E ffi cient Regulation of the Behaviors of Silk Fibroin Hydrogel via Enzyme-Catalyzed Coupling of Hyaluronic Acid - Wang et al. - 2021 -

《E ffi cient Regulation of the Behaviors of Silk Fibroin Hydrogel via Enzyme-Catalyzed Coupling of Hyaluronic Acid - Wang et al. - 2021 -》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

pubs.acs.org/LangmuirArticleEfficientRegulationoftheBehaviorsofSilkFibroinHydrogelviaEnzyme-CatalyzedCouplingofHyaluronicAcidLinWang,FeiyuWang,BoXu,ManZhou,YuanyuanYu,PingWang,*andQiangWangCiteThis:Langmuir2021,37,478−489ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Puresilkfibroin(SF)hydrogelexhibitspoorelasticityandlowwaterretentionability,owingtothecompactcrystallinestructureandhighcontentofhydrophobicaminoacids.Herein,acompositedouble-networkhydrogelofSFandtyramine-modifiedhyaluronicacid(mHA)wasconstructed,viathelaccase-catalyzedcouplingreactionsbetweenthephenolichydroxylgroupsfromSFandmHAchains.TheobtainedhydrogelexhibitsimprovedstructuralstabilityandflexibilitycomparedtopureSFhydrogel.Meanwhile,theswellingratio,mechanicalproperty,drugloading,andreleasebehaviorscanbereadilyregulatedbyalcoholization,alteringpHvalue,andionicstrengthofsoakingsolutions.IncreasingpHvaluespromotedtheswellingcapacityofSF/mHAhydrogel,resultinginanefficientloadingofcationicdrugsandsustainedreleaseofanionicdrugsaswell.Theadditionofinorganicsaltsreducedelectrostaticrepulsioninthehydrogelscaffold,accompanyingwithanoticeableimprovementoftoughness.Furthermore,alcoholtreatmentinducedconformationchangesoffibroinprotein,andthecompositehydrogelachievedahigherfractureandimprovedelasticity.Thepresentworkprovidesabiologicalalternativetoregulatethemechanicalbehavior,drugloading,andsustainedreleasecapacityoftheSF-basedhydrogel.■INTRODUCTIONgastrointestinaltract,inflamedtissue/wounds,andtheextrac-18Asamaterialwithexcellentbiocompatibilityandbiodegrada-ellulartumorenvironment.Utilizationofhydrogelascarrierstion,1,2silkfibroin(SF)isobtainedfromdegummedBombyxnotonlyachievesaslow-releaseeffectbutalsoprotectsthedrugmorisilkwormsilkandmainlycomposedofglycine,alanine,fromharmfulenvironments,suchasthepresenceofenzymesin3,4theintestineandthelowpHvalueinthestomach.19serine,andtyrosine.AnumberofsilkpolymorphsconstituteDownloadedviaUNIVOFCAPETOWNonMay14,2021at17:34:15(UTC).thecrystallineregionofSFcontainingα-helixandβ-sheetNevertheless,thehydrogelspreparedfrompureSFhavethe5shortcomingssuchasunsatisfactorymechanicalpropertiesandstructures,whichendowstheSFmaterialswithcertain6poorswellingperformance,mainlyowingtothehighcontentofmechanicalproperties.Inaddition,molecularconformation7ofSFiseasilyaffectedbyexternalconditions,suchaspH,hydrophobicaminoacidsandcompactnessofcrystallineSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.8920organicsolvents,andsalt.Forinstance,theadditionofethanolregions,whichlimitstheirfurtherapplications.intosilksolutionwouldinducesilkmoleculestochangeintoβ-Hyaluronicacid(HA),asanaturalglycosaminoglycanthatsheetstructures,10whichisbeneficialtotheformationofSF21ubiquitouslyexistsintheextracellularmatrix,hasbeenwidelymaterialswithincreasedstiffness.Morerecently,SFhasbeenusedinhydrogelpreparationbecauseofitsinherentwidelyutilizedinthebiomedicalfieldsuchasderivedscaffoldsbiocompatibility,biodegradability,andgelationproperty.2211foradhesionandgrowthofcells,hydrogelsfortreatmentofMoreover,carboxylgroupsinHAchainsareavailableforionic12spinalcordinjury,andflexiblebiosensorsforthemonitoringofinteractions,bioconjugation,targeteddrugrelease,23and13metabolites.hydrogenbondingtocombinewithlargeamountsofwater.24Amongthereportedbiomaterials,hydrogelsplayanMeanwhile,pHvalue,ionicstrength,andothersoakingimportantroleinthecurrentdrug-deliverytechniqueswitha14,15three-dimensionalnetworkfordrugloading.Inaddition,therateofthetherapeuticagentreleasecanbecontrolledbytheReceived:October28,2020structuralchangesofhydrogels.16Inparticular,pH-andsalt-Revised:December11,2020responsivehydrogelsaremostlystudiedbecausebothPublished:December24,2020parametersareimportantenvironmentalfactorsinphysiological17andchemicalsystems,especiallyindrugdeliveryfields.ThepHvariationsexistwithinthehumanbodysuchasthe©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c03136478Langmuir2021,37,478−489

1Langmuirpubs.acs.org/LangmuirArticleFigure1.SchematicdiagramoftheenzymaticpreparationofSFhydrogelanditsresponsestoacid,alkali,andalcohols[(A)SFsolutionpreparation;(B)HAmodificationusingTAandEDC/NHS;(C)enzymaticcouplingofmHAontoSF;and(D)responsestosoakingconditions].conditionscanaffectthestatusofHAhydrogelsbecausetheofCaCl2/CH3CH2OH/H2Oatamolarratioof1:2:8and70°Cfor3h.carboxylgroupsareionizedathighpHandprotonatedatlowAftercompletedissolution,theobtainedSFsolutionwasdialyzedpH.25,26againstdeionizedwatertoremovetheresidualsaltanddetermineitsconcentrationusingaweighingmethod.Morerecently,greatprogressonenzyme-mediatedregulationTyramine-modifiedhyaluronicacid(mHA)waspreparedviaEDC-ofhydrogelsisachieved,likeperoxidase-assisteddevelopingsilk3227andNHS(EDC/NHS)-mediatedgraftingofTAontoHAchains.Aproteinhydrogelinbiofunctionalizedmicrofluidicsystems,compositehydrogelofSFandmHAwaspreparedusing0.2U/mLphosphorylase-catalyzedpreparationofpH-responsiveampho-laccase,30g/LSF,and15g/LmHAintheratioof2:1.Eachmixture28tericglycogenhydrogels,andphosphatase-regulatedhealablesolutioncontainingdifferentratiosofSFtomHAwastransferredinto29polymerichydrogelsforself-repairing.Laccase(EC.1.10.3.2)theTefloncontainerswithholesof2cminheightand2cmindiameter,cancatalyzetheoxidationofphenolichydroxylgroupsintothenincubatedat50°CandpH5toformSF/mHAhydrogels.Foractivefreeradicals,resultingintheformationofcross-linkscomparison,single-componenthydrogelsofSForHAcatalyzedbybetweenphenolicsubstrates.30AsshowninFigure1,tyrosinelaccasewerealsopreparedaccordingtotheabovementionedmethod.residuesinSFandtyramineinmHAareenzymaticallyoxidizedExplorationoftheMolecularWeightChangesduringtheLaccase-CatalyzedProcess.TA+Lac,mHA+Lac,SF+Lac,andSFinthepresenceofoxygen,thenthegeneratedradicalscouple+mHA+LacaredefinedasthemixturesoflaccasewithTA,mHA,SF,togetherandformdifferentcross-linksbetweenSFchains(SF-SF,andmHA,respectively.MolecularweightdistributionofTA,TA+SF),mHAchains(mHA−mHA),andtwodifferentchains(SF-Lac,mHA,mHA+Lac,andHAat10g/Lwasmeasuredbysize31mHA).Basedonthepreviouswork,theresponsivenessoftheexclusionchromatography(SEC),respectively,usingaHPLCobtainedhydrogelstoanumberofphysiologicalstimulus,suchinstrument(WatersE2695,Waters,USA)andBioSuite450SECaspH,ionicstrength,andalcoholization,iswellinvestigated.column(Waters,USA).Themobilephasecontaining0.3mol/LNaClWiththesecharacteristics,thedrugloadingandreleasebehaviorand50mmol/LsodiumphosphatebuffersolutionsatpH7.0wasusedofhydrogelcanberegulatedbychangingenvironmentalastheeluent,withaflowrateof0.5mL/min.SECprofilesofthedifferentsampleswereascertainedat214nm/280nmwithanUV/visconditions,whichexpandstheapplicationperformancesofthedetectorconnectedtotheSECsystem.SF-basedhydrogels.ThesamplesofSF,SF+Lac,SF+mHA,andSF+mHA+Lacat5g/LwerealsoexaminedbySECattheabsorbanceof280nm.Sodium■MATERIALSANDMETHODSdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)wasMaterials.DegummedrawB.morisilkfibersweresuppliedbyfurtherusedtoevaluatethechangesinmolecularweightoftheseXinyuanSilkCo.,Ltd.(Nantong,China).LaccasefromTrametessamples.Eachfibroinsolutionwasmixedwithloadingbuffer(4x/2-versicolor,withanactivityof0.5U/mg,wasprovidedbySigma-Aldrich.thioethanol=9:1)atthevolumeratioof3:1,thenheatedfor10minatSodiumhyaluronate,withthemolecularweightof3−10and10−10090°Cpriortothemeasurement.SDS-PAGEproteinstandardkDa,wasprovidedbyBloomageBiotechnologyCo.,Ltd.(Jinan,(Beyotime,China)wasusedasamolecularweightmarker.ProteinChina).Tyraminehydrochloride(TA),1-(3-dimethylaminopropyl)-3-bandswerestainedwithCoomassieBrilliantBlueR250forvisual-ethylcarbodiimidehydrochloride(EDC),andN-hydroxysuccinimideization.(NHS)werepurchasedfromAladdinReagentCo.Ltd.(Shanghai,MorphologicalObservation.Morphologiesofthefreeze-driedChina).TrypanblueandmethylenebluewerepurchasedfromMacklinhydrogelsampleswerecharacterizedusingaSU-1510scanning(Shanghai,China).electronmicroscope(HITACHI,Japan).EachsamplewasmountedPreparationofSFSolution,Modified-HASolution,andSF-g-onanaluminumdevicefilledwithnitrogenandsputter-coatedwithamHAHydrogels.Degummedsilkfibersweredissolvedinthesolutionthinlayerofgoldpriortoscanning.Theporosityoffreeze-dried479https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

2Langmuirpubs.acs.org/LangmuirArticleFigure2.MolecularweightdistributionofTAandmHAat214nm(a)and280nm(b)beforeandafterlaccasetreatments.33sampleswasdeterminedbyliquiddisplacement.Briefly,asamplewasafteralcoholtreatments,withCuKαradiationoperatingat2.2kWinimmersedintheknownvolume(V1)ofhexaneinagraduatedcylindertherange5−45°atascanrateof5°/min.forapproximately30min,andthetotalvolumeofthehexaneandtheLoadingandReleasingDrugBehaviorsofHydrogels.hexane-impregnatedscaffoldwasrecordedasV2.Finally,thehexane-Spectrophotometryisasimplewaytoevaluatethedrugloadingandimpregnatedscaffoldwasremovedfromthecylinder,andtheresidualreleaseeffectsofthecompositehydrogels.ThealcoholizedandhexanevolumewasrecordedasV3.Theporositywascalculatedbyeq1untreatedhydrogelswiththeoriginalmassofmwereplacedintheacidictrypanbluesolution(pH2)andalkalinemethylenebluesolutionVV13−(pH12),respectively.TheoriginalvolumeofV1wasrecorded,andtheporosity(%)=×100%VV23−(1)massconcentrationofρ1wascalculatedaccordingtoeqs3and4,respectively.After24hofincubation,theremainingvolumeandtheSwellingRatioandMechanicalPropertyTestingofthecorrespondingconcentrationofthemodeldrug(i.e.,trypanblueorHydrogels.TodeterminetheeffectsofthepHvalue,ionicstrength,methyleneblue)weremeasuredandrecordedasV2andρ2,respectively.andalcoholconcentrationontheswellingratiosandmechanicalTheloadingamountofmodeldrugsinthehydrogelscanbecalculatedpropertiesofthehydrogels,eachsamplewastreatedwithacid,alkali,accordingtoeq5.CaCl2,NaCl,andalcohol,respectively.Theswellingratioofthe2preparedhydrogelwasmeasuredbyagravitationalmethod.Briefly,they1=+57.01429xR10.00037429(=0.9999)(3)hydrogelsamplewiththeinitialweightofm1wasimmersedinthe2preparedsolutionsat25°Cfor24h,thentakenoutandweigheditsy=+172.5xR0.0228(=0.9999)(4)22massasm2aftercompletelyremovingthesurfacewaterusingtheabsorbentpaper.Theswellingratiosweresubsequentlycalculatedwherey1andy2aretheabsorbanceat580nmand665nmandx1andx2accordingtoeq2.representtheconcentrationoftrypanblueandmethyleneblue,respectively.mm21−swellingratio(%)=×100%ρρVV−m(2)Theloadingamountofmodeldrug(mg/g)=11221m(5)Mechanicalpropertiesoftheuntreated,swollen,andalcoholizedwhereρ1andρ2arethemassconcentrationofthesolutionbeforeandhydrogelsweretestedusingaMIT-1universalmaterialtestingmachineafterdrugloading.(SanfengCo.,China),andthecorrespondingcompressivepropertiesToinvestigatethedrugreleasebehavioroftheSF/mHAhydrogels,wererecordedatroomtemperature,withacompressionspeedof5thehydrogelsamplescarryingtrypanblueormethyleneblueweremm/min.Furthermore,cycliccompressionexperimentswerealsoplacedin15mLofPBSsolutionsunderdifferentreleasingconditions,performedonthealcoholizedanduntreatedhydrogels.TheslopeofasshowninTableS1(SupportingInformation).Subsequently,2mLofstress−straincurveswithintheelongationof10−15%isrecordedasthethesamplesolutioncontainingthereleaseddrugwastakenatregularinitialcompressivemodulus,whilethefractureenergyisobtainedbyintervals,andanother2mLoffreshPBSbufferwasaddedintothebath.calculatingtheareacoveredbythecurveatthebreakingpoint.Thepercentageofdrugreleasewascalculatedbyeq6,andthetotalRheologicalCharacterization.RheometerPhysicaMCR301releaseamountofmodeldrugwascalculatedaccordingtoeq7.(AntonPaar,Austria)wasusedtotesttherheologyofSF-g-mHAhydrogelsaftertreatmentwithdifferentconcentrationsofsaltsolutions.n−121×∑1CCin+×5Thetestusedaparallelplatewithadiameterof25mmwiththesampleDrugreleasepercentage(%)=×100%wplacedonthesampletable,whilethedistancebetweenthesampletable(6)andtheplatewas1.5mm.Thetestmethodsusedwerefrequencysweep34whereCandCaretheconcentrationsofmodeldrugforthetaken(ω=0.1−100rad/s,γ=5%).Thestoragemodulus(G′)andlossinmodulus(G″)wererecorded.solutionandtheremainingsolution,respectively,nrepresentstheDSCandXRDAnalyses.Thermalpropertiesofthefreeze-driedsamplingtime,andw(mg)meansthetotaldrugloadingamount.hydrogelswererecordedonadifferentialscanningcalorimeter(TAm2Q200,America)ataheatingrateof10°C/minintherangeof50−400Thereleaseamountofmodeldrug(mg/g)=×100%m(7)°C.AD8X-raydiffractometer(BrukerAXS,Germany)wasusedtoexplorethecrystalstructurechangesofthehydrogelsamplesbeforeandwherem2isthetotalreleaseamountandmisthequalityofthehydrogel.480https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

3Langmuirpubs.acs.org/LangmuirArticleFigure3.MolecularweightdistributionofSFandmHAbasedondifferenttreatments[(a)SEC;(b)SDS-PAGE:(1)SF,(2)SF+Lac,(3)SF+mHA,and(4)SF+mHA+Lac].Figure4.SwellingratiosofthehydrogelsunderdifferentpHconditions(a)andthecorrespondingmechanicalproperties[(b)SF+Lac,(c)SF+mHA+Lac,and(d)mHA+Lac].■RESULTSANDDISCUSSIONbedeterminedbySECanalysis,accordingtothenewlyformedMolecularWeightDistributionofSFandmHAafteramidebondsat214nmandthephenolicringstructureat280LaccaseTreatment.Afterlaccase-catalyzedoxidationofTAnm,respectively.Theearlierthepeaktimeappears,thelarger35andmHA,thechangesinthemolecularweightdistributioncanthemolecularweightitis.ItcanbeseenfromFigure2thatthe481https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

4Langmuirpubs.acs.org/LangmuirArticleFigure5.Swellingratiosofthehydrogelsafterincubationwithdifferentconcentrationsofsalt,andthecorrespondingmechanicalpropertyofSF+mHA+Lac[(a,b)NaCl;(c,d)CaCl2].sampleofmHA+Lachasthelargestmolecularweight,followedroleoflaccaseintheformationofcompositehydrogels.ThanksbymHA,HA,TA+Lac,andTA.TheearlierpeakofTA+Lactothefacilepreparation,thecompositehydrogelofSFandmHAcomparedwithTAshowsthatthemolecularweightofTA+Laccanbefabricatedintodiverseformsusingdifferentmoldsincreasedafterlaccasecatalysis.Inparticular,HAinFigure2b(FigureS1c).Furthermore,itseemsthattheobtainedhydrogelshasnodetectableabsorptionpeak,whilemHAexhibitsanaretoughandflexible,whichcanendurelarge-scaledeformation,obviouspeakat280nm,revealingthatTAmoleculeswereasshowninMoviesS1andS2(SupportingInformation).successfullygraftedontoHAchainsduringthelaccaseSurfaceMorphologyandPorosityoftheSFHydrogelstreatment.BasedonDifferentTreatments.ToobservethestructuralFigure3adepictstheretentiontimecurvesofdifferentSFmorphologyofthecompositehydrogels,thefreeze-driedsamples.ForthesamplesofSFandSF+mHA,theinitialpeaksamplesweresubjectedtoscanningelectronmicroscopytimeobviouslymovesforwardafterlaccasetreatment,especially(SEM)observationandporositymeasurements.Itcanbeseenfortheformer,indicatingtheoccurrenceofefficientcross-fromFigureS2athatthesampleswithdifferentcompositionslinkingbetweenSFandmHAmolecules.TheresultsfromSDS-revealdistinctmicrostructures.ThesamplesofSF+LacandSFPAGEareconsistentwiththoseofSEC,asshowninFigure3b,+mHA+LacrepresentmoreregularporestructuresandtighterthetopofSF+LacandSF+mHA+LaclanesexhibitsadarkerstructurescomparedtoSFandSF+mHA,owingtotheappearancecomparedtoothers,implyingthattheyhavelargermolecularweight.36Therefore,itisfeasibletoprepareadouble-formationofcross-linkingstructuresduringthelaccasenetworkhydrogelbylaccase-catalyzedcross-linkingofHAandtreatment,asshowninFigure1.Specially,mHAhasnostableSFchains.secondaryandtertiarystructuresasSF,indicatingthatitcan37FormabilityoftheSFhydrogelswithandwithoutlaccasehardlyformthescaffoldwithuniformporousstructures.catalysiswastestedbythevialinversionmethod.AscanbeseenFigureS2balsoprovidestheporosityoftheabovementionedinFigureS1a,allmixturesolutionsformsolid-likehydrogelsinmaterials.TheporosityofpuremHAisonly30%,whichisthepresenceoflaccase,regardlessoftheratiosofSFtomHA.InsignificantlylowerthanotherscaffoldscontainingSF.Thecontrast,themixedsolutionsremainintheoriginalliquidstateporosityofSF+LacandSF+mHA+Lacisalmostthesame,withoutlaccase(FigureS1b).TheresultsemphasizethecrucialexhibitingabitlowerthanthoseofSFandSF+mHA,indicating482https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

5Langmuirpubs.acs.org/LangmuirArticleFigure6.DSC(a,b)andX-raypatterns(c,d)ofscaffoldslyophilizedfromuntreatedandalcoholizedhydrogels.thatformationofcross-linkingstructuresslightlyreducedthestructuresinthecompositehydrogel.Thecompressivemodulusporosity.andfractureenergyforthehydrogelsamplesarecalculatedandEffectsofpHConditionsontheSwellingBehaviorandshowninFigureS3(SupportingInformation).Aboveall,theMechanicalPropertyoftheHydrogels.ThecompositethreehydrogelsachieveacertainpHresponsiveness,amonghydrogelscontainionizablecarboxylandaminogroups,whichSF+mHA+Lacdisplaysthebestmechanicalpropertiesdifferentdegreesofswellingratioscanbeobtainedwhenonaccountofthedoublecross-linkedstructures.incubatinginacidicoralkalinesolutions.AsshowninFigure4a,EffectsofSaltsontheSwellingBehaviorandmHA+Lachydrogelhasthemaximumswellingratio,whiletheMechanicalPropertyoftheHydrogels.ChangingionicsampleofSF+LacachievestheminimumvalueunderthesamestrengthofincubationsolutionsmightinfluencetheswellingpHconditions,mainlybecauseofthetighterstructureofthebehaviorandmechanicalpropertyofthehydrogels.Asshowninfibroinprotein.Moreover,theswellingratiosofhydrogelsareFigure5a,c,theswellingratiosofhydrogelsgraduallydecreasestronglydependentonpHofthesurroundingsolutions.Forwiththeincreaseofionconcentrations,andalloftheswellingexample,thevolumeofhydrogelssignificantlyincreasedatpHratiosarelowerthanthosetreatedatpH7,especiallyforthe12,especiallyforthemHA+Lachydrogel,whichisattributedtosampleofmHA+Lacwhichillustratestheinhibitoryswellingthemutualrepulsionofnegativechargesofcarboxylgroupsinabilityofsaltions.TheswellingratiosofhydrogelsinCaCl238mHAchains.Onthecontrary,theionizationofcarboxylsolutionarelowerthanthoseinNaClsolution,owingtothatthegroupsinSFandmHAwasinhibitedatpH2,whichreducedthepositivelychargedCa2+canmoreefficientlyinteractwiththeswellingdegreeofthehydrogels.39negativelychargedfunctionalgroups(COO−)thanNa+.InFurthermore,theincreasingofswellingratiossignificantlyaddition,Ca2+andNa+ionscangraduallyinducethepartialdeterioratesthemechanicalpropertyofmHA+Lachydrogel.AsconformationtransitionofSFfromhelixformtoβ-sheet,and2+40,41canbeseeninFigure4b−d,fracturepointsofSF+LacandtheinductionabilityofCaismoreremarkable,mainlymHA+Lachydrogelsarebothreducedcomparedtothatoftheattributedtothereducedrepulsiveforcesandthenewlyformed42untreatedbecauseoftheincreasedswellingratios.Compara-chelatingstructures.tively,thehydrogelsofSF+mHA+LactreatedatpH2andpHAscanbeseeninFigure5b,d,withtheincreaseofsalt7revealbettertoughnessfortheirincreasedstrainsatfractureconcentrations,thestrainsatthebreakingpointofthehydrogelspoint,probablyowingtotheexistenceofdouble-networkincreaseaccordingly.Thestressandstrainatthefracturepoint483https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

6Langmuirpubs.acs.org/LangmuirArticleFigure7.Swellingratiosofthealcohol-treatedhydrogels(a),correspondingmechanicalpropertyofsampleSF+mHA+Lac(b),andtheconformationofmolecularchainsbeforeandafteralcoholization(c).ofthehydrogelstreatedwithCa2+aresignificantlyhigherthanfrom175.5to177.8°Ccanbedetected,whichcanbeascribedtothatwithNa+,exhibitingbettermechanicalproperties.FigurestheprecipitationofthemHAmolecularchains45andneedtobeS4andS5showthestress−straincurvesoftheothersalt-treatedfurtherexploredthroughX-raydiffraction(XRD)analysis.samples,themodulus,andfractureenergycorrespondingtoAccordingtothestudyofCuKα-irradiatedSFstructure,thestress−straincurves,respectively.ItcanbefoundthatthemaindiffractionpeaksofSilkIareataround12.2°,19.7°,24.7°,fractureenergyandsaltconcentrationstendtoincreaseand28.2°,andthemaindiffractionpeaksofSilkIIareataround46synchronously,mainlybecauseoftheincreaseinthecontent9.1°,18.9°,and20.7°.AscanbeseeninFigure6c,d,theofβ-sheetstructuresinducedbysaltionsandthedecreaseinuntreatedhydrogelsofSF+LacandSF+mHA+LaccontainmutualrepulsionbetweenHAmolecules.thediffractionpeaksofSilkIandSilkII,andthecalculatedFigureS6depictsthestoragemodulus(G′)andlossmoduluscrystallinityisabout5.15and8.80%,respectively.After(G″)ofthesalt-treatedhydrogelswiththefrequencysweep.Thealcoholization,thediffractionpeaksofSF+mHA+LacandresultsindicatethattheobtainedG′ofallcompositehydrogelsisSF+Lacareconcentratedat20.7°,whichrepresenttheβ-sheet43greaterthanG″,revealingtheelasticcharacteristics.Mean-structureofSilkII.Thecalculatedcrystallinitiesforthetwowhile,increasingsaltconcentrationsleadstocorrespondingsamplessignificantlyincreaseto17.15and21.79%,respectively,increaseinthestoragemodulus,whichissimilartotheverifyingthatalcoholtreatmenthasobviousinfluenceontheSF47abovementionedresultsoffractureenergy.Thisisbecauseconformation.Comparatively,theeffectsofalcoholizationonthatthenewlyformedβ-sheetstructurereducesthefluidityofthepatternofmHA+Lacarenotobvious,whichisconsistenttheentirechainsegmentandincreasesthestoragemoduluswiththeresultsoflittlechangesinthermodynamicproperties48simultaneously.mentionedabove.EffectsofAlcoholTreatmentontheThermodynamicsEffectsofAlcoholTreatmentontheSwellingBehaviorandCrystallinityoftheHydrogels.Figure6a,bshowstheandMechanicalPropertyoftheHydrogels.Swellingratiosdifferentialscanningcalorimetry(DSC)resultsoftheuntreatedofthehydrogelsamplestreatedwithdifferentmassfractionsofandalcoholizedhydrogels.Theendothermicpeaksfortheethanolwerealsomeasured,andtheresultsareshowninFiguresamplesofSF+LacandSF+mHA+Lacappearingat140.5°C7a.Itcanbeseenthatincreasingethanolconcentrationdoesand145°Cshifttohighertemperaturesat163.0°Cand182.4evidentlyaffecttheswellingbehaviorofthehydrogels,andthe°C,respectively,mainlyowingtotheconformationaltransitionswellingratioevendecreasestoanegativevalueafterimmersion44ofSFfromarandomcoiltoβ-sheetcrystallization.Asforthein75%ethanolsolution.ThisisbecauseethanolcanattractthesampleofmHA+Lac,aslightshiftoftheendothermicpeakwaterfromSFandmHAchainsbecauseofitsstrongpolarityand484https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

7Langmuirpubs.acs.org/LangmuirArticleFigure8.Loading−unloadingcyclesofSF+mHA+Lachydrogelbefore(a,b)andafterimmersionin75%ethanol(c,d).furtherinitiatestheaggregationofSFchainsandformβ-sheetapproaching60%,whilethealcoholizedsamplecanfullycrystallizationviatheinteractionsbetweenhydrophobicaminoreboundevenafterbeingcompressedto80%strain,revealing49,50acidsegments.Meanwhile,themechanicalpropertyofSF+thatalcoholizationendowsthecompositehydrogelwithmHA+Lachydrogeldisplaysasignificantimprovementattheimprovedmechanicalpropertybytheformationofenzymaticbreakingpointwhentheethanolconcentrationexceeds50%cross-links.(Figure7b).Stress−straincurvesofSF+LacandmHA+LacDrugLoadingandReleaseBehaviorsfortheHydrogelandthecorrespondingmodulusandfractureenergyarealsoofSF+mHA+LacunderDifferentConditions.BasedonshowninFigureS7.WiththeincreaseoftheethanolmasstheswellingbehaviorsofSF+mHA+Lachydrogelunderfraction,themechanicalpropertiesofhydrogelscontainingSFdifferentconditionslikeacid,alkali,salt,andalcohol,wetooktheareobviouslyenhanced,whichismainlymanifestedintheadvantagesofitspH-andion-sensitivepropertiestocarryoutincreaseoffractureenergy.Theconformationchangesbeforedrugloadingandreleasingexperiments.ToincreasetheamountandafteralcoholizationareproposedinFigure7c,andtheblueofthemodeldrugintothecompositehydrogel,drugloadingoffoldintheellipsedottedlineindicatesthenewlyformedβ-sheetanionictrypanblueandcationicmethylenebluewasperformedstructureofSFwhiletheredfoldindicatesthedehydrationandatpH2andpH12,respectively,ensuringthatmoredrugscanaggregationofthemHA.bindtothehydrogelswithoppositechargesviaelectrostaticTofurtherexplorethemechanicalpropertyofthealcoholized51forces.sampleofSF+mHA+Lac,cycliccompressionexperimentsFigure9a,bshowsthechangesintheconcentrationrateswereperformedundertheconditionsofthespecifiedstrain(50%)andgraduallyincreasingstrains(20%−80%),respec-duringthedrug-loadingprocess.Ascanbeseen,extendingtively.Whenthemaximumstrainissetto50%,asshowninincubationtimeleadstoaremarkabledecreaseintheresidualFigure8a,c,thedissipatedenergyfromthefirstcycleisconcentrationratesoftrypanblueandmethyleneblue,significant;meanwhile,thestressforthealcoholizedsampleisespeciallyfortheuntreatedhydrogels,reachingapproximatelynoticeablyhigherthanthatoftheuntreated,reaching15%and5%,respectively.Theamountofdrugloadedintheapproximately250kPa.Figure8b,dshowsthecyclicstress−hydrogelscanbedepictedinFigure9c.Forthealcoholizedstraincurvesbasedonthestrainfrom20to80%.Itcanbeseenhydrogelsample,theloadingamountofthemodeldrugisabitthattheuntreatedhydrogelbreakswhenthestrainislowerthanthatoftheuntreated,mainlybecauseofthe485https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

8Langmuirpubs.acs.org/LangmuirArticleFigure9.Drugloadingeffectsoftrypanblue(a)andmethyleneblue(b)intothehydrogelofSF+mHA+Lac,andtheloadingamountofmodeldrugs(c).formationofβ-sheetsinthecrystallineregionsandthetighterpatternsundertheacidicoralkalineconditions.Theamountsofstructurecausedbyalcoholization.themodeldrugsreleasedfromthecompositehydrogelareDrugreleaseexperimentswereconductedbycontrollingthesummarizedinFigure10c,ascanbeseen,thesustainedreleasepHvaluesandsaltconcentrations,andtheresultsareshownineffectdecreasestoacertainextentaftersaltandalcoholFigure10.ItcanbeseeninFigure10a,bthatthepHvalueofthetreatments.Furthermore,thepercentagesofdrugreleasetothesoakingsolutionshasagreatinfluenceontheamountofdrugdrugloadingfordifferentsamplesaredepictedinFigure10d,released.Theamountoftrypanbluereleasedfromthehydrogelanditisdifficultforallloadeddrugstobereleasedsustainably,reachedthemaximumatpH12,owingtothatthetotalamountwhichisrelatedtotheintermolecularforcesbetweenthemodelofcarboxylgroupsinthecompositehydrogelincreasesunderdrugandSFormHAmolecules.Accordingtotheresultsofalkalineconditions,whichaccordinglyincreasestherepulsionloadingandreleasebehaviorsforthedifferentmodeldrugs,itbetweenthehydrogelandtheloadeddrugs(Figure10e).ForcanbedrawnthattheloadingandreleaseeffectsofcationicthehydrogelsampleundertheconditionofpH12+NaCl,thedrugsaremoreremarkable,whichisrelatedtothehighcontentelectrostaticrepulsionslightlydecreasesinthepresenceofsalt,ofnegativechargesinthecompositehydrogel.Therefore,itcanthusthedrugreleasepercentageisabitlowerthanthatofthebeusedasacarriertoloadcationicdrugslikeranitidinesampletreatedatpH12alone,reachingapproximately50%afterhydrochloride,whichmightprolongthereleasetimeofdrugin72hofincubation.stomachforbettertreatment.Asforthemodeldrugofmethyleneblue,anacidicconditionisconducivetothesustainedreleasefromthecompositehydrogel.■CONCLUSIONSThehighestreleaseeffectisobtainedatpH2,whichissuperiorWehavesuccessfullypreparedadouble-networkfibroin-basedtothesampletreatedwithpH2+NaClcondition.Thiscanbehydrogelsviathelaccase-catalyzedcouplingofmHA.Theinterpretedasthattheaminogroupsoffibroininthecompositecompositehydrogelhasgoodformability,porosity,andhydrogelareprotonatedandpositivelycharged,whichincreasestoughnesscomparedtopureSFandmHAhydrogelsandtheelectrostaticrepulsionbetweenthehydrogelandthecationicexhibitsdifferentresponsestoacid,alkali,salt,andalcoholdrug.AsshowninFigure10e,thecompositehydrogelsloadedsolutions.Briefly,thehydrogelswellsinalkalineorlow-withdifferenttypesofmodeldrugsexhibitdifferentreleaseconcentrationalcoholsolutionsandshrinksinacidic,salt486https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

9Langmuirpubs.acs.org/LangmuirArticleFigure10.Drugreleasepercentageoftrypanblue(a)andmethyleneblue(b)fromthedrug-loadedSF+mHA+Lac,releasedamountofmodeldrugs(c,d),andtheproposedloadingandreleasingmechanismunderdifferentpHvalues(e).solutions,orhigh-concentrationalcoholsolutions.Mechanicalfibroin-basedhydrogels,whichwillexpandtheapplicationsofandthermodynamicpropertiesofthecompositehydrogelaresilkfibroininthefieldsofbiomedicalmaterials.evidentlyimprovedafterincubatingwithethanol,owingtotheformationoftheβ-sheetstructureofSF.Thedrugloadingand■ASSOCIATEDCONTENTreleasingbehaviorsforthecompositehydrogelcanbewell*sıSupportingInformationcontrolledbychangingthepHvalueandsaltconcentrationofTheSupportingInformationisavailablefreeofchargeatsoakingsolutions.Undertheacidicconditions,thehydrogelhttps://pubs.acs.org/doi/10.1021/acs.langmuir.0c03136.exhibitshighefficacyofanionicdrugloadingandcationicdrugDifferenttreatmentsofhydrogelsforloadingandreleaseeffects.Conversely,thealkalineconditionsarefavorablereleasingdrugs;moldabilityofthemixtureofSFandforthecompositehydrogelstoloadcationicdrugsandreleasemHAandthemultipleshapesofthehydrogel;SEManionicdrugs.Thepresentworkprovidesanefficientwaytoimagesandporosityofthefreeze-driedscaffolds;modulusregulatethemechanicalanddrug-releasebehaviorsoftheandfractureenergycorrespondingtothestress−strain487https://dx.doi.org/10.1021/acs.langmuir.0c03136Langmuir2021,37,478−489

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