E ff ect of Detergents on Morphology, Size Distribution, and Concentration of Copolymer-Based Polymersomes - Antenucci et al. - 2021 - U

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pubs.acs.org/LangmuirArticleEffectofDetergentsonMorphology,SizeDistribution,andConcentrationofCopolymer-BasedPolymersomesRadosławGórecki,*FabioAntenucci,KarolisNorinkevicius,LineElmstrømChristiansen,ScottTrevenMyers,KrzysztofTrzaskus,andClausH́élix-NielsenCiteThis:Langmuir2021,37,2079−2090ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Polymersomesmadeofamphiphilicdiblockco-polymersaregenerallyregardedashavinghigherphysicalandchemicalstabilitythanliposomescomposedofphospholipids.Thisenhancedstabilityarisesfromthehighermolecularweightofpolymerconstituents.Despitetheirincreasedstability,polymerbilayersaresolubilizedbydetergentsinasimilarmannertolipidbilayers.Inthiswork,weevaluatedthestabilityofpoly(ethyleneglycol)-block-poly(ε-caprolactone)(PEG−PCL)-basedpolymer-somesexposedtothreedifferentdetergents:N-octyl-β-D-glucopyranoside(OG),lauryldimethylamineN-oxide(LDAO),andTritonX-100(TX-100).Changesinmorphology,particlesizedistribution,andconcentrationsofthepolymersomeswereevaluatedduringthetitrationofthedetergentsintothepolymersomesolutions.Furthermore,wediscussedtheeffectofdetergentfeaturesonthesolubilizationofthepolymericbilayerandcomparedittotheresultsreportedintheliteratureforliposomesandpolymersomes.ThisinformationcanbeusedfortuningthepropertiesofPEG−PCLpolymersomesforuseinapplicationssuchasdrugdeliveryorproteinreconstitutionstudies.9,12,13■INTRODUCTIONbilayerpermeability,andparticlesizedistribution.Despitetheenhancedstabilityofpolymersomes,higherAmphiphilicdiblockandtriblockcopolymermacromolecules,membraneviscosity,andalargerbendingmodulus(ascomprisinghydrophilicandhydrophobicblocks,areknownto14−17comparedtoliposomes),polymersomescanalsobeassembleintosupramolecularstructuressuchasmicelles,solubilizedbydetergents.Thedetergentmoleculesareinsertedpolymervesicles(polymersomes),rod-likemicelles,orplanar1,2intothebilayerstructure,whichcausesachangeinthemembranes.Thereisastronginteresttousetheseamphiphilepacking,rearrangementofthebilayercurvature,DownloadedviaUNIVOFCONNECTICUTonMay16,2021at07:55:53(UTC).supramolecularassembliesinvariousresearchandindustrialandultimately,atahighenoughdetergentconcentration,purposesastheycloselymimictheirbiologicalequivalents,9,14,18,19completedisassemblyofthevesicles.amphiphilicphospholipids,andtheirself-assemblies(lip-Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.Severalbilayersolubilizationmechanismshavebeenosomes).Targetedandcontrolledreleaseofanencapsulateddescribed,andwhiletheyallhaveminorvariations,thedrugpayloadisthemostcommonlystudiedmedicalgeneralconsensusisthatthesolubilizationisathree-stageapplication,butpolymersomesarealsousedtostudythe19−22process.Inthefirststage,thedetergentsmigrateintothereconstitutionoftransmembraneproteinswithinthemem-bilayer,resultinginanincreaseinvesiclesize.Inthesecondbranelayeroftheassemblyforthepreparationofselective1,3−9stage,whenthesaturationpointofdetergentsinthebilayerisseparationdevicesandsensors.Polymersomesareaachieved,thetransitionfromvesiculartomixedmicellarpromisingnanoscaleassemblyfortargetedtherapyand2structuresisinitiated.Inthethirdstageofsolubilization,atreatmentofseveredisorders,includingcancer,duetotheir10completetransitionofthevesiclestomixedmicelles,consistingimprovedstabilityincomparisontomicellarstructuresand1ofamphiphilesanddetergents,takesplaceandfurtheradditionliposomes.Thesuperiorchemicalandphysicalstabilityofpolymersomesoverliposomesisbecauseofthehighermolecularweight(MW)ofamphiphilicblockcopolymerReceived:October19,2020chains,whichformathickerandmorerigidbilayer1,11whenRevised:January19,2021theyself-assembleintovesicles.Published:February3,2021Anothertypeofamphiphilicmoleculedetergentscanbeintroducedtotheliposomeorpolymersomesolutiontochangethepropertiesoftheself-assemblies;suchasmorphology,©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c030442079Langmuir2021,37,2079−2090

1Langmuirpubs.acs.org/LangmuirArticleFigure1.Chemicalstructuresofthedetergentsusedinthestudy,(A)OG(MW=292g/mol,cmc≈20.0mM);(B)LDAO(MW=229g/mol,cmc≈2.0mM);(C)TX-100(n=10)(MW=625g/molcmc≈0.24mM).ofdetergentonlyleadstothereductionofthephospholipid/compositionswillnotbehaveinthesamemannerupon9,19,20,22−24polymerconcentrationwithinthemixedmicelles.introductiontodifferentdetergenttypes,similartomembranesMultiplestudiesinvestigatingthemechanismofliposomecomposedofdifferentphospholipids.Thiscould,theoretically,solubilizationhaveshownthattheconcentrationofdetergentsallowonetomodifytheresultingbilayermembraneandrequiredtosolubilizeliposomevesiclesisdependentontwopolymericnanostructurepropertiesforaspecificapplicationby13factors,including(1)thecompositionofthephospholipidscombiningtherightcopolymeranddetergenttype.usedtoconstructtheliposomallipidbilayerand(2)thetypeInthepresentedwork,weinvestigatedthedetailedeffectsof9,14,18,20−29ofdetergentusedforsolubilization.Incontrasttothethreedetergentsonapoly(ethyleneglycol)-block-poly(ε-publishedstudiesregardingthesolubilizationofliposomes,caprolactone)(PEG−PCL)-basedpolymersomesystem.thereisalimitednumberofpublicationsthatfocusonthePEG−PCLconsistsofanontoxicPEGsectionandanin-40−42explanationofthesolubilizationeffectontheblockcopolymer-vivobiodegradablePCLpart.Theycanbeeasily30,31basedvesiclesystems.Moreover,thestudiesareoftensynthesizedinlargescale,withtheuseofnontoxiccatalysts4344limitedtoopticaldensity(OD)measurementsanddynamicsuchascalciumorenzymes,andself-assembledstructures26lightscattering(DLS)analysis,withvisualizationmadeoncanbepreparedviascalablemethods,suchasdirect24,30,32hydration.2,40ThismakesPEG−PCL-basedsystemsattractivegiantunilamellarvesicles.DLSisknowntohave40,45significantdrawbacksforproperanalysisofpolydisperseforindustrialapplicationssuchasdrugdelivery.33−38solutions,asoccurswithvesiclesmixedwithdetergents.Furthermore,PEG−PCLproductsarealreadyapprovedasTherefore,DLSanalysisasastand-alonemethodonlypartiallydrugdeliverysystemsbyhealthcareauthorities,suchasthe2,46describestheprocessesofvesiclesolubilizationanddoesnotU.S.FoodandDrugAdministration(FDA).MultipleprovideanyinformationonparticleconcentrationsorchangesstudieshaveinvestigatedPEG−PCLsystemsforconjugationinthevesiclemorphology.Asaresult,conclusionsbasedsolelywithproteins(e.g.,glycoproteinsorantibodies),cellbinding40,47−49onDLSmeasurementsareofteninaccurateandvague.Very(targetingstudies),andcellviability;however,thereisfewstudiescomplementODandDLSanalysesofnanostruc-nostudyfocusingonthePEG−PCLbilayersolubilization.Totureswithcryogenictransmissionelectronmicroscopythebestofourknowledge,thisisthefirststudytoinvestigate(cryoTEM),whichallowsthevisualizationofthechangingthedetergent-inducedvesiclesolubilizationofapolymersome9,21,27,39systemthatcombinesODanalysis,DLS,tunableresistivepulsemorphologiesoftheself-assemblies.However,whentheconcentrationsofthestudiedparticlesdrop,itbecomessensing(TRPS),andcryoTEM.Becauseofthejointmoredifficulttovisualizethesystemproperly,asveryfewofapplicationoftheabove-mentionedanalyticalmethods,thetheparticleswillberetainedontheTEMgrid.Additionally,effectofsolubilizationwascharacterized,notonlyqualitativelycryoTEMprovidesonlyqualitativeandnotquantitative(byODandaverageparticlesize)butalsoquantitatively(byinformationabouttheparticlesizedistributionandconcen-TRPSmeasurementsprovidingparticlesizedistributionandtrations,thus,onlypartiallyexplainingthesolubilizationparticleconcentrationsinthesolution).Furtherunderstanding9oftheprocesshasbeenprovidedbytheprecisemorphologymechanism.Inthemostdetailedstudyonthedetergentsolubilizationofpolymericbilayers,Pataetal.investigatedtheanalysisusingcryoTEM.Thisstudyexplains,indetail,thesolubilizationofpolymericmembranesmadeofpoly(ethylenemechanismofsolubilizationofPEG−PCLpolymersomesglycol)-block-poly(ethylethylene)(PEG−PEE)andpoly-inducedbythreedifferentdetergents.(ethyleneglycol)-block-poly(butadiene)(PEG−PB)block30copolymerswithTritonX-100(TX-100).Theauthors■EXPERIMENTALSECTIONbasedtheirconclusionsonODandDLSmeasurementsthatMaterials.ThethreedetergentsusedN-octyl-β-D-glucopyrano-werecombinedwiththevisualizedsolubilizationofgiantside(OG),lauryldimethylamineN-oxide(LDAO),andTritonX-100unilamellarvesicles.Asaresult,themorphologicaltrans-(TX-100)inthestudyarecommonlyemployedinbilayerformationsoccurringinthenanostructures,thechangesinmembraneresearchforsolubilization,ortheextractionand23,27,50,51particleconcentration,andthenanoparticlesizedistributionreconstitutionoftransmembraneproteins.Thedetergentsanalysiswereneglected,aswasthequantitativeoverviewonhavedifferentchemicalstructures,MWs,andcriticalmicellethesolubilizationprocess.Furthermore,thestudywaslimitedconcentrations(cmc)inpureH2O,andthosearepresentedinFigure1.toonedetergenttype(TX-100),thusthereisstillampleN-LauryldimethylamineN-oxide(30%aqueoussolution)wasopportunitytobroadentheunderstandingoftheeffectoftheobtainedfromSigma-Aldrich,Denmarkandusedasreceived.OGdetergenttypeontheprocessofthesolubilizationofpolymer-(≥99%)wasobtainedfromAnatrace,USA,andusedas30%aqueousbasedbilayers.Finally,thementionedstudiesweremadeonsolution.TX-100(≥99.8%)wasobtainedfromSigma-Aldrich,PEG−PEE-andPEG−PB-basedsystems,anditcanbeDenmark,andusedas20%aqueoussolution.Phosphatebufferedexpectedthattheblockcopolymerbilayersofdifferentsaline(PBS)waspreparedbydissolvingthesalts(8mg/mLNaCl,0.22080https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

