Control of Lipid Bilayer Phases of Cell-Sized Liposomes by Surface-Engineered Plasmonic Nanoparticles - Nobeyama et al. - Unknown - Unkn

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Supportinginformationto:ControlofLipidBilayerPhasesofCell-SizedLiposomesbySurface-EngineeredPlasmonicNanoparticlesTomohiroNobeyama$,KazukiShigyou#,HirotakaNakatsujiǂ,HiroshiSugiyamaǁ,∞,KomuraNaoko¶,※,HiromuneAndo¶,※,TsutomuHamada†andTatsuyaMurakami$,∞,§,*$GraduateSchoolofEngineering,ToyamaPrefecturalUniversity,5180Kurokawa,Toyama939–0393,Japan#NanoLifeScienceInstitute(WPI-NanoLSI),KanazawaUniversity,Kakuma-machi,Kanazawa920-1192,JapanǂDepartmentofAppliedChemistry,GraduateSchoolofEngineering,OsakaUniversity,2-1Yamadaoka,Osaka565-0871,JapanǁDepartmentofChemistry,GraduateSchoolofScience,KyotoUniversity,Sakyo-ku,Kyoto606-8502,Japan∞InstituteforIntegratedCell-MaterialSciences(iCeMS),KyotoUniversityInstituteofAdvancedStudy(KUIAS),Yoshida-honmachi,Sakyo-ku,Kyoto606–8501,Japan¶InstituteforGlyco-coreResearch(iGCORE),TokaiNationalHigherEducationandResearchSystem,Furo-cho,Chikusa-ku,Nagoya464-8601,Japan※CenterforHighlyAdvancedIntegrationofNanoandLifeSciences(G-CHAIN),GifuUniversity,1-1Yanagido,Gifu501-1193,Japan†SchoolofMaterialScience,JapanAdvancedInstituteScienceandTechnology,1–1Asahidai,Ishikawa923–1212,Japan§DepartmentofPharmaceuticalEngineering,FacultyofEngineering,ToyamaPrefecturalUniversity,5180Kurokawa,Toyama939–0393,Japan1

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2SupportingMaterialsandMethodsGeneralInformationGeneralreagentswerepurchasedfromNakalaiTesque(Kyoto,Japan).Gold(ΙΙΙ)chlorideandsodiumborohydride(NaBH4)wereobtainedfromSigma-Aldrich(SaintLouis,MO,USA).Silvernitrate,L-(+)-ascorbicacid,andsodiumoleatewerepurchasedfromWako(Osaka,Japan).1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)wasobtainedfromNOF(Tokyo,Japan).1,2-Dioleoyl-3-trimethylammonium-propane(DOTAP),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(NBD-PE),and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissaminerhodamineBsulfonyl)(Rhodamine-PE)werepurchasedfromAvantiPolarLipids,Inc(Alabaster,AL,USA).Methyl-beta-cyclodextrin(MβCD)andcholesterol(chol)wereobtainedfromSigma-Aldrich(SaintLouis,MO,USA).ITOglasseswerepurchasedfromSigma,andthefluorescentgangliosideprobe(ATTO-GM3)wasobtainedfromProf.HiromuneAndo1.Visible/near-infrared(Vis/NIR)spectraweremeasuredusingtheV-630spectrometer(JASCOCooperation,Tokyo,Japan).Giantunilamellarvesicles(GUV)imageswereobtainedusingZeissLSM800confocalfluorescentmicroscopeorNikonTieclipsefluorescentmicroscope.Theconcentrationofthecationichigh-densitylipoproteinmutant(catHDL)onaproteinbasiswasmeasuredwithaDCProteinAssaykit(Bio-Rad,CA,USA).ThehydrodynamicdiameterofcatHDLwasmeasuredutilizingNanotrackUPA-UT151apparatus(MicrotracBEL,Osaka,Japan).catHDLpreparationThepreparationofcatHDLwasdescribedpreviously.2,3Briefly,POPCandDOTAPweremixedinethanolinaround-tableflaskatamolarratioof7:3.A6mol%solutionofNBD-PEinethanolwasaddedifnecessary.Thereactionmixturewasconcentratedanddriedunderreducedpressuretoremoveallsolvents.Thelipidfilmformedatthebottomoftheflaskwasdispersedusing30mg/mLsodiumcholatesolutioninphosphate-bufferedsaline(PBS,pH7.4).Followingincubationat37°Cforatleast2h,arecombinantapoA-ImutantwithadeletedNterminal43aminoacid,andacell-penetratingTATpeptidefusedattheCterminusweremixedataprotein-to-lipidmolarratioof100inPBScontaining4Murea.Thereactionmixturewasincubatedatroomtemperature(r.t.)overnight.ThemixturecontainingcatHDLwasdialyzedagainst3LPBSforatleast4h,changingthedialysatethreetimestoremoveurea,sodiumcholate,andunreactedproteins.Subsequently,thesamplewascentrifugedtoremovedebris.ThehydrodynamicdiameterofcatHDLwasdeterminedatapproximately37nmusingNanotrackUPA-UT151apparatus.SynthesisofCTAB-coatedAuNRsAuNRsweresynthesizedutilizingtheseedlessmethod.4Typically,1200µLofa4mMaqoueous3

