockanda pre-cipitantfor thePEOblock,it is presumedthatreverse micelles wereformedinthestartingsolutioninacetone.Theself-assembled structuresofblockandgraftcopolymersstronglydependonthe solventtypeandconcentration:directmicelles,reversemicelles, ormoreorlessrigidorganizedstructures(cylinders,lamellae)[29]. Formationofreversemicelleswasmorelikelybecausethe solu-tioninacetonewasfluidandisotropic.Formationofdirectmicelles bymixingwaterandthesolutioninacetoneinvolvedastructural inversionfromreversetodirectmicelles.
3.2. Emulsionsstabilizedbyblockcopolymernanoparticles
O/w emulsions were prepared using medium chain triglyc-erides(MCT)asoil.Thepreparationofemulsionswasinvestigated using increasing amounts of oil using particles suspensions of concentrations1.5,3or6%(w/v).Emulsificationwascarriedout bymechanicalstirringwithaconventionalrotor-statorshearing device(Ultra-Turrax).Emulsificationwassuccessfulupto50%oil content:thefulloilwasdispersedasdropletsattheendofthe emul-sificationprocess.Partialfailurewasobservedfor60%oilcontent:
Table2
Nanoparticlemeandiametersmeasuredbydynamiclightscatteringasafunction ofthecompositionoftheacetone/watermixedsolvent.
Acetone (%) Water (%) PCL-b-PEO (%)
z-Averagediameterbeforesolvent evaporation(nm),andPdIinbrackets
76 23 1 26(0.2)
69 30 1 26(0.2)
49 50 1 35(0.12)
29 70 1 35(0.13)
0 95 5 30(0.12)a
aAfterevaporationofacetone.
ano/wemulsionwasindeedformed,butfreeoilwasstillpresent asamacroscopiclayerofpureoilabovetheo/wemulsion. Cream-ingwasobservedbecausethecontinuousphasewasfluid;itsrate wasdependingonthedropletdiameter.Themeandropletdiameter dependedontheconcentrationsofnanoparticlesandoilasshown inFig.3a.Systematic experimentswhere eithertheoil content wasvariedatconstantconcentrationofPCL-b-PEOnanoparticlesor thenanoparticlesconcentrationwasvariedatconstantoilcontent, mergedintoasinglemastercurvewhenthemeandropletdiameter wasplottedasafunctionofthemassratioM(oil)/M(nanoparticles) (Fig.3b).Thisbehaviorshowedthatthedropletsizewascontrolled bythemassratioM(oil)/M(nanoparticles).Suchphenomenonhas previouslybeenobservedwithPickeringemulsionsstabilizedwith inorganicsolidparticlessuchassilica[8].Thelineardependenceof dropletdiameterwithrespecttoM(oil)/M(nanoparticles)isinfull agreementwiththelimitedcoalescencephenomenonintroduced byArdittyetal.[30].Thelinearrelationshipreads[8]
D= 6
oilananoparticles
M(oil)
M(nanoparticles) (1)
whereoilisthedensityofoilandananopariclesistheinterfacialarea coveredbyoneblockcopolymernanoparticleatdensecoverage oftheoildroplets.Thediameterofdropletsdidnotdecreasebut remainedconstantformassratios below5 wheretheexpected droplet size was the smallest.The present mechanical stirring devicewasnotpowerfulenoughtobreakupsmalloildroplets.It wasdifficulttoobtaindropletemulsionssmallerthan2mdespite usinghighnanoparticleconcentrationand/orlowamountsof inter-nalphase.Theemulsionsobtainedunderwentreversiblecreaming andshowedexcellentstabilityuponstorageat25◦Coverseveral monthsevenforhighinternalphaseratioemulsions(>50%,v/v). Thedropletsizemeasuredduring3monthsstorageat4,20and 40◦Cremainedconstant.
3.3. Investigationofthephysicalstateofnanoparticles:1HNMR studies
Directmicellesof PCL-b-PEOformedinD2O.ThePCLblocks formedacentralhydrophobiccore,whilethePEOblocksformed
Fig.3.(a)EmulsionsmeandiameterasafunctionofMCToilandnanoparticlescontents.(b)EmulsionsmeandiameterasafunctionoftheM(oil)/M(nanoparticles)mass ratio.
ahydrophiliccoronalayerswollenbywater.1HNMRspectraof nanoparticleswere run inorder to assessthe physicalstate of