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Signal Transduction and Targeted Therapy

MiR146a-loadedengineeredexosomesreleasedfromsilk ﬁbroinpatchpromotediabeticwoundhealingbytargeting IRAK1

QiankunLi1,2,WenzhiHu2,QilinHuang2,JieYang2,BingminLi2,3,KuiMa2,4,5,QianWei2,YaxiWang2,JianlongSu2,6,MengliSun2, ShengnanCui2,RungongYang1,HaihongLi7 ✉,XiaobingFu1,2,4,5 ✉ andCuipingZhang2,4,5 ✉

Unhealablediabeticwoundsneedtobeaddressedwiththehelpofnewer,moreefficaciousstrategies.Exosomescombinedwith biomaterialsforsustaineddeliveryoftherapeuticagentsareexpectedtobringnewhopeforchronicwoundtreatment.Here,the engineeredexosomesmodifiedforefficientlyloadingmiR146aandattachingtosilk fibroinpatch(SFP)weredemonstratedto promotediabeticwoundhealing.Silk fibroinbindingpeptide(SFBP)wasscreenedthroughphagedisplay,andSFBP-Gluc-MS2 (SGM)andpac-miR146a-pacfusionproteinwereconstructed.Thedesignedexosomes(SGM-Exos,miR146a-Exos,andSGMmiR146a-Exos)wereisolatedfromtheengineeredplacentalmesenchymalstemcells(PMSCs)transducedwithSGMor/andpacmiR146a-pacprotein.GlucsignalsindicatedSGM-Exo@SFPmarkedlyincreasedthebindingrateandthestabilityofSGM-Exo. Moreover,theloadingefficiencyofmiR146ainSGM-miR146a-Exoswasten-foldhigherthanthatinmiR146a-Exos.Superiorto untreated,SGM-miR146a-Exo-onlytreated,andSFP-onlytreatedgroups,SGM-miR146a-Exo@SFPdrivedwoundhealingassociated withlessinflammation,collagendeposition,andneovascularization.Thetranscriptomicsanalysissuggestedanti-inflammatoryand regenerativeeffectswithSGM-miR146a-Exo@SFPtreatment.Here,weshowefficientexosome@biomaterial-basedmiRNAdelivery systemsforregenerativemedicineandtissueengineering.

SignalTransductionandTargetedTherapy (2023)8:62

INTRODUCTION

Extracellularvesicles(EVs)orexosomes,membrane-enclosedand secretedbynumerouscelltypes,1 havebecomeanincreasingly attractive fieldofresearchinrecentyears,withapplicationsin treatmentforawiderangeofdiseases.Initially,EVswerethought as “cellulardust” andnothavinganyvalue,primarilytofunctionin themaintenanceofcellularhomeostasisbythewasteremoval fromcells.In1996,Raposoetal.2 describedthe firstevidenceof EVsplayingimportantbiologicalfunctions.In2005,Valadietal.3 demonstratedthatEVscantransferfunctionalmRNAmoleculesto recipientcellsandtherebymediatecell–cellcommunication. Subsequently,awealthofevidenceindicatedthatEVsplayed significantphysiologicalroles,inadditiontotheirpathological roles,throughthedeliveryofbioactivemolecules,suchasRNA, proteins,lipids,andothers.4 Withanincreasingunderstandingof EVcharacteristics,thepotentialutilityofEVsintissueengineering andregenerativemedicinehasbeenexploredthroughdirect applicationorviaincorporationintobiomaterialscaffolds. Invivo,EVshaveahighpotentialforabsorptionbyneighboring cellsandthereforeaveryshorthalf-life,instability,andlowlongtermretentionaftertransplantation.IncorporationofEVsinto