3solutionofAgNO3,400µLof50mMaqoueoussolutionofHAuCl4,and258µLof100mMsolutionofaqueousL-ascorbicacidwereaddedto40mLof0.1Maqueoussolutionofhexadecyltrimethylammoniumbromide(CTAB).Afterthecolorofthereactionmixtureturnedfromyellowtocolorless,48µLof35%HCland30µLofafreshlypreparedice-coldaqueoussolutionofNaBH4wereaddedwhilestirring.AuNRsformedinthemixturewerepellettedbycentrifugingat20,000×gfor40minat25˚C,followedbyincubationat30˚Covernight.Finally,theAuNRswereredispersedin0.1MCTABatca.1mg/mL.Preparationofpm-AuNRpm-AuNRsweresynthesizedaccordingtoourpreviousreport.2,3Briefly,1mLofCTAB-coatedAuNR(ca.1mg/mL)wascentrifugedat20,000×gfor20minat25˚Cfollowedbyremovalof900µLofsupernatants.Thesamplewasredispersedwiththeresting100µLsupernatants,dilutedwith900µLofdeionizedwater,thencentrifugedat20,000×gfor20minat25˚C.Subsequently,750µLofthesupernatantwasremovedand250µLof16mg/mLsodiumoleatesolutionwasaddedtothesamples.Thismixturewasheatedfor1hat50˚Candtheproducts(oleate-coatedAuNRs)werepurifiedusingtheNap5gelcolumn(GEHealthcare).Oleate-coatedAuNRsweremixedwithcatHDLina1:0.4weightratioandthenheatedfor1hat50˚Cina1.5mLmicrotube.Theproducts(pm-AuNRs)werecentrifugedat20,000×gfor20minat25˚Candallsupernatantswerecarefullyremoved.pm-AuNRsweresuspendedina200mMsucrosesolutionatca.1mg/mL.PreparationofGUVsGUVswerepreparedusingtheelectro-formationmethod.540mMofDPPC,DOPC,andcholesterolinchloroformweremixedinaDurhamtube.1mg/mLsolutionofRhodamine-PEorATTO594-GM3inethanolwasaddedifnecessary.Aportionofthemixture(5.7mmollipids)wasdroppedandspreadonanITOglass(8–12Ω/sq,Sigma-Aldrich,MO).TheratiosoflipidsandcholesterolforLo,Ld,Lo/Ld,andSo/LdGUVswereDPPC:DOPC:Chol=2:0:1,0:1:0,2:2:1or1:1:0,respectively.Afterheatingfor5minat50˚C,thelipidfilmwasfurtherspreadbyaddingasingledropofhotchloroform,thendryinginvacuofor≥2h.ThesampleglasswascoveredwithanotherITOglassusingasiliconsheet(ca.1mmthickness)witha1.5cmsquare-shapedholetocreateasmallchamberonthelipidfilm.0.3×102µLof200mMsucrosesolutionwasaddedintothechamberandahome-madeelectriccircuitwasmadeofthinfoil,thechamber,andanarbitraryfunctiongeneratorAFG-2005(GWInstek).ForSo/LdGUVs,theprocedurewasslightlymodified.ThelipidfilmforSo/LdGUVswasheatedat70˚Cfor5mininsteadof50˚C,andthe200mMsucrosesolutionandotherapparatuswereheatedat70˚Cpriortoelectrification.Electricpower(10Hz,1.4vpp,0.35mV)wasappliedtothechamberwiththearbitraryfunctiongeneratorsat50˚CinanICI-1incubator(AS4

