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SCIENCECHINAMaterialsARTICLES
:///s40843-022-2107-0
Nanoplatformswithdonor-acceptorStenhouseadductmolecularswitch
forenzymaticreactionsremotelycontrolledwithnear-infraredlight
KeLi,Miao-DengLiu,Qian-XiaoHuang,Chuan-JunLiu*andXian-ZhengZhang*
ABSTRACTEnzymeshavebeenwidelyusedinbiomedicalistheprecisecontroloftheenzymaticreactioninvivo.
However,theprecisecontrolofenzymaticreactionsremainsaofbiologicalfunctionsowingtoitsfavorablespatiotemporal
,,lighthasbeenincreasinglyappliedinthe
upconversionnanoparticles(UCNPs)asthecoreandadonor-biomedicalfield,suchasphototherapyandphoto-triggereddrug
acceptorStenhouseadductasamolecularswitch(MS)wasrelease[19–22].However,mostphotoresponsivesystemscanbe
designedfornear-infrared(NIR)-controlledenzymaticreac-activatedonlybyhigh-energyphotons[23],suchasthosein
,theUCNPscouldemitbrightultraviolet(UV)orvisiblelight,whichhavepoorphotoactiva-
greenlighttoisomerizetheMSandthentransformtheper-tionefficiencyinbiologicaltissuesbecauseoftheirpoortissue
meabilityoftheMS-gatedpolymerlayer,allowingsmallmo-penetrationdepth[24–27].Upconversionnanoparticles
,the(UCNPs)areanewgenerationofanti-Stokes-shift-typephoto-
contactbetweentheenzymeandsubstratecouldberegulatedluminescentnanoconverters[28],whichcanefficientlyconvert
-long-wavelengthnear-infrared(NIR)lightintohigh-energyUV
isomerismofMSisreversibleandtheenzymeactivityisnotorvisiblelightwhileminimizingthesideeffectsofUVlight[29].
changed,theenzymereactorbasedonthisnanoplatformisOwingtotheiruniqueopticalconversioncapacity,UCNPshave
alsoreversible,whichcanachieveprecisetemporalandspatialbeenusedindeeposmoticphotodynamictherapyandtoacti-
(GOX),lac-vatephotoresponsivesystemsforspecificbiomedicalapplica-
tateoxidase(LOX),urateoxidase(UOX),andluciferaseweretions[30–32].
,molecular
Boththesubstratesinandoutofthenanoplatformweresa-photoswitches,suchasstilbenes,spiropyrans,azobenzenes,and
tisfactorilycontrolled,indicatingthatthegatednanoplatformdiarylethenes,havebeenemployedinopticalelectronicdevices
equippedwithvalve-likecharacteristicscouldrealizeremotely[33–35],energyconversionandbiomedicalfields[8,36].How-
,mostphotoswitchescanbeisomerizedonlybyusinghigh-
energyphotons,suchasthoseinUVlight[23].Thesephotons
Keywords:enzymaticreaction,molecularswitch,near-infrared,notonlyhavepoortissuepenetrationdepth[24–27],butarealso
remotecontrol,,anewclassofvisible-light-
responsivephotochromiccompounds,calleddonor-acceptor
Stenhouseadducts(DASA)[37],
INTRODUCTIONmolecule,whichcanswitchfromahydrophobicformtoa
Owingtotheirexcellentbiocatalysisfunctions,enzymescanhydrophiliczwitterionicstructureuponvisible-lightirradiation
catalyzeadiverserangeofchemicalreactionsandhavebeen[38],issuitableforbiomedicalapplications[22].However,tothe
widelyusedinthebiomedicalfield[1,2].Theconversionofanbestofourknowledge,NIRregionsaremoresuitablethanUV
endogenicmoleculetogeneratetoxicmoleculesfordiseaseorvisiblelightforbiomedicalapplications[7,39].AnNIR-
treatmentisacommonstrategyinenzymetherapy[3–5].AsaresponsiveDASAsystemneedstobedevelopedtobroadenthe
powerfulendogenousoxidoreductasebiocatalyst,glucoseoxi-applicationofDASAinthebiomedicalfield.
dase(GOX)hasbeenemployedtocatalyzeglucosetogenerateHere,weproposeafunctionalizednanoplatformequipped
hydrogenperoxide(H2O2)[6–8],whichcanbeconvertedintowithaUCNPcoreandDASAmolecularswitch(MS)-con-
oxygentoimprovetheefficiencyofphotodynamictherapyorjugatedpolymermembraneforNIR-triggeredenzymaticreac-
,suchasGOX,wereimmobilizedonthesurface
,urateoxidase(UOX)(980nm)
catalyzetheoxidizationofuricacid(UA)torelievethepainirradiation,thestimulatedUCNPscouldemitbrightgreenlight
causedbytheaccumulationofsodiumurate[9,10].StudieshavetoisomerizetheMSandtransformthepermeabilityofMS
beenconductedtodevelopefficientmethodstoconstructmembranes,whichcouldallowtheenzymetocontactthesub-
thermo-[11–13],pH-[14,15],orlight-,byswitchingthe
[16–18],theprimarygatedmembraneonandoff,theoccurrenceoftheexperimental
limitationofcurrentenzymeapplicationsinthebiomedicalfieldenzymaticreactionswasremotelycontrolledbyNIR(Scheme1).
KeyLaboratoryofBiomedicalPolymersofMinistryofEducation&DepartmentofChemistry,WuhanUniversity,Wuhan430072,China
*Correspondingauthors(emails:******@(LiuC);xz-******@(ZhangX))
©ScienceChinaPressandSpringer-VerlagGmbHGermany,partofSpringerNature20221
ARTICLESSCIENCECHINAMaterials
Scheme1Schematicofthecontrolledenzymaticreaction.(a)PenetrationofthesubstrateintothenanoplatformfortheenzymaticreactionunderNIR
irradiation.(b)ReleaseofD-luciferinasanenzymesubstrateunderNIRirradiationandsubsequentoxidizationbyluciferase.
BenefitingfromtheupconversioncapacityofUCNPsandthespectrometer(PerkinElmer).UV-visabsorbancedatawere
favorablepenetrabilityofNIR,thenanoplatformcanbroadenrecordedbyUV-visspectroscopy(LambdaBio40).Thermo-
theapplicationofDASAaswellasbeappliedinenzymegravimetricanalysis(TGA)datawererecordedbyaPyris1
replacementtherapyandprovideanewperspectiveondrugthermogravimetricanalyzer(PerkinElmer).Confocallaser
(CLSM)imageswereobservedonasuper-
resolutionlaserconfocal(Leica)scanningconfocalmicroscope.
EXPERIMENTALSECTIONAninvivoimagingsystem(IVIS,PerkinElmer)wasusedto
observetheinvivocontrolledenzymaticreactionofthenano-
Materialsplatforms.
Ttrifluoroaceticacid(TFA),R2O3(R:Y,Yb,andEr),penta-
3+3+
fluorophenol,4-cyano-4-(thiobenzoylthio)pentanoicacid,hex-PreparationofcoreUCNPs(β-NaYF4:Yb,Er)
ylamine,triethylamine,azodiisobutyronitrile;Beforecore-shellUCNPsynthesis,Y(CF3COO)3,Yb(CF3COO)3,
azobisisobutyronitrile(AIBN)werepurchasedfromSigma-andEr(CF3COO)3werefirstlysynthesized,
-Dimethylaminopyridine(DMAP),ethylbenzoylace-(20mmol;R=Y,Yb,orEr)wasaddedintoaflaskcontaining
tate,meldrum’sacid,4-iodoanisole,cuprouschloride,potassiumTFA(150mmol),theobtained
hydroxideandfurfural,oleicacid(OA)and1-octadecene(ODE)Y(CF3COO)3(),Yb(CF3COO)3(),and
-Er(CF3COO)3()weremixedwithOA()and
poly(ethyleneglycol)(mPEG,Mw5000)waspurchasedfromODE(25mL)ina100-mLthree-neckedflaskandheatedupto
ShanghaiPonsureBiotech,°,
(PI)°Cfor30minundervacuum.
peroxide(H2O2)assaykitwasobtainedfromSolarbioScience&Thereafter,themixturewascooledto50°Cunderargonatmo-
TechnologyCo.,′,7′-,methanolsolution(8mL)containingNH4F
(DCFH-DA)waspurchasedfromYiShengBiologicalTech-(8mmol)andNaOH(5mmol)wasaddedintothemixtureand
nologyCo.,’sphosphatebufferedsaline(PBS),stirredat50°,the
trypsin,RMPI1640,3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-mixturewasheatedto305°C(20°Cmin−1)andstirredfor1h
nyltetrazolium-bromide(MTT),fetalbovineserum(FBS),
penicillin-streptomycinwereobtainedfromLonzaGroupLtd.(RT),ethanol(50mL)waspouredintotheflasktoprecipitate
3+3+
AllotherreagentsandsolventswereanalyticalgradeprovidedbythecoreUCNPs(β-NaYF4:Yb,Er),whichwerecollectedby
SinopharmChemicalReagentCo.,,000rmin−1for8minandwashedwith
ethanolforfivetimes,andredispersedincyclohexane(20mL).