;https://doi.org/10.1038/s41392-022-01263-w

biomaterialsmaybeapowerfultoolforenhancingEVstabilityand realizingacontrolledsustainedreleasewithinthebody.5 This premiseissupportedbyseveralstudiesonincorporatingEVsinto biomaterialconstructsfordelivery.6 Butit’sworthnotingthatthe abilityofbiomaterialshostingEVsisdifferent,whichmaybe dependonthebindingefﬁciencyofEVstobiomaterials.Scientists havefoundevidencethatEVscancombinewithextracellular matrix(ECM)components7 inasimilarmannertothesequestrationofgrowthfactorsinECM.Adhesionreceptorspresentedon theexosomalmembranearelikelyresponsiblefortheinteractions betweenEVsandECM,implyingthatintegratinginstructive biomolecularsignalsintoexosomalmembranetargetingbiomaterialscanenhancethebindingrateofEVstobiomaterials.

MicroRNAs(miRNAs),aspartofexosomalcargo,canbindto theirtargetmRNAsnegativelyregulatingmRNAexpression posttranscriptionally. 8 Duetotheinstabilityandsusceptibilityto degradation,miRNAsaredifﬁculttobetransferredintotarget cells,warrantingthedevelopmentofaneffectivemiRNAdelivery system.9,10 EVshavebeenrecognizedaspromisingvehiclesfor biomoleculesdeliveryowingtotheirabilitytoprotectthemfrom serumproteasesandimmuneresponses.Fordeliveryofthe

1DepartmentofTissueRepairandRegeneration,TheFirstMedicalCenter,ChinesePLAGeneralHospital,Beijing100853,China; 2ResearchCenterforTissueRepairand Regeneration,MedicalInnovationResearchDepartmentandtheFourthMedicalCenter,ChinesePLAGeneralHospital,Beijing100048,China; 3DermatologyDepartment,The FourthMedicalCenter,ChinesePLAGeneralHospital,Beijing100048,China; 4ResearchUnitofTraumaCare,TissueRepairandRegeneration,ChineseAcademyofMedical Sciences,2019RU051,Beijing100048,China; 5PLAKeyLaboratoryofTissueRepairandRegenerativeMedicineandBeijingKeyResearchLaboratoryofSkinInjury,Repairand Regeneration,Beijing100048,China; 6SchoolofMedicine,NanKaiUniversity,Tianjing300071,Chinaand 7DepartmentofWoundRepairandDermatologicSurgery,Taihe Hospital,HubeiUniversityofMedicine,Shiyan442000,China

Correspondence:HaihongLi(lihaihong1051@126.com)orXiaobingFu(fuxiaobing@vip.sina.com)orCuipingZhang(zcp666666@sohu.com) Theseauthorscontributedequally:QiankunLi,WenzhiHu

Received:9May2022Revised:3November2022Accepted:20November2022

nucleicacids(i.e.,siRNAs,miRNAsetc.),differentmethodshave beendevelopedtopackagethemintoEVs,suchaselectroporation,sonication,chemicaltransfection,etc.11 However,certain limitationsassociatedwiththesemethodsmayincludecompromisedintegrityandstabilityoftheengineeredEVsaswellas decreasedbiologicalactivityofthecargo.12,13 Biologicalmethods employedformiRNA-loadedexosomesfromgeneticallymodified hostcellshavegainedsignificantattentionduringthepastfew years,14 withlackingundesiredsideeffectsandmaintainingthe structuralintegrityoftheexosomalmembrane.Buthowto encapsulatemiRNAsefficientlyinengineeredexosomesby engineeringparentcellsisabigchallengeforbettertherapeutic effects.