4One)overnight.Aftercoolingthechambertoroomtemperature,thedispersionofGUVswasrecollectedfromthechamberwithadisposablesyringeandstoredin1.5mLtubesatroomtemperature.ObservationofGUVsAchamberforGUVobservationwaspreparedbysandwichingasingle0.1mmthicksiliconsheetwithaca.6mmholebetweentwo0.13–0.17mmthickcoverglasses(24mm×50mmand18mm×18mm,respectively).Typically,aftermixing5µLoftheGUVdispersionand5µLofthepm-AuNRsdispersioninamicrotube,3.5µLofthemixturewasdroppedwithintheholeononecoverglass.Aftertheholewascoveredwithanothercoverglasstoclosethechamber,thephaseseparationpatternofGUVswasobservedatroomtemperaturewiththefollowingtwotypesofmicroscopes.ForaNiconECLIPSETiepifluorescencemicroscope(Nikon,Tokyo,Japan),theexcitation/emissionwavelengthswere455/485nmforNBD-PEand535/575nmforRhodamin-PE,anda×60objectivelens(Nikon)wasused.ForaZeissLSM-800confocalmicroscopy(Zeiss,Oberkochen,Germany),theexcitationwavelengthwas488nmforNBD-PEand560nmforRhodamin-PE.TheacquiredrangeoftheemissionwavelengthswasadjustedwithaZensoftware(Zeiss),anda×40objectivelenswasused.Theexposuretimewasadjustedsoastoobservetheimagesallowingtoidentifythedomainstructuremostclearlybutfor<1stoavoidphotobleaching.ThecontentsofGUVswithaspecificphasepatterninFigures5,S3,S6,S7,S9,S12werecalculatedwith≥30independentimagesofmorethan30individualGUVs67.GUVswithpartiallycircularandpartiallynoncirculardomainsand/orverysmalldomainswereclassifiedas“unclear”.Observationofthephasetransitionprocess2µLofpm-AuNRsand2µLofLo/LdGUVs(DPPC:DOPC:chol=2:2:1)weredroppedwithintheabove-mentionedholeononecoverglass.Placinganothercoverglassontopallowedmixingthetwosubstances.TheareaneartheboundarywasthenobservedusinganLSM800microscope.CholesteroldepletionusingMβCDMβCDwasdissolvedina200mMsucrosesolutionat20mMandstoredat4˚C.TheMβCDsolutionwaswarmedatr.t.andmixedwiththeGUVdispersant.Microscopicobservationwasperformedatr.t.Preparationofpm-AuNR-cholspm-AuNRsdispersionwasmixedwith1/10volumeof32mg/mLcholin2-propanolandcentrifugedat20,000×gfor20minat25˚C.Allsupernatantswereremovedcarefullyandthepellet5

5wasre-dispersedina200mMsucrosesolutionatca.1mg/mL.Observationoftheheating-inducedSo-to-Lophasetransitionpm-AuNR-cholsandSo/LdGUVs(DPPC:DOPC:Chol=1:1:0),inwhichtheLdphasewaslabeledwith0.1mol%Rhodamine-DPPE,weremixedina1:1volumeratioandheatedat50˚Cfor10mininamicrotube.ThemixturewasplacedintothechamberandthedomainpatternsofGUVswereobservedusingtheECLIPSETimicroscope.ObservationoftheNIRlaser-inducedSo-to-LophasetransitionThechambercontainingtheabove-mentionedmixturewassetonaglass-bottomdish(IWAKI)andilluminatedat852nmwithaChameleonfemtosecond-pulsedlaser(Coherent).Then,thechamberwascooledtor.t.foratleast10minpriortotheanalysis.Observationofthephasetransitioninducedbysaturatedchol-loadedMβCD(MβCD-chol)MβCD-cholpreparationwasdescribedpreviously.8MβCDandcholweredissolvedin200mMsucrosesolutionat5mMandina1:1chloroform:2-propanolsolutionat64.7mM,respectively.1mLoftheMβCDsolutionand19.3µLofthecholsolutionweremixed,thenrotatedat37˚Covernighttoobtainaclearsolution.Themixturewasthenfiltratedwitha0.45µmporesizefilter(Millipore)andmixedwithGUVsina1:1volumeratio.6

6FigureS1.LophaseselectiveadhesionofNBD-PElabeledpm-AuNRs.Lo-phaseGUVswerepreparedwithDPPCandcholesterolina2:1molarratio,andLd-phaseGUVswerepreparedwithDOPCwith1%Rhodamine-PE,afluorescentlipidselectivelylocatedontheLdphase.Scalebar,20µm.7