2Langmuirpubs.acs.org/LangmuirArticleTable1.DLSResultsofUnfilteredandFiltered(0.2μmPES)PEG−PCLSolution,PresentingthePDIandHydrodynamicRadiuses(Dh)oftheTwoParticlePopulationsMeasuredPDIZ-avgpeak1Dh(nm)/intensity(%)peak2Dh(nm)/intensity(%)unfiltered0.45±0.02196±2nm285±220nm/92%3200±1140nm/8%filteredvia0.2μmPES0.25±0.01160±2nm192±60nm/79%63±15nm/21%Figure2.CryoTEMmicrographsofunfilteredPEG−PCLpolymersomesolution(left)andfilteredsolutionvia0.2μmPESfilter(right),scalebar:500nm.mg/mLKCl,1.44mg/mLNa2HPO4,0.24gKH2PO4)inMilli-Qcuvettes(Sarstedt,Germany).TheDLS-measuredsampleswerewater,pHwasadjustedto7.4withHCl.AllchemicalsforthefurtheranalyzedbycryoTEMvisualizationandTRPSmeasurements.preparationofPBSwereofanalyticalgradeandwereobtainedfromMorphologyCryogenicTransmissionElectronMicrosco-MerckKGaA,Germany.Poly(ethyleneglycol)methylether(Mn∼py.Samplesforvisualizationwerevitrifiedonaglow-dischargedlacey550)(PEGME)wasobtainedfromMerckKGaA,Germany.formvarfilmenforcedbysiliconmonoxidecoatingandsupportedbyaPoly(ethyleneglycol)45-block-poly(ε-caprolactone)44(PEG45−coppermeshgrid(TedPellaInc.,USA).ThesampleswerevitrifiedinPCL44)(PDI1.2)waspurchasedfromAdvancedPolymerMaterials,liquidethanewiththeuseofVitrobotMarkIV(FEI,USA)andCanada.Protonnuclearmagneticresonance(HNMR)andsize-subsequentlymountedinaGatancryoholder(FEI,USA).Theimagesexclusionliquidchromatography(SEC)analysesforthediblockwereacquiredincryogenicmodeusingaTecnaiG220TWIN200kVcopolymerarepresentedintheSupportingInformationinFiguresS1TEMequippedwithaFEIHigh-Sensitive4k×4kEaglecamera.andS2,respectively.TheresultsofdifferentialscanningcalorimetrySizeDistributionandParticleConcentrationsTRPS.Virgin(DSC)arepresentedinFiguresS3andS4.sampleandthreesamplescorrespondingtoeachstageofPreparationofPolymersomes.Polymersomeswerepreparedsolubilization,basedontheresultsfromOD,DLS,andcryoTEMviadirecthydrationaspreviouslyreportedinSuietal.,inPBS(pHanalyses,foreachofthedetergentswerechosenforTRPSanalysis,7.4,136mMNaCl,2.6mMKCl).40Inbrief,1gofPEG−PCLwassizedistribution,andparticleconcentrationsmeasurementwereanalyzedwithaTRPSqNanoGoldsystem(IzonScience,Newmixedwith10gofPEGME,topromotethehydrationoftheZealand),equippedwithaNP150polyurethanenanopore(Izoncopolymer,asPEGMEisbothsolubleinthepolymeraswellasinScience,NewZealand)andbyfollowingthestandardprotocolwater.Themixturewasheatedto60°Candstirredat300rpmfor3052recommendedbythemanufacturer.Theanalyticalwindowformin.Subsequently,thetemperaturewasloweredto40°C,and100TRPSmeasurementswasoptimizedfortheparticleswithdiametermLofPBSbufferwasadded.Theobtainedsolutionwasstoredatbetween50and550nm.ToperformtheTRPSmeasurements,theroomtemperatureandfilteredthrougha0.2μmpolyethersulfonesampleswerediluted50timeswithPBSbufferandanalyzedundera(PES)filter(Sarstedt,Germany)priortouse.voltageof0.96Vandapressureof13mbar.DataanalysiswasCharacterizationofPolymersomesandtheSolubilizationperformedusingthedatacaptureandanalysissoftware,IzonControlProcessStabilityoftheParticlesintheDetergentSolutionOD.ODmeasurementsweremadewithaVarioskanmicroplateSuiteV3.4(IzonScience,NewZealand).reader(ThermoFisherScientific,MA,USA)todeterminethevesicle-solubilizingconcentrationsforeachdetergent.Toperformthe■RESULTSANDDISCUSSIONtitrationexperiment,200μLofthefilteredpolymersomesolutionwasmixedwiththedetergenttoobtainthedesiredconcentrationinCharacterizationofthePolymersomeSolution.Thethewellofapolystyreneflat-bottom,clearmicroplate(GreinerBio-polymersomesolution,withoutdetergentaddition,wasOne,Austria).KineticODmeasurementsatawavelengthof400nmanalyzedwithDLS,cryoTEM,andTRPSasastandardforweremadeatspecifiedtimepointsat20°C.Allsampleswerecomparisontosolutionswiththethreechosendetergentspreparedintriplicate.WeobservednofurtherchangesinODafter90throughouttherestofthisstudy.TheresultsoftheDLSminofdetergentincubationandthereforechose2hforthedetergentmeasurementsofthepolymersomesolutionbeforeandafterincubationtimeintheanalyzedsamples.filtrationthrougha0.2μmPESfilterarelistedinTable1.ThePolydispersityIndexandAverageHydrodynamicDiame-cryoTEMimagesofthesamesolutionsbeforeandafterterDLS.TopreparethesamplesforDLSmeasurements,thefiltrationareshowninFigure2.filteredpolymersomesolutionwasmixedwithdetergentstoreachtheCryoTEMvisualizationrevealedthattheunfilteredPEG−desiredconcentrationsandwasstirredfor2hat750rpmatroomtemperature.Averagehydrodynamicdiameter(Z-avg)andpoly-PCLpolymersomesolutionconsistedofvesicleswithadispersityindex(PDI)measurementsweremadebyaDLSmembranethicknessvaryingbetween15and20nm(majorityinstrument,theZetasizerNano(Malvern,UK).Allmeasurementsunilamellar,thoughsomemultilamellarvesicleswerepresent).weremadeinbatchmodeat20°Cusingpoly(methylmethacrylate)Additionally,thesamplescontainedsinglemicelles,rod-like2081https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

3Langmuirpubs.acs.org/LangmuirArticleFigure3.ParticlesizedistributionhistogramsobtainedfromTRPSmeasurements:(A)startingmaterialofthepolymersomesolution;(B)solutionswithOG;(C)solutionswithLDAO;and(D)solutionswithTritonX-100(TX-100).Theparticleconcentrationdropsasthedetergentconcentrationisincreased.TRPShasalimitedresolutionwindowandforthechosensettingsparticles≤50nmindiameterwerenotdetectable.micelles,andmicrometer-sizedmultilamellarvesicles.FiltrationC12alkyltail.Itiswidelyusedforthesolubilizationofremovesthelatter;however,rod-likemicellesarestillpresentbacterialmembranesfortheisolationofintegralmembrane53−56inthefilteredsolution,togetherwithsub-micronvesiclesandproteins.TX-100isanonionicdetergentoftenusedforsinglemicelles.Likewise,thePDIofthefilteredsolutionwastransmembraneproteinreconstitution,anditisknownto0.25andtheZ-avgwas160nm,comparedtoaPDIof0.45penetratethelipidmembraneattheonsetofsolubilization31,39andZ-avgof196nmfortheunfilteredsample.ThroughTRPSwithoutaffectingthemembranebilayerstructure.Themeasurements,theconcentrationoftheparticlesinthefilteredselecteddetergentswerechosenbasedontheirfrequentuseinPEG−PCLsolutionwasmeasuredtobeintherangeof2×membraneproteinsolubilizationandreconstitutionstudies.1013particles/mL,withameanphysicaldiameterof115nm.AODmeasurementsofthePEG−PCLpolymersomesolutionshistogramoftheparticledistributionforthefilteredsolutioniswithtitrateddetergentsarepresentedinFigure4.presentedinFigure3.BecauseofporeblockagefromtheTheODmeasurementswereobtainedtodefinethepresenceofthemicrometer-sizedvesiclesintheunfiltereddetergentconcentrationsatwhichthetransitionbetweenthePEG−PCLpolymersomesample,itwasnotpossibletosolubilizationstagesofPEG−PCLpolymersomesoccurfor21measureparticledistributionwithTRPS.Throughouttheeachdetergent.AsshowninFigure4A,atlowconcentrationsrestofthearticle,werefertothefilteredPEG−PCLofOG(1.7−8.5mM),thenormalizedODincreasedby0.1polymersomesolutionasthevirginsample.(for120mincontacttime).Incontrast,upontheintroductionThree-StageSolubilizationofPEG−PCLPolymer-ofLDAOandTX-100,normalizedODincreasedby0.4andsomesinMixtureswithDetergents.OGisaglucopyrano-0.3(seeFigure4B,C),respectively.Theincreaseinturbiditysideandoneofthemostcommonlyappliednonionicforalloftheinvestigatedsamplesuponexposuretolowdetergentusedforthesolubilizationofintegralmembranedetergentconcentrationscanbeexplainedbytheincreaseofproteinsandtheirreconstitutionintoproteoliposomes,duetothevesiclediameter.Thisisinalignmentwiththefirststageof23,50,51,53,54itshighcmcvalueandnondenaturatingproperties.bilayersolubilization.Atthelowconcentrationsofdetergents,LDAOisazwitterionicamineoxide-baseddetergentwithaso-calledsub-solubilizingconcentrations,anincreaseofthe2082https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

4Langmuirpubs.acs.org/LangmuirArticle(startingfrom10.2,1.1,and2.4mM,forOG,LDAO,andTX-100,respectively)indicatestheinitiationofthesecondstageofsolubilization,wherethedisintegrationofthevesiclesoccursbecauseoftheirbilayerbecomingsaturatedwithdetergentmolecules.Fullsolubilization(stage3)occurredat23.4mMforOG,8.7mMforLDAO,and6.4mMforTX-100,whenthenormalizedODreached0.13,0.22,and0.43forOG,LDAO,andTX-100,respectively.ThisobserveddropinODisrelatedtothethirdstageofsolubilizationformationofsmallersinglemicelleswithahigherdetergent-to-copolymerratio.ThelimiteddropinODforTX-100comparedtoOGandLDAOcanbelinkedtothehigherMWofTX-100andincomplete21bilayerpenetrationbythedetergent.Nonetheless,TX-100solutionsareknowntohaveincreasedturbidityincomparisontoLDAOandOGbecauseofitslowercloudpointinaqueous9solution.Moreover,Figure4showsthatbetweenthesecondandthirdstagesofsolubilization,ODmeasurementsaremoretimedependentforalldetergents,becausethedisintegrationofthevesiclesisakineticprocess.EffectofOGonthePolymersomeMorphology.AfterdeterminingthechangesinODofthepolymersomesolutionswiththethreedetergentsDLSmeasurementsweremadeasthefirststeptoexplainthemorphologicalchangeshappeningupondetergenttitration.Namely,theaveragediameterofparticles(Z-avg)andthePDIweremeasured.TheresultsoftheDLSmeasurementsforthesampleswithdifferentOGconcen-trationsarepresentedinFigure5A.AtconcentrationsofOGbetween1.7and13.5mM,theZ-avgremainsunchangedatroughly200nm,indicatingthatthevesicleswerestablewithinthisrange.However,cryoTEMmicrographs(presentedinFigure6)showthatfrom5.1mMOG,thenumberofrod-likemicellesandsinglemicelleswassignificantlyhigherthanforthevirginsample.Interestingly,ataconcentrationof5.1mMOG,thepresenceofrod-likemicelleswasthemostprominentmorphology,incomparisontotheothersamplescontainingOG.ThisexplainstheincreasedODofthesolutions(seeFigure4A),astheenlargedvesicleswithdetergent-saturatedbilayersarepresentwith19,21singlemicellesandrod-likemicellesinthesample.Thesignificantincreaseofthepopulationofrod-likemicellesisbarelydetectablebyDLS,asZ-avgremainsunchangedandPDIonlyslightlyincreasesbecauseofthelargevesiclesscatteringlightwithahigherintensitythanthesmallerparticlespresentinthesolution.At13.5mMOG,adetergentconcentrationinthemiddleofthesecondstageofsolubilization,thetotalparticleconcentrationmeasuredbyTRPSdroppedfrom2×1013to8×1012particles/mL(fortheparticlesabove50nm).Moreover,thenumberoftheparticlesintherangeof100−200nminphysicaldiameter,correspondingtovesicles,droppedbyhalf(seeFigure3A),from3×1012to1.5×1012particles/mL.Afterreachingconcentrationsof20.1mMOG,Z-avgdropsto167±17nmandPDIincreasesto0.43±0.03,indicatinganincreasednumberofsinglemicellesinthesolutionandadecreasednumberofvesicles.AsshownincryoTEMmicrographs(Figure6),mostlysinglemicellesandrod-likeFigure4.ResultsoftheODmeasurementsofthePEG−PCLpolymersomeuponintroductionofthedetergents:(A)OG;(B)micelleswerepresentinthesolution,withafewvesiclesvisibleLDAO;and(C)TritonX-100(TX-100).Concentrationsarewithradiusesbelow100nm.providedinmMaswellasfractionsofthecmc.Atconcentrationsreaching23.4mMOG,thebilayerofthevesicleswascompletelysolubilized.TheZ-avgdroppedto50vesiclediameteroccursbecauseofthepenetrationofthe±2nmandthePDIwasreducedto0.10±0.01.Thisisalso25,28,57detergentmoleculesintothebilayer.ThesubsequentconfirmedbyTRPSmeasurements,asthemeasuredtotalobserveddropinODatincreasingdetergentconcentrationsparticleconcentrationdroppedto2.04×1012(seeFigure3A).2083https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

5Langmuirpubs.acs.org/LangmuirArticleFigure5.DLS-measuredaveragehydrodynamicdiameter(Z-avg)andPDIofparticlesupontheintroductionofthedetergents:(A)OG;(B)LDAO;and(C)TritonX-100(TX-100).TheZ-avgandPDIdropssharplywhenthedetergentconcentrationcapableofsolubilizingthemajorityofthevesiclesisreached.Noporeformationinthevesiclebilayer,typicallyobservedforEffectofLDAOonthePolymersomeMorphology.21,25liposomes,wasobservedwithcryoTEMatanyoftheOGTheZ-avgandPDIofpolymersomesolutionsexposedtotheconcentrations.increasingLDAOconcentrationsarepresentedinFigure5B.2084https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

6Langmuirpubs.acs.org/LangmuirArticleFigure6.CryogenictransmissionelectronmicroscopymicrographsofvesiclesampleswithdifferentconcentrationsofOG,scalebar:500nm.Figure7.CryogenictransmissionelectronmicroscopymicrographsofvesiclesampleswithdifferentconcentrationsofLDAO,scalebar:500nm.Atconcentrationsupto2.2mMLDAO,theZ-avgandPDI1.1mMLDAO(seeFigure4B).ThecryoTEMmicrographsofremainedunaffected,averagingatsizesof210−230nmwithathesampleswith0.2and1.1mMLDAOexhibitenlargedPDIof0.40−0.44.Thesenumbersindicatestabilityofthevesicles(Figure7)confirmingtheDLSandODreadings.polymersomesinthepresenceofthedetergentastheyshowaHowever,forthesamplewith2.2mMLDAO,theODslightincreaseinsize,butarestillsimilartotheresultsfromdecreasedincomparisontothesampleswithlowerLDAOthevirginsolution(seeTable1andFigure2).Thesechangesconcentrations.ThisisbecauseofthedecreasedvesiclealsocorrelatetotheincreaseinOD,uptoconcentrationsofconcentrationandsizewhichisobservableonthecryoTEM2085https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

7Langmuirpubs.acs.org/LangmuirArticleFigure8.CryogenictransmissionelectronmicroscopymicrographsofthevesiclesampleswithdifferentconcentrationsofTritonX-100(TX-100),scalebar:500nm.micrograph(Figure7).TheDLSmeasurementisnotsensitiveH2O)comparedtoOG(∼20mMinpureH2O).Thus,LDAOtothesechanges,asPDIandZ-avgremainunchangedatmoleculesarrangedintomicellesatlowerconcentrationsthanLDAOconcentrationsupto2.2mM.OG,andincombinationwithPEG−PCLchains,aremoreAt4.4mMLDAO,theZ-avgdecreasedsharplyto157±37pronetoformsinglemicellesthanlongrods,therebynmandPDIincreasedto0.52±0.02.Additionally,theerrorsolubilizingthebilayeratlowerconcentrationsthanOG.Atofthemeasurementswasalsoincreasedatthisconcentration6.5mMLDAO,thePDIdroppedto0.24±0.12andZ-avgto(seeFigure5B).Theseresultsindicatethattheaveragesizeof54±16nm.WhiletheerrorofthePDIreadingswasmoretheparticlesdecreased,whiledifferencesintheparticlepronounced,theseDLSresultscorrelatewiththeobservedpopulationsbecamemoreprominent.ThecryoTEMmicro-dropintheconcentrationofpolymersomes,andanincreasedgraphofthesamplewith4.4mMLDAO,showsthepresenceconcentrationofsinglemicellesalsoconfirmedbycryoTEMofpredominantlyunilamellarvesicles.Thesevesicleswereimaging(Figure7).Completesolubilizationwasreachedat8.7smallerinsizeincomparisontothepolymersomesampleswithmMLDAO,wherethemeasuredPDIwas0.16±0.01andZ-0.2and1.1mMLDAO(Figure7).Thesizeofthevesiclesinavgreached44±1nm.CryoTEMconfirmedthatthesamplethepolymersomesamplewith4.4mMLDAOwascomparablemostlycontainedsinglemicellesandsub-200nmlengthrod-tothesamplecontaining2.2mMLDAO;however,thelikemicelles,whiletheTRPSmeasurementshowedapopulationofrod-likemicelleswasdecreased.Theyweremostreductioninthetotalparticleconcentrationto1.69×1012likelysolubilizedtosinglemicelles,whichwerepresentintheparticles/mL.SimilartoOG,noporeformationwasobservedsolutionwiththevesicles.ThesemorphologicalfindingswithinthevesiclebilayerwasobservedoncryoTEMcorrelatetotheODmeasurements,asthepolymersomemicrographsatanypointduringthevesiclesolubilizationsamplewith4.4mMLDAOexperiencedasharpdropinOD(seeFigure7).(seeFigure4B).Thedropinconcentrationofvesiclesat4.4EffectofTritonX-100onthePolymersomeMorphol-mMLDAOwasquantifiedwithTRPS(seeFigure3B),astheogy.ThePDIandZ-avgmeasuredbyDLSforpolymersomestotalconcentrationofparticlesdroppedfrom2×1013mixedwithTX-100arepresentedinFigure5C.TX-100didparticles/mLinthevirginsolutionto8.65×1012particles/notinducesignificantchangestotheZ-avgatconcentrationsmL.Furthermore,theconcentrationofparticlesbetween100upto3.2mMTX-100.However,thecryoTEMmicrographofand200nmwasreducedbyhalf,whichisidenticaltotheeffectthesamplewith0.4mMTX-100(presentedinFigure8),observedat13.5mMofOG,whichistheconcentrationwhereshowedlargervesiclesincomparisontothevirginsolution(seestage3ofthesolubilizationprocessoccurs(seeFigure3A).Figure2),whichwasalsoobservedasanincreaseintheODIncontrasttoOG,therod-likemicellemorphologytendstomeasurements(seeFigure4C).decayatincreasedLDAOconcentrations(above4.4mM),ThePDIdidincreaseatconcentrationsupto3.2mMofwithmostoftheparticlesbeingsinglemicelles.EventhoughTX-100.ThisrelatestothemorphologicalchangesobservedthetwodetergentshavesimilarMW229g/molforLDAOwithcryoTEM.UpontheadditionofTX-100,longrod-likeand292g/molforOG,theircmcisdifferent.LDAOismicelleswereformedwithlengthsexceeding2μm.Theircharacterizedbya10-foldlowercmcvalue(∼2mMinpurepopulationandlengthsincreaseduntilaconcentrationof4.02086https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

8Langmuirpubs.acs.org/LangmuirArticlemMofTX-100wasreached,wheremostofthestructureswerephenomenaisknowntooccurinliposomesandisconsidered21,25solubilizedintosinglemicelles(seeFigure8).Whentheaprerequisiteforthevesiclesolubilization.Itcanbeconcentrationreached4.0mMTX-100,itcouldbeelucidatedspeculatedthattheabsenceoftheporeformationisaresultoffromDLSmeasurementsthatacompletevesiclesolubilizationasignificantmismatchinthedetergentlengthincomparisontotookplacebecausetheZ-avgdroppedto48±1nmandthethediblockchain.EvenforthelargestdetergentmoleculeinPDIdroppedto0.25±0.02.Somevesicleswerestillvisibleonthisstudy,TX-100,themassofthePEG45−PCL44chainthecryoTEMmicrographsat4.0mMTX-100,thoughtheir(averageMW=7000g/mol)ismorethan10timeslarger.Inpopulationwasgreatlyreduced.Whileafurtherincreaseinthecomparison,forthecommonlyusedvesicle-formingphospho-59detergentconcentrationresultedinacontinueddecreaseoflipids,MWrangesbetween700and800g/mol,whichisOD(seeFigure4C),theZ-avgandPDIremainedconstant.AsignificantlyclosertotheMWofthedetergents.Additionally,PDIbelow0.2wasnotreachedevenatthehighesttestedpolymershavecertaincharacteristicsthatareabsentinthelipidconcentrationof6.4mMTX-100,asinthecaseofOGandconstituents.Polymersarenotperfectlypuresystems,evenLDAO,whencompletesolubilizationwasachieved.ThiscouldwhenthePDIislow.TheblockcopolymerusedinthisstudyindicatethatasmallportionoftheinsolubilizedvesicleswaswascharacterizedwithaPDIof1.2.Nevertheless,incontraststillpresent.Nevertheless,cryoTEMmicrographsofthe4.8topurelipidmixtures,apolymermixturewillalwaysconsistof60mMTX-100samplerevealedunusualaggregateswithapolymerchainswithavarietyofblocklengths.Thesemorphologybetweenlargemicellesandrod-likemicelles(seepolymerswithnonuniformchainlengthsbuildthevesicleFigure8).TheresultsofTRPSmeasurements(presentedinbilayer,andbecauseofthemembraneliquidityandlateralFigure3D)showedthatthetotalparticleconcentrationwasdiffusion,theywillbeabletoreshufflewithinthelayer.Thus,1.60×1013,1.49×1013,and6.78×1012particles/mLfor0.4,enablingchainconfigurationstoalterwithinthebilayerand1.6,and4.8mMTX-100,respectively.Asharpdecreaseinthemodifyingthepackingofthechains.Infact,molecularconcentrationofparticlesinthesizerangeof100−200nm,indynamicsmodelinghasindicatedthatblockcopolymercomparisontothevirginsolution,wasrecordedonlyforthemembranesarecapableofadjustingthethicknessofthesamplewith4.8mMTX-100.Nevertheless,theparticlebilayerwhenfacingmembraneconstituentswithahydro-concentrationsdidnotdroptothelevelsobservedforthephobicmismatch.Asaresult,polymerchainsnotonlymovesampleswithOGandLDAO(Figure3B,C).Theconcen-withinthebilayer,buttheyarealsoabletocompressinthetrationof100nmparticlesat4.8mMTX-100wastwiceasvicinityofthemoleculecausingthehydrophobicmismatch,7,8,61,62highasthehighestconcentrationsofOG(23.4mM)andsuchasmembraneproteins.SimilarbehaviorcanbeLDAO(8.7mM)with1×1012particles/mLforTX-100,inexpectedwhendetergents,withmuchshorterhydrophobiccomparisonto5×1011particles/mLforbothOGandLDAO.chainsthanthepolymermolecules,areincorporatedintotheWespeculatethatthisdiscrepancyintheparticleconcen-bilayer.trationisbecauseofthepresenceoftheamorphousaggregatesBecauseoftheMWofthePEG−PCLdiblockcopolymer,aformedwhenTX-100wasusedforthepolymersomefullyextended,hydrophilicPEGblockwillalwaysbelongersolubilization.Thephenomenonoftheformationofthanahydrophilicheadofalipidmolecule,andtheamorphousaggregatescanbeexplainedbythesignificantlyhydrophilicconstituentofthedetergent.BecauseofthehigherMWofTX-100(615g/mol)incomparisontoOGandsignificantlylongerhydrophilicregionofthedetergent,itLDAOandthedifferenceinthestructureofthehydrophilicshouldbemoredifficultforthedetergenttopenetratethroughandhydrophobicmoietiesofthedetergents.TX-100consiststhehydrophilicmoietyoftheamphiphilicpolymerin1,8,11,19,63ofalonghydrophilicchainofPEGandahydrophobiccomparisontoitslipidcounterpart.Thus,ahigheralkylphenylgroup,incomparisontoLDAOandOG,whichstabilityofthebilayertowarddetergentattackwouldbebothhavehydrophobicchainsandhydrophilicgroups.LDAOexpected.Nevertheless,inthisworkwehavefoundthatthehasthesmallesthydrophilicregioncomposedofamineoxideconcentrationsrequiredtosolubilizePEG-PCL-basedpoly-andOGhasalargeglucoseheadgroup(seeFigure1).TX-100mersomescanbesignificantlylowerthanthosereportedforhasthelowestcmcofthethreetesteddetergents;however,itliposomes,suchasareportedconcentrationof80mMOG64wasfoundtobetheleasteffective,solubilizingthebilayeratrequiredtosolubilize15mg/mLofegglecithinvesicles,orcmcfractionsreaching2000%eventhoughTX-100haseggsphingomyelin(SM)/cholesterolvesiclesthatwerepreviouslybeendescribed,inanearlyworkonliposomereportedtowithstandconcentrationsashighas5mMof24solubilization,toresidewithinthegroupof“fast-solubilizing”TX-100.ThehighestTX-100concentrationusedinthis21,26,58detergents.TX-100insteadslowlyandpartiallystudywas6.4mM,whichislessthanhalfoftheconcentrationsolubilizesthebilayer,whichleadstotheformationoflargerreportedbyNallanietal.tosolubilize10mg/mLofegg-aggregatesandmixedmicelles.Thebreakdownofthebilayerphosphatidylcholinelipidvesicles(16mMTX-100),wherethe65shouldresultinthepartialdestabilizationofthevesiclesandanalysiswasbasedonDLSandODmeasurements.Theconsequentlytotheleakageofvesicles,butnovesicleswithanbilayerstabilitytowardthedetergentisverymaterial22openbilayerwereobservedwithcryoTEM.dependent,asotherliposomalformulationshavebeenComparisonoftheObservedSolubilizationProcessreportedtobecompletelysolubilizedatconcentrationsastotheLiterature.Thethreestagesofsolubilizationwerelowas0.3or0.4mMTX-100,forSM-and1-palmitoyl-2-definedandstudiedforPEG-PCLpolymersomesexposedtooleoyl-sn-glycero-3-phosphocholine-basedsystems,respec-24increasingconcentrationsofthethreeinvestigateddetergentstively.TheanalyzedPEG−PCLsystemismorepronetoauntilthecompletesolubilizationandbreakdownofthevesiclesdetergentattackthanpreviouslydescribedpolymersomesofwereconfirmed.AcrossthewholestudiedconcentrationPMOXA-PDMS(5mg/mL),whichwerereportedtofully31spectraoftheinvestigateddetergents,openedporeswerenotwithstandconcentrationsof16mMofTX-100.Higherobservedwithinthevesiclebilayer.Theformationofporeswassensitivitytothedetergentpresencemaybecausedbythelessexpectedatthesolubilizingconcentrationsbecausesuchflexiblehydrophobicbuildingblockofthecopolymer(PCL)in2087https://dx.doi.org/10.1021/acs.langmuir.0c03044Langmuir2021,37,2079−2090

