Mixed Polyplexes Micelles with Thermoresponsive and Lysine-based Zwitterionic Shells Derived From Two Poly ( Vinyl Amine ) -Based Block

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SupportingInformationMixedPolyplexesMicelleswithThermoresponsiveandLysine-basedZwitterionicShellsDerivedFromTwoPoly(VinylAmine)-BasedBlockCopolymersRyosukeKanto,†RyoYonenuma,†MizukiYamamoto,§HiroyukiFurusawa,‡ShigekazuYano,‡MitsuruHaruki,§HideharuMori*††GraduateSchoolofOrganicMaterialsScience,‡GraduateSchoolofScienceandEngineering,YamagataUniversity,4-3-16,Jonan,Yonezawa,992-8510,Japan§DepartmentofChemicalBiologyandAppliedChemistry,CollegeofEngineering,Nihon1Nakagawara,Tokusada,Tamuramachi,Koriyama,Fukushima963-8642,Japan*Towhomcorrespondenceshouldbeaddressed.e-mail:h.mori@yz.yamagata-u.ac.jp

1ExperimentalSectionMaterials.2,2′-Azobis(isobutyronitrile)(AIBN,KantoChemical,97%)wasrecrystallizedfrommethanol.N-Acryloyl-N-carbobenzoxy-L-lysine(ALys(Cbz)OH)usedasalysine-containingmonomer,1N-vinylphthalimide(NVPI)usedasaprotectedmonomer,2,3andbenzyl1-pyrrolecarbodithioate(dithiocarbamate-typechaintransferagent;CTA),4,5weresynthesized,asdescribedpreviously.NIPAM(TCI,>98%)wasrecrystallizedtwicefromhexane.Trimethylsilyldiazomethane(TCI,10%solutioninhexane),N,N-dimethylformamide(DMF,KantoChemical,99.5%),andallothermaterialswereusedasreceived.Polyethyleneimine(PEI)MAX40K(weightaveragemolecularweightsof40,000)waspurchasedfromPolyscience(Warrington,PA,USA).Helacells(JCRB90046)werefromJCRBcellbank(Shinjuku,Tokyo,Japan).Eagle'sminimalessentialmedium(EMEM)wasfromSigma-Aldrich(St.Louis,MO,USA).Fetalbovineserum(FBS)andpenicillin/streptomycinwerefromLifeTechnologies(SouthSanFrancisco,CA,USA).NonessentialaminoacidswerefromMPBiomedicals(Irvine,CA,USA).WST-8kitwasfromKishidaChemical(Osaka,Japan).SynthesisofPVAm-b-PALysOH.Asyntheticrouteemployedforthepreparationofcationic-zwitterionicblockcopolymerisshowninScheme1a.NVPI(3.37g,17.8mmol),CTA(46.8mg,0.201mmol),AIBN(10.4mg,0.065mmol),andDMF(9.75mL)wereplacedinadryglasstubeequippedwithamagneticstirringbar.Afterdegassingthemixedsolutionbythreefreeze-evacuate-thawcycles,thetubewassealed.Thereafter,themixedsolutionwasstirredat60°Cfor16h.Aftercooling,thepolymerizationmixturewasprecipitatedintodiethylether,collectedbyfiltration,anddriedundervacuumtoaffordPNVPIasayellowpowder(Mn=4,900,Mw/Mn=1.35).Thedithiocarbamate-terminatedPNVPI(0.294g,0.06mmol,Mn=4,900),AIBN(3.2mg,0.02mmol),ALys(Cbz)OH(0.990g,3.0mmol),andDMF(3mL)wereplacedinadryglasstube.Afterdegassingthemixedsolutionbythreefreeze-thawcycles,thetubewassealed.Thereafter,themixedsolutionwasstirredat60°Cfor16h.Aftercooling,thepolymerizationmixturewasprecipitatedintodiethylether,isolatedbyfiltration,anddriedundervacuumtoaffordPNVPI-b-PALys(Cbz)OHasapaleyellowsolid(1.03g,yield=80%).ThedeterminationofNVPI/ALys(Cbz)OHcompositionwasachievedusing1HNMRspectroscopybycomparisonofrelativeareaofthearomaticprotons(4H)ofNVPIunit,andaromaticandamidoN-Hprotons(5Hand2H)ofALys(Cbz)OHunit(δ7.0-8.2ppm),incomparisontothemethyleneprotons(2H)ofALys(Cbz)OHunit(δ4.9–5.1ppm),usingtheequation1,

27-3xIntegralat7.0-8.2ppm=(1)2(1-x)Integralat4.9-5.1ppmwherexrepresentsthefractionoftheNVPIand1-xrepresentsthefractionofALys(Cbz)OH,respectively.TheresultingPNVPI-b-PALys(Cbz)OHshowedreasonablesolubilityinorganicsolventssuchaschloroformandDMSO,whilebeinginsolubleinwater(TableS2).Forsize-exclusionchromatography(SEC)measurements,thePALys(Cbz)OHsegmentinthecrudeblockcopolymerwasmodifiedusingtrimethylsilyldiazomethane,asdescribedpreviously.7,8Notethattrimethylsilyldiazomethaneshouldberegardedextremelytoxic,andallofitshandlingandtheevaporationprocessshouldbeconductedwithproperventilation.9ThedetailedpolymerizationresultsandcharacterizationaregiveninTableS1andFiguresS1-S2.ToremovetheN-carbobenzoxy(Cbz)-protectinggroup,PNVPI-b-PALys(Cbz)OHwastreatedwithtrifluoroaceticacid(TFA)/HBrusingthereportedprocedure1,10withslightmodifications.Typically,1.00gofPNVPI-b-PALys(Cbz)OH(Cbzcontent=1.9mmol,Mn=10,600,Mw/Mn=1.45NVPI/ALys(Cbz)OHcomposition=70/30)and10mLofTFAwereintroducedinaglassvial.Afterthemixturewasstirredatroomtemperaturefor1hundernitrogen,afour-foldmolarexcessofa33wt%solutionofHBrinaceticacid(1.5mL,7.6mmol)wasaddeddropwise,andthenthereactionwasperformedfor1.5hunderstirring.Thereafter,theproductwasprecipitatedintodiethylether,followedbyfiltration.Theproductwasthenpurifiedbydialysisagainstdistilledwaterinadialysisbag(MWCO1000Da,SpectraPore)over2days.Afterremovingthesolventbyreducedpressure,theproductwasdriedundervacuumtoaffordPNVPI-b-PALysOHasawhitesolid(0.510g,62%).Toremovethephthalimideunit,PNVPI-b-PALysOHwastreatedfollowingthereportedprocedure2,11withslightmodifications.PNVPI-b-PALysOH(0.500g,0.14mmol,Mw/Mn=1.45NVPI/ALysOHcomposition=70/30)andhydrazinemonohydrate(1.75mL,35mmol,ca.25equiv.withrespecttothephthalimide)weredissolvedinmethanol(3.2mL)and1,4-dioxane(1.6mL)inaflask.Thereactionwasconductedbyrefluxat90°Cundernitrogenfor4h.Thereafter,theproductwasdialyzedinadialysisbag(MWCO1000Da)against0.02MNaOHaqueoussolutionfor2daysandthendistilledwaterfor2days.Finally,theresultingproductwasdriedundervacuumtoaffordPVAm27-b-PALysOH63(0.22g,yield=78%).ThesolubilityoftheblockcopolymersislistedinTableS2.

3SynthesisofPVAm-b-PNIPAM.Cationic-thermoresponsiveblockcopolymerswerepreparedbyRAFTpolymerizationofNIPAMusingdithiocarbamate-terminatedPNVPImacro-CTA,followedbythedeprotection(Scheme1b),asreportedpreviously.2,3,12ThedetailedpolymerizationresultsandcharacterizationaregiveninTableS3andFiguresS4-S5.ThedeterminationofNVPI/NIPAMcompositionwasachievedusing1HNMRspectroscopybycomparisonofrelativeareaofthearomaticprotons(4H)ofNVPIunitandtheamidoN-Hproton(1H)ofNIPAMunit(δ7.0-8.0ppm),incomparisontothemethylprotons(6H)oftheNIPAMunit(δ0.9–1.2ppm),usingtheequation2.3x+1Integralat7.0-8.0ppm=(2)6(1-x)Integralat0.9-1.2ppmwherexrepresentsthefractionoftheNVPIand1-xrepresentsthefractionofNIPAM,respectively.TheresultingPVAm-b-PNIPAMwassolubleinwateratroomtemperature,irrespectiveofthepH(TableS2).Blockcopolymer/DNAPolyplexes.PolyplexeswerepreparedbymixingthestocksolutionsofDNAandtheblockcopolymerinpotassiumphosphatebufferatvaryingfeedratios.Theconditionsofthepolyplexformationwasstandardizedtopermitthecomparisons,asdescribedpreviously.1,11Thecation(-NH+3group)/anion(phosphategroup)ratio(N/P)isgiveninthenumberratiooftheprimaryamine-basednitrogenatoms(protonableVAmunits)inblockcopolymertothephosphategroupsinDNA.Asastocksolution,theblockcopolymer(1.0mg/mL)wasdissolvedin10mMpotassiumphosphatebuffer(pH=7.4)atroomtemperature.Independently,DNA(DNAsodiumsaltfromsalmontestes,Sigma-Aldrich(D1626)(2000bp),0.2mg/mL)stocksolutionwaspreparedusingthesameprocedure.Apre-determinedamountofthepolymersolutionwasaddedgraduallytotheDNAsolutionandmixedatroomtemperature,inordertoachievedesiredN/Pratio.ThemixturewasthendilutedbyaddingpotassiumphosphatebuffertoattainatargetedDNAconcentration(50mg/L).Inthecasesofmixedpolyplexmicelles,thestocksolutionsofPVAm-b-PALysOHandPVAm-b-PNIPAMweremixedinitiallyatthedifferentmoleratiosof1:5-5:5.Thereafter,themixedpolymersolutionwasaddedtotheDNAsolutiontoaffordthemixedpolyplexmicellesatroomtemperatureataconstantNinPVAm-b-PNIPAM/Pratio=5/1,followedbytheadditionofpotassiumphosphatebuffertoattainatargetDNAconcentration(50mg/L).Thesameprotocolwasutilizedforthepreparationofthemixedpolyplexesatdifferentfeedratios.

4Instrumentation.Protonandcabon-13nuclearmagneticresonance(1Hand13CNMR)spectrawererecordedusingaJEOLJNM-ECX400usingD2OorDMSO-d6.SECwasperformedonaTosohHPLCHLC-8220system,comprisingaguardcolumn(TSK-guardcolumnα]andfourgelcolumns(TSK-GELs:α-M,α-4000,α-3000,α-2500),andrefractiveindexandultravioletdetectors.TheeluentwasDMFcontaining10mMLiBrat40oCwithaflowrateof1.0mL/min.Thesystemwascalibratedusingpolystyrenestandards(Tosoh)fordeterminationofnumber-averagemolecularweight(Mn)anddispersity(Mw/Mn).Thetemperature-dependentturbiditymeasurementwasperformedonaJASCOV-630BIOUV-visspectrophotometer(JASCOEHC-716)equippedwithatemperaturecontroller(EHC-717)bymeasuringtheturbidityat500nmbetween10and70°Cata1.0°C/minscanrate.Thecirculardichroism(CD)measurementwasconductedusingaJASCOJ-720spectropolarimeter.DynamicLightScattering(DLS)wasperformedatadetectionangleof173°usingaZetasizerNano(Sysmex)withaHe-Nelaser.Thesamplesolutionswerepreparedunderappropriateconditionsandfiltratedthrough2μmfilter.Themeasurementswereconductedatspecifictemperatures(25,37,and50°C),after5minthermalequilibriumateachtemperature.Theintensity-averagedhydrodynamicdiameters(Dh)wereprovidedbytheinstrument.ZetapotentialmeasurementwasperformedonaZetasiserNano-ZSwiththesamesamplesusedforDLSmeasurement.ThepHofeachsamplewasadjustedusingNaOHandHCl.Atomicforcemicroscopy(AFM)measurementswereperformedusingNanoscopeIIIa(Veeco)inambientconditions.Thepolyplexsolutions([DNA]=0.001mg/mL)wereinitiallypreparedusing2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonicacid(HEPES)buffer(10mM,pH=7.4),anditwasdroppedontheMgCl-treatedmica,asdescribedpreviously.1Transmissionelectronmicroscopy2(TEM,JEOLTEM-2100Ffieldemissionelectronmicroscope)operatedat200kVwasemployedtocharacterizepolyplexes.Eachsamplewaspreparedfrompotassiumphosphatebuffer,andadropofthesolution(0.05mg/mL)wasplacedoncarbon-coatedCugridsandallowedtodryinginair.Adropofosmiumtetroxide(OsO4)aqueoussolutionwasplacedonthesample,andthenthesamplewasdriedatroomtemperature.Theresultingpolyplexeswereelectrophoresedon0.8wt%agarosegelcontainingtrisacetateEDTA(TAE)buffersolution(pH=8),asdescribedpreviously.1Briefly,thepolyplexsolution(8µL)wasmixedwithloadingbuffer(2µL),andeachpolyplexsolutionwasthenloadedontotheagarosegel.EthidiumbromidewasincubatedinthegeltoshowthelocationofDNAunderUVillumination(312nm)usingATTOPrintgraph(AE-6905CFCCDCameraController).Experimentsusinganelectrophoresistank(Mupid)wereconductedat50Vfor1h.

5CellviabilityassayThecytotoxicityofthepolymerswasdeterminedusingtheWST-8kitinHeLacells.ThisassayisbasedonthecleavageofthetetrazoliumsaltWST-8bymitochondrialdehydrogenaseinviablecells.13HeLacellswereculturedona96-wellplateatadensityof1×104cellsperwellin100µLofEMEMcontaining10%(v/v)FBS,nonessentialaminoacids,100units/mLpenicillin,and100μg/mLstreptomycinina5%CO2incubatorat37°C.Aftertwodays,theculturemediumwasreplacedby100µLofthemediumcontainingthepolymers,andthecellswereincubatedfor24h.Followingthis,theculturemediumwasreplacedwith100μLofthemedium,andthen10μLofWST-8reagentwasaddedtoeachwell.Followingincubationfor2hat37°C,theabsorbanceofeachwellwasmeasuredat450nmontheiMarkmicroplatereader(Bio-RadLaboratories,Hercules,CA,USA).Thecellviability(%)wascomparedwiththecontrolwellswithoutthepolymer(100%)andcalculatedbyOD450(samples)/OD450(control)×100.Experimentswereperformedintriplicate.

6TableS1.SynthesisofPNVPI-b-PALys(Cbz)OHbyRAFTpolymerizationofALys(Cbz)OHusingPNVPImacro-CTAat60°Cfor16ha)[M]/[macro-CTA]Conv.b)Mnn:mb)RunMd)w/Mn/[AIBN](%)Theoryc)NMRb)SECd)1100/4/18714,70011,10011,8001.3379:212200/5/19318,50025,80013,6001.6937:633400/5/19330,90049,90015,1001.7821:794150/3/18819,60025,90010,6001.4530:70a)PolymerizationofALys(Cbz)OHwasconductedusingPNVPImacro-CTA(Run1:MnSEC=7500,Mw/Mn=1.43,Runs2-3:MnSEC=5900,Mw/Mn=1.43,Run4:MnSEC=4900,Mw/Mn=1.35)inDMF(conc.=2M).b)Calculatedby1HNMRinDMSO-dc)6.Thetheoreticalmolecularweight=(MWofALys(Cbz)OMe)×[ALys(Cbz)OH]d)0/[macro-CTA]0×conv.+(Mnofmacro-CTA).Methylatedsamplesweremeasuredbysize-exclusionchromatography(SEC)usingpolystyrenestandardsinN,N-dimethylformamide(DMF,10mMLiBr).macro-CTARun1Run2Run3Run424262830323436Time(min)FigureS1.SECcurvesofPNVPI-b-PALys(Cbz)OMesandPNVPImacro-CTA.

7HODMSO2(a)c,c',i,ka,b,a',b'e~gaa'bbb'nmONOc'ONHOdfhjHOegNOHOijckhd(b)aa'bbb'nmONOc'ONHdfhHOegNH2Oic1086420ppmFigureS2.1HNMRspectraof(a)PNVPI-b-PALys(Cbz)OHand(b)PNVPI-b-PALysOHinDMSO-d6.

8(a)acbbdnmNH2DOONHgi2ea,b,c,dHONH2f~hfhOie86420ppm(b)acbbdH2ODMSOnmNH2ONHefgggf,ba,c,de86420ppmFigureS3.1HNMRspectraof(a)PVAm-b-PALysOHinD2Oand(b)PVAm-b-PNIPAMinDMSO-d6.

9bbnmnmNH2NH2ONHONHHONH2OPVAm-b-PALysOHPVAm-b-PNIPAMTableS2SolubilityofPVAm-b-PNIPAMandPVAm-b-PALysOHsolventPVAm-b-PNIPAMPVAm-b-PALysOHPNVPI-b-PALys(Cbz)OHEt2O---CHCl3--+DMSO+-+MeOH+--H2O(pH2)++-H2O(pH7)++-H2O(pH12)++-+:solubleatroomtemperature,-:insolubleatroomtemperature.

10TableS3SynthesisofPVAm-b-PNIPAMbyRAFTpolymerizationofNIPAMusingPNVPImacro-CTAat60°Cfor24ha)[M]/Conv.b)Mnn:mb)RunMd)w/Mn[macro-CTA](%)Theoryc)NMRb)SECd)1100/1961500011400125001.3437:632200/1912480030000208001.4814:86a)PolymerizationofNIPAMwasconductedusingPNVPImacro-CTA(Mn,SEC=4200,Mw/Mn=1.36)at[macro-CTA]/[AIBN]=2inDMF(conc.=1M).b)Calculatedby1HNMRinDMSO-dc)6.Thetheoreticalmolecularweight=(MWofNIPAM)×[NIPAM]0/[macro-CTA]0×conv.+(Mnofmacro-CTA).d)Measuredbysize-exclusionchromatography(SEC)usingpolystyrenestandardsinN,N-dimethylformamide(DMF,10mMLiBr).macro-CTARun1Run224262830323436Time(min)FigureS4.SECcurvesofPNVPI-b-PNIPAMsandPNVPImacro-CTA.

11H2ODMSOacdbnmgONOONHegfghe,hb,fa,c,d876543210ppmFigureS5.1HNMRspectrumofPNVPI-b-PNIPAMinDMSO-d6.cadH2ObbDMSOnmgNH2ONHegfgf,ba,c,de(a)(b)1086420ppmFigureS6.1HNMRspectraofPVAm-b-PNIPAMinDMSO-doo6at(a)25Cand(b)120C.

12gabhbmnicNOOONHOqdHOjlnrkmoNpOeHOsuftd~f,r~uDMSOa,b,g,hl~nk,qcpijio200150100500ppmgabbhmncNiOOONHdDMSOkkejfa,b,g,h,jicd~f200150100500ppmFigureS7.13CNMRspectraof(a)PNVPI-b-PALys(Cbz)OHand(b)PNVPI-b-PNIPAMinDMSO-d6.

133020100-10oZetapotential(mV)25C-20o37C-3024681012pHFigureS8.pH-DependentzetapotentialofPVAmhomopolymerinwater(conc.=2.0mg/mL).

14(a)20(b)200M0M0.1M150.1M150.5M0.5M1M1M1010Intensity(%)Intensity(%)550011010010001101001000D(nm)D(nm)hh(c)20(d)200M0M150.1M150.1M0.5M0.5M1M1M1010Intensity(%)5Intensity(%)50011010010001101001000D(nm)D(nm)hh(e)200M0.1M150.5M1M10Intensity(%)501101001000D(nm)hFigureS9.Salt-dependentDLStracesofPALysOHinwater(pH=7);polymerconc.=(a)0.5mg/mL,(b)1.0mg/mL,(c)2.0mg/mL,(d)5.0mg/mL,and(e)10mg/mL,respectively.

15(a)(b)(c)(d)FigureS10.PhotographsoftheaqueousPALysOHsolutions(pH=7)atdifferentpolymerandsaltconcentrations;polymerconc.=(a,b)0.5mg/mLand(c,d)10mg/mL,NaClconc=(a,c)0Mand(c,d)1.0M.

16(a)N/P=1(b)N/P=51035o25C25oC30837oCo37Co2550C50oC620f(Is)f(Is)1541025004110100100011010010001x10D(nm)D(nm)hhFigureS11.DLSmeasurementsofpolyplexesbetweenDNAandPVAm23-b-PNIPAM39inpotassiumphosphatebuffer(pH7.4,[DNA]=50mg/L,(a)N/P=1and(b)N/P=5)at25oC,37oC,and50oC.41x10/dmol)250000o25C-5000o37Co50C4-1x10Molarelipiticitydeg(cm220240260280300Wavelength(nm)FigureS12.CDspectraofpolyplexesbetweenDNAandPVAm23-b-PNIPAM39inpotassiumphosphatebuffer(pH7.4,[DNA]=50mg/L,N/P=1)at25oC,37oC,and50oC.

17(a)0.5µm(b)1µm(c)1µmFigureS13.AFMphaseimagesofpolyplexeswithPVAm23-b-PNIPAM39atN/P=(e)0.5,(f)1,and(g)10onZnCl2-coatedmica([DNA]=1mg/LinHEPESbuffer(10mM,pH7.4))at25°C.Z-range=(a)40deg,(b)50deg,and(c)110deg.SeeFigure3e-fforcorrespondingheightimages.

18(a)(b)(c)FigureS14.TEMimagesofDNA/cationic-thermoresponsiveblockcopolymer,PVAm23-b-PNIPAM39,polyplexesobtainedat(a,b)N/P=1and(c)N/P=10.

19(a)(b)FigureS15.TEMimagesofPVAm-b-PALysOH/PVAm-b-PNIPAM/DNApolyplexinpotassiumphosphatebuffer(pH=7.4)atN/N/Pratios=1/5/1.

20mpp1mpp22020oo25C25Coo1537C1537Coo50C50C1010Intensity(%)5Intensity(%)5004411010010001x1011010010001x10D(nm)D(nm)hhmpp5mpp6406035o25oC25C503037oC37oC4025o50C20301520intensity(%)intensity(%)10105004411010010001x1011010010001x10D(nm)D(nm)hhFigureS16.DLStracesofthemixedpolyplexesobtainedatdifferentN/N/Pratios(seeTable3fordetailedsampleinformation)inpotassiumphosphatebuffer(pH7.4,[DNA]=50mg/L)at25°C,37°C,and50°C.

21TableS4.Effectofsaltonthesizeandzetapotentialofmixedpolyplex(mPP4)obtainedfromthezwitterioniclysine-basedblockcopolymer/thermoresponsiveblockcopolymer/DNA=1/5/1a)Dh(nm)CodeSalt25°C37°C50°C10.01M108,51230026520.1M130,75035449930.5M56,34043,405366a)Conditions:solvent=potassiumphosphatebuffer(pH7.4),temperature=25,37and50°C,[DNA]=50mg/L.

2225oCo37C10150.01M0.01M80.1M0.1M0.5M0.5M10645Intensity(%)Intensity(%)20012340012341x101x101x101x101x101x101x101x101x101x10D(nm)D(nm)hho50C150.01M0.1M100.5M5Intensity(%)0012341x101x101x101x101x10D(nm)hFigureS17.Salt-dependentDLSmeasurementsofPVAm-b-PALysOH/PVAm-b-PNIPAM/DNApolyplexobtainedatN/N/P=1/5/1(mPP4,Table3)inpotassiumphosphatebuffer(pH=7.4)at25,37,and50°C.

23TableS5.EffectofpHonthesizeandzetapotentialofmixedpolyplex(mPP3)obtainedfromthezwitterioniclysine-basedblockcopolymer/thermoresponsiveblockcopolymer/DNA=1/2.5/1a)Dh(nm)Zetapotential(mV)CodepH25°C50°C25°C50°C15.01465145.44.326.54,1172643.4-1.137.4593770.1-2.049.0175446-3.6-15.9a)Conditions:solvent=potassiumphosphatebuffer,temperature=25and50°C,[DNA]=50mg/L.

24pH=6.5mpp320o25Co1537Co50C10intensity(%)50411010010001x10D(nm)hpH=9.0mpp320o25C15o37Co50C10intensity(%)50411010010001x10D(nm)hFigureS18.pH-dependentDLStracesofthemixedpolyplex(mPP3,N/N/P=1/2.5/1,seeTable3fordetailedsampleinformation)inpotassiumphosphatebuffer([DNA]=50mg/L)at25°C,37°C,and50°C.

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