7FigureS2:TypicalconfocalfluorescenceimagesofGUVsfollowingthepm-AuNRstreatment.TheLdphasedomainswerelabeledwithRhodamine-PE(red)andpm-AuNRswerelabeledwithNBD-PE(green).ThefocuswassetaroundtheuppersurfacesofmanyGUVstoobservetheirdomainpatternsclearly(A)andaroundtheequatorialplaneofmanyGUVstodetecttheirinsidefluorescence(B).In(A),non-circular(string-shaped)domainstructuresingreenareobservedinGUVsthroughouttheimage,whilethepatternlooksdifferentlyinmuchsmallerGUVsforsomeunknownreason.In(B),theirinsidefluorescenceinnotsignificantlydetected.Scalebar,20µm.8

8FigureS3.pm-AuNRsconcentrationdependencyofthephasetransitionjudgedbythefluorescencesignalpatternofRhodamine-DPPE(A)(seeFigure2(B),(C))orATTO-GM3fluorescence(B)(seeFigure4(B),(C)).Theright-handbarin(A),indicatedas“10%PBS”,meanstheGUVcontentinthepresenceof10%PBS.Thiscontrolexperimentistoeliminatetheeffectsofthesaltinvolvedinpm-AuNRsdispersiononthephasetransition.9

9FigureS4.Theschematicimageoftheobservationoftheboundaryofpm-AuNRsandGUVsdispersants.AdropletofeachGUVsandpm-AuNRsdispersantswereloadedonacoverglasschamber,asshownontheleft.Aboundarywasformedonthecoverglasschamber,asshownontheright.10

10FigureS5.TypicalmicroscopicimageofGUVsneartheboundaryofGUVsandpm-AuNRsrichregions.(A)Fluorescentimagenearthecontactareabetweenthepm-AuNRsdispersionandtheLo/LdGUVsdispersion.Thewhitelineindicatestheboundarybetweenthetwoareas.(B)MagnifiedGUVimagesof(A),whichisintheGUVsrichregion(left),ontheboundary(middle),andinthepm-AuNRsrichregion(right).Scalebar,10µm.11

11FigureS6.(A)CationicHDLdependencyoftheLo-to-Sophasetransition.HDLswerepreviouslydilutedin200mMsucrosesolutions.(B)RepresentativeimageofHDLattachedLo/LdGUV.HDLconcentrationwas40μg/mLonaproteinbasis.Scalebar,5μm.12

12FigureS7.EffectofMβCDontheLo-to-SophasetransitioninLo/Ld-mixedGUVs(Lo/Ld-GUVs).(A)TheratiooftheGUVdomainpatterns.Observationswereperformed0(middle)or3h(left)aftertheMCDtreatment.(B)Representativeimageofeachdomainpattern.‘Microdomains’weredefinedasGUVswithdomainsmovingandlargerthanthoseinvolvedin‘unlcear’.Scalebar,5μm.13

13FigureS8.NormalizedtypicalVis/NIRspectraofcholesterol-loadedpm-AuNRs(pm-AuNRs-chol)preparedatvariousconcentrationsofchol.14

14FigureS9.Heat-inducedSo-to-Lophasetransitionwithpm-AuNRs-chol.(A)TypicalimagesofSo/Ld-mixedGUVspriortothetreatment(left)andafterthetreatmentwithpm-AuNRs(topright)orpm-AuNRs-chol(bottomright)at50˚C.Scalebar,10μm.(B)TheratiooftheGUVsphaseseparationpatternafterheatingwithpm-AuNR(light)orpm-AuNR-chol(left).15

15FigureS10.RepresentativeimageofasingleSo/Ld-mixedGUVtreatedwithpm-AuNR-chols.TheLdphaseoftheGUVandpm-AuNRs-cholwerefluorescentlylabeledwithRhodamine-PEandNBD-PE,respectively.Scalebar,5μm.16

16FigureS11.Laserexperimentsetup.(A)ExperimentalapparatusforNIRlaserirradiationofthemixtureofpm-AuNR-cholsandSo/LdGUVs.TheopticalpathoftheNIRlaserisindicatedwithredarrows.(B)Enlargedviewofthebluerectanglein(A).Acoverglasschamberissetonaglass-bottomdish(IWAKI).(C)Schematicimageofthesideviewof(B).17

17FigureS12.TheSo-to-Lophasetransitioninducedwithcholesterol-loadedMβCD(MβCD-chol).TheMβCD-cholsolutionusedincludedca.1.3mMcholesterol.ThenumberofhorizontallinesindicatesthedilutiondegreeofMβCD-cholbya200mMsucrosesolution.18

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