9Langmuirpubs.acs.org/LangmuirArticle66comparisontoPDMS.FlexiblePDMSchainsmaybeLineElmstrømChristiansen−AquaporinA/S,2800expectedtoaccommodatedetergentmoleculesmoreeasilyKongensLyngby,DenmarkthanstifferPCL,resultinginhigherstabilityofthebilayerScottTrevenMyers−AquaporinA/S,2800KongensLyngby,8towardthedetergentattack.Obviously,theconcentrationofDenmarktheparticleswillbeanessentialfactoraffectingthedetergentKrzysztofTrzaskuś−AquaporinA/S,2800KongensLyngby,concentrationatwhichsolubilizationoccurs.ThementionedDenmarkstudiesdonotincludeparticleconcentrationmeasurements,ClausHélix-Nielsen−DepartmentofEnvironmentalthusthequantitativecomparisonoftheresultsislimited.Engineering,TechnicalUniversityofDenmark,2800Kongens■Lyngby,DenmarkSUMMARYANDCONCLUSIONSCompletecontactinformationisavailableat:Thisworkpresentsthefirstanalysisofdetergents’effectonhttps://pubs.acs.org/10.1021/acs.langmuir.0c03044PEG−PCL-basedpolymersomes.ItisthefirstthoroughanalysiscombiningODandDLSmeasurementswithdetailedNotesaqualitativecryoTEManalysisandquantitativeTRPSparticleTheauthorsdeclarenocompetingfinancialinterest.concentrationmeasurements.Wehavedemonstratedtheeffectofanincreasingconcentrationofthreedetergents:OG,■ACKNOWLEDGMENTSLDAO,andTX-100onPEG−PCLpolymersomes.TheThisworkwasapartoftheIndustrialPhDprojecttitrationofthesedetergentsintothepolymersomesolutions‘DevelopmentofNextGenerationofAquaporinInsidewascarriedouttothepointtowhichnopolymersomeswerebiomimeticmembranes’,co-foundedbyInnovationFundobservedoncryoTEMmicrographs.IthasbeenfoundthatDenmarkandAquaporinA/S.TheauthorswouldliketodespitethehigherMWofpolymersincomparisontolipidacknowledgethestaffoftheCoreFacilityforIntegratedmolecules(thusexpectedhigherstabilityofpolymersomesinMicroscopyatUniversityofCopenhagenKlausQvortrup,general),significantlylowerconcentrationsofdetergentscanTillmannPape,andMichaelJohnson,andthankthemforhelpdisruptthevesicularbilayerofPEG−PCLthanwaspreviouslyandsupportinobtainingcryogenictransmissionelectronreportedforliposomes.Thesolubilizationmechanismofmicroscopyimagesofthesamples.polymersomesiscomparabletothatofliposomes;however,theexpectedporeformationwithinthebilayerwasnot■REFERENCESobservedforthePEG-PCLpolymersomesatanystageofthe(1)Palivan,C.G.;Goers,R.;Najer,A.;Zhang,X.;Car,A.;Meier,solubilizationwiththetesteddetergents.ThebehavioroftheW.BioinspiredPolymerVesiclesandMembranesforBiologicalandsolubilizationalsodiffersdependingonthedetergentused.MedicalApplications.Chem.Soc.Rev.2016,45,377−411.EventhoughLDAOandOGhavetenandonehundredfold(2)Grossen,P.;Witzigmann,D.;Sieber,S.;Huwyler,J.PEG-PCL-highercmcthanTX-100,respectively,itwasshowninthisBasedNanomedicines:ABiodegradableDrugDeliverySystemandItsworkthattheyarecapableofreducingtheconcentrationofApplication.J.ControlledRelease2017,260,46−60.polymersomesatlowercmcfractions.Thiseffectiscausedby(3)Nielsen,C.H.BiomimeticMembranesforSensorandthesignificantlyhigherMWanddifferenceinstructuralSeparationApplications.Anal.Bioanal.Chem.2009,395,697−718.propertiesofTX-100incomparisontoLDAOandOG.(4)Kowal,J.;Zhang,X.;Dinu,I.A.;Palivan,C.G.;Meier,W.PlanarBiomimeticMembranesBasedonAmphiphilicBlockCopolymers.■ACSMacroLett.2014,3,59−63.ASSOCIATEDCONTENT(5)Gunkel-Grabole,G.;Sigg,S.;Lomora,M.;Lörcher,S.;Palivan,*sıSupportingInformationC.G.;Meier,W.P.Polymeric3DNano-ArchitecturesforTransportTheSupportingInformationisavailablefreeofchargeatandDeliveryofTherapeuticallyRelevantBiomacromolecules.https://pubs.acs.org/doi/10.1021/acs.langmuir.0c03044.Biomater.Sci.2015,3,25−40.(6)Goers,R.;Thoma,J.;Ritzmann,N.;DiSilvestro,A.;Alter,C.;ProtonnuclearmagneticresonanceanalysisofPEG−Gunkel-Grabole,G.;Fotiadis,D.;Müller,D.J.;Meier,W.OptimizedPCLdiblockandchemicalstructure,SECofthePEG−ReconstitutionofMembraneProteinsintoSyntheticMembranes.PCLdiblock,andDSCanalysisofPEG−PCLdiblockCommun.Chem.2018,1,35.(PDF)(7)Itel,F.;Najer,A.;Palivan,C.G.;Meier,W.DynamicsofMembraneProteinswithinSyntheticPolymerMembraneswithLarge■HydrophobicMismatch.NanoLett.2015,15,3871.AUTHORINFORMATION(8)Itel,F.;Chami,M.;Najer,A.;Lörcher,S.;Wu,D.;Dinu,I.A.;CorrespondingAuthorMeier,W.MolecularOrganizationandDynamicsinPolymersomeRadosławGórecki−DepartmentofEnvironmentalMembranes:ALateralDiffusionStudy.Macromolecules2014,47,Engineering,TechnicalUniversityofDenmark,2800Kongens7588−7596.Lyngby,Denmark;AquaporinA/S,2800KongensLyngby,(9)Lichtenberg,D.;Ahyayauch,H.;Alonso,A.;Goñi,F.M.Denmark;orcid.org/0000-0003-0149-6880;DetergentSolubilizationofLipidBilayers:ABalanceofDrivingEmail:rgor@env.dtu.dk,rgo@aquaporin.comForces.TrendsBiochem.Sci.2013,38,85−93.(10)Alibolandi,M.;Ramezani,M.;Abnous,K.;Sadeghi,F.;AuthorsHadizadeh,F.ComparativeEvaluationofPolymersomeversusFabioAntenucci−DepartmentofVeterinaryandAnimalMicelleStructuresasVehiclesfortheControlledReleaseofDrugs.J.NanoparticleRes.2015,17,76.Sciences,UniversityofCopenhagen,1870FrederiksbergC,(11)Discher,D.E.;Ahmed,F.Polymersomes.Annu.Rev.Biomed.DenmarkEng.2006,8,323−341.KarolisNorinkevicius−AquaporinA/S,2800Kongens(12)Patra,S.K.;Alonso,A.;G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