Withtheadventoftheglobalagingsociety,thenumberof patientswithdiabetesisincreasing,andrefractorywoundssuchas diabeticfootanddiabeticulceraregettingmoreandmoreattention asthecomplicationsofpatientswithdiabetes.15 Thenormalwound healingprocessinvolvesthephasesofhemostasis,inflammation, proliferation,andremodeling.Achronicwoundusuallyresults owningtohaltinthephaseofinflammationfollowedbyimpaired angiogenesisanddelayedre-epithelialization.16,17 Thebasiccauseof itispersistent/chronicbacterialinfectionandbiofilmformation. Therefore,preventinginfectionandcontrollinginflammationis criticaltochronicwoundtreatment.Amongwounddressing biomaterials,silk fibroin(SF)hasattractedsignificantattentionfor woundhealingbecauseofantimicrobialpropertiestoprevent pathogeninvasionandproliferation,therebyreducingtheriskof woundinfection.18 Exosomesderivedfromvariousstemcellshave beenindicatedtopromoteskinrepairandregeneration,19 but seldomofthemhaveantimicrobialfunction,whichsuggests incorporatingEVsintoSFscaffoldforthetreatmentofchronic wounds.However,mostofnativeexosomesusuallycontainless anti-inflammationactivecomponents,thereforehavinglimited therapeuticeffectsoninfectivewounds.Onepossiblesolutionto thislimitationisengineeredexosomes,whichcouldbeusedto encapsulateanti-inflammatoryregulatorsuchasmiRNA-146a (miR146a)20,21 viageneticengineering.

Here,wereportanovelandefficientmiRNAdeliverysystem basedonengineeredexosomesandSFscaffoldtopromote diabeticwoundhealing.Silk fibroinbindingpeptide(SFBP)-GlucMS2(SGM)andpac-miR146a-pacfusionproteinswereconstructedandtransducedintohumanplacenta-derivedMSCs (PMSCs).Subsequently,theSGMproteincouldbeintegratedinto plasmamembraneorendosomalmembraneofPMSCs.When exosomesareshedfromthemembranes,miR146awillbe encapsulatedefficientlyinexosomesbythespecificbindingof phageMS2capsidproteinandpacsite.22 Furthermore,duringthe processofexosomebiogenesis,SFBPtargetingSFwasexpressed intoexosomalmembrane,thereforeenhancingthebindingrateof SGM-miR146a-ExostoSFP.Thesystemdevelopedherecould improvetheefficiencyofengineeredexosomescarryingtarget miRNAthroughMS2inordertoplayaregulatoryrolemore effectively.Besides,silk fibroinaffinityexosomesboundtoSF couldbebetterpreservedandmaintainlastingtherapeutic effects.Comparedwithuntreated,SGM-miR146a-Exo-onlytreated, andSFP-onlytreatedgroups,SGM-miR146a-Exo@SFPachieveda bettertherapeuticeffectondiabeticwoundhealing.

RESULTS

Characterizationoftheengineeredexosomes SFBPwasselectedthroughscreeningthe12-phagepeptidelibrary andvalidatedbyELISA(SupplementaryFig.S1,TablesS1–S9). Next,weinsertedSFBPbetweenSPandC1C2domainsof exosomalmembraneproteinLactadherin,andfusedabioluminescencereportsystemGaussialuciferase(Gluc)23 followingSFBP tomonitorexosomesinreal-time.MS2domainwasfusedtotheC terminalofLactadherintocargodesignedmiRNAwhichhavetwo