Characterization
3+3+
Scanningelectronmicroscopy(SEM)imageswerecollectedbyPreparationofcore-shellUCNPs(β-NaYF4:Yb,******@NaYF4)
usingscanningelectronmicroscope(Sigma).TransmissionY(CF3COO)3(1mmol)wasaddedintoa100-mLthree-necked
electronmicroscopy(TEM)imageswereobtainedbyTecnaiG2flaskcontainingOA()andODE(25mL),andheatedup
-ZSto80°,themixturewas
ZEN3600particlesizer(MalvernInstruments)wasappliedtostirredat156°
°C,theobtainedUCNPcyclohexanesolution
FluorescencespectrumwasmeasuredonanLS55luminescence(10mL)wasaddedintothemixtureunderargonatmosphere
2©ScienceChinaPressandSpringer-VerlagGmbHGermany,partofSpringerNature2022
SCIENCECHINAMaterialsARTICLES
,aftercyclohexanewasevaporated,inPBS(2mL)forfurtheruse.
methanolsolution(8mL)containingNH4F(8mmol)and
NaOH(5mmol)wasaddedintothemixtureandstirredat50°CInvitroenzymaticreactionevaluation
,themixturewasBeforeenzymaticreactionevaluation,DCFH-DA(2μL)was
heatedto305°C(20°Cmin−1)-mLvialcontainingNaOH(20mmolL−1,80μL)
AfterthemixturewascooledtoRT,ethanol(50mL),PBS(918μL)was
intotheflasktoprecipitatethecore-shellUCNPs(β-NaYF4:addedintothevialtoprepareaDCFHstocksolutionfor
3+3+
Yb,******@NaYF4),,theDCFHstocksolution
10,000rmin−1for8minandwashedwithethanolforfivetimes,(20μL)wasaddedintoavialcontainingPBSsolution(980μL)
andredispersedincyclohexane(20mL)(−1).Simultaneously,thesame
samplewaspreparedandtreatedwith540-nmirradiation
−1
******@SiO2(UCS)andcargo-loadedUCS().Afterirradiationfordifferenttimes,thefluores-
Firstly,IgepalCO-520(2mL)wasdispersedina100-mLthree-,analiquotofDCFH
neckedflaskcontainingcyclohexane(40mL)(20μL)wasaddedintoPBSsolution(980μL)
Afterwards,OA-coatedUCNPcyclohexanesolution(4mL)wascontainingglucose(10mmolL−1)andUCGM(100mgL−1),
,ammonialactate(10mmolL−1)andUCLM(100mgL−1),orUA
(100μL,30%)wasaddedintothemixtureandstirredfor(10mmolL−1)andUCUM(100mgL−1).Thefluorescenceof
,tetraethoxysilane(TEOS,200μL)wasdrop-themixturetreatedwithNIR,540nm,orindark,respectively,at
wiseaddedintothemixtureatarateof200μLh−.
wasstirredatRTfor48hbeforeaddingethanoltocollectthe
-Endocytosisandcolocalization
hexaneandethanoltoremoveexcessIgepalCO-
collectedUCSwasdispersedindeionizedwater(20mL).After24hat37°
fabricationofUCS,D-luciferin(10mg)dissolvedinwatermediumcontainingCy5labelledUCGM(100mgL−1)was
(1mL)-culturedintheincubatorat
(1mgmL−1,4mL)°,thecellswerewashedwithPBSfor
ofthenanoparticles,******@UCSwasredispersedindeio-threetimesandobservedbyCLSM.
nizedwaterforfurtheruse.
Intracellularenzymaticreactionevaluation
PreparationofDASA-functionalizedblockcopolymersThelight-regulatedintracellularGOXcatalyticreactionwas
TheDASA-functionalizedblockcopolymersweresynthesizedindirectlyevaluatedbyobservingtheproductionofintracellular
accordingtoasimilarmethoddescribedbythelitera