pacsitesat ﬂanks.Thecodingsequencesoffusionproteinswere showedinSupplementaryTablesS10–S15andtheconstructionof thefunctionalfusionproteinSGMwasdisplayedinSupplementaryTableS16.Theconstructedlentiviralplasmids(pLV)were Gluc-pLV(G-pLV),SFBP-Gluc-MS2-pLV(SGM-pLV),pac-miR146apac-pLV(miR146a-pLV)andSGM-miR146a-pLVrespectively.The sequencesofthoselentiviraltransferplasmidswereshowedin SupplementaryFig.S3a.Thelentiviral(Lv)particleswereobtained from293Tcellstransfectedwithrecombinantlentiviralplasmids. TheobtainedlentiviralparticleswereusedtoinfectPMSCs (SupplementaryFig.S3b)andthestabletransfectedcelllineswere establishedafterscreeningbypuromycin(SupplementaryFig. S3c).Subsequently,thedesignedengineeredexosomes(SGM-Exo, miR146a-Exo,andSGM-miR146a-Exo)wereisolatedfromsupernatantoftheirparentPMSCsinfectedwithSGMor/andpacmiR146a-pacLv.TheGluc-labeledexosomes(G-Exo)wereusedas thecontrolexosomesinluminescenceimaginganalysis(Fig. 1a). ThentheengineeredexosomeswereincorporatedintotheSF scaffoldforfurthertreatment(Fig. 1b).ThroughTEM,DLSandWB analysis,wefoundthatthecharacteristicsofSGM-Exoswere similartothatofExos.AsshowninFig. 1candd,bothExosand SGM-Exosdisplayedatypicalcuporroundshapeappearancewith asizeofapproximately85nm.ExosomalmarkersCD9,CD63,and TST101werehighlyexpressedinExosandSGM-Exosdetectedby westernblot.ERmarkerCalnexin,asamonitorofcellular contaminationduringexosomeisolation,wasonlyexpressedin PMSCswhilerarelydetectableinExosandSGM-Exos(Fig. 1e). Besides,thebioluminescenceimaging(BLI)analysis(Fig. 1f) showedthatthebioluminescencesignalsofSGM-Exoincreased graduallywithadose-dependentmanner(Fig. 1g)andSGM-Exos weresteadilyinternalizedovertimebyHaCaTcellswitha concentrationof1 μg/mL(Fig. 1h).

PreparationandcharacterizationofSFPservedasexosome-laden scaffolds

ThepreparationprocesstoobtainSFPwasshownschematicallyin Fig. 2a.AsshowninFig. 2b,theSFPwasalight,softandwaterabsorbingmaterialwithsufficientmechanicalstrengthtopulland bend.TheSFPwaspreparedaccordingtothesizeofthewound. ThefavorablebiologicalsafetyofSFPwereevaluatedtoensure securityofapplicationbythebiocompatibilityandcytotoxicity testsinvitroandinvivo(SupplementaryFig.S2).SFPhadno influenceoncellviabilitydetectedbyCCK8test.Theblood biochemistryindicatinghepatorenalfunctioninSFPgroupwerein thenormalreferencerange.Thehistologicalsectionsofliverand kidneytissueshowednoobviousabnormalitiesinSFPandcontrol groups.Toobservethestructurecharacterizationsofsilk fibroin patch-exosome,ExosandSGM-Exoswererespectivelyloadedon SFPandwashedwithdeionizedwater.TheSFP,Exo@SFP,and SGM-Exo@SFPwerepreparedforscanningelectronmicroscope (SEM)detection.TheimagesshowedSFPhadaloosetexturewith irregularorribbon-likeporesofdifferentsizebeingapproximately 10–50 μm.Comparedwiththenaturalexosomeswithoutthe capacityofSF-affinity,theSF-affinityexosomes,SGM-Exos, distributedmoredenselyonSFP(Fig. 2c).Next,thestructuresof SF,SFP,andSGM-Exo@SFPwereanalyzedbyFTIR.SFhadamideI andIIpeaksat1651.25and1537.06cm 1 representingrandom coiland α-helixstructuresrespectively.However,theabsorbance ofSFPshiftedtolowerwavenumbers(1625.46and1529.16cm 1) whichrepresentthecharacteristicsofprotein β-sheetstructure.24,25 Furthermore,weobservedaslightlyspectralshifttohigh wavelengthsinSGM-Exo@SFPgroup,implyingtheengineered exosomesweresuccessfullyloadedontothepatch(Fig. 2d).

Evaluationofbindingrate,stability,andsustainedreleaseofSGMExosinSFP

Duringtheprocessofexosomebiogenesis,Gaussialuciferaseis expressedintoexosomalmembranetomarkexosomes.Thenwe

MiR146a-loadedengineeredexosomesreleasedfromsilk ﬁbroinpatch... Lietal.

Fig.1 Generationandcharacterizationofengineeredexosomes. a Schematicdiagramrepresentingthegenerationofdesignedengineered exosomesincludingG-Exo,SGM-Exo,miR146a-Exo,andSGM-miR146a-Exo. b Schematicofdonorcells(left).SchematicofDNAconstructsused fortheproductionofSGM-miR146a-Exo(middle).Schematicofsilk fibroinpatch(right). c Representativetransmissionelectronmicroscope imagesforExosandSGM-Exos(scalebar:100nm). d ParticlesizedistributionsofExosandSGM-Exosmeasuredbydynamiclightscattering (n = 4/group). e Westernblotanalysisofexosomemarkers. f RepresentativeimagingofbioluminescencesignalsofSGM-Exosatdifferent concentrations(upper).RepresentativeimagingofbioluminescencesignalsofSGM-ExosinternalizedbyHaCaTcells(lower). g Graphshowing quantificationofSGM-Exosatdifferentconcentrations(n = 4/group, R2 = 0.9684). h GraphshowingquantificationofSGM-Exosinternalizedby HaCaTcells(n = 4/group, R2 = 0.9807)

SignalTransductionandTargetedTherapy(2023)

MiR146a-loadedengineeredexosomesreleasedfromsilk ﬁbroinpatch...

Fig.2 Thepreparationandcharacterizationofthesilk ﬁbroinpatch(SFP)servedasexosome-ladenscaffolds. a Schematicillustrationofthe preparationprocedureforSFscaffolds. b Physicalpicturesofsilk ﬁbroinsolution,SFscaffold,andSFP. c ThestructurecharacterizationsofSFP, Exo@SFP,andSGM-Exo@SFPdetectedbyscanningelectronmicroscope(upperscalebars:100 μm,lowerscalebars:1 μm).Theredarrows indicatedtheexosomesloadedontheSFP. d FTIRspectraanalysisofSF,SFP,andSGM-Exo@SFP(n = 4/group)

comparedthebindingratesofSGM-ExosandG-ExoswithSFPby measuringGlucsignals.ItwasfoundthatthebindingrateofSGMExoswithSFPwasincreasedaswellasG-Exoswiththeincreasing exosomeconcentration.Atthesameexosomeconcentration,the bindingrateofSGM-ExoswithSFPwasthreetimeshigherthan thatofG-Exos(Fig. 3a).Next,weinvestigatedthestabilityof exosomesundertwostoragetemperatures(RTand4°C)and foundthattheGlucsignalslastedforalongertimeinSGMExo@SFPgroupcomparedwiththatinSGM-Exogroupwhetherat RTor4°C,indicatingthatSFPcouldenhancethestabilityofSGMExos(Fig. 3bandc).Furthermore,thebasicexpressionofmiR146a inSGM-Exo@SFPgroupwasalsohigherthanthatinSGM-Exo group,showingtheprotectiveeffectsofSGM-Exo@SFPon miR146a(SupplementaryFig.S3).ThroughinternalizationexperimentofHaCaTcells,weconﬁrmedthatSGM-Exo@SFPcould continuouslyprovideSGM-Exoforreceptorcellsinvitro(Fig. 3d).

Finally,weobservedthesustainedreleaseofSGM-ExosfromSFP invivo.TheBLIdatarevealedthatrobustGlucsignalsofthepoints werewithin12hinbothgroups.Incomparisonwithquickly descendingsignalsinthegroupofSGM-Exo,theGlucsignalsin thegroupofSGM-Exo@SFPcouldbeacquireduntil72h(Fig. 3e). Together,theseresultsindicatedthatSFBPenhancedthebinding rateofSGM-ExosontoSFPandSFPcouldimprovesustained releaseandstabilityofSGM-Exos.

EncapsulationofmiR146aandsilencingeffects InordertoevaluatetheloadingefﬁciencyofmiR146ainSGMmiR146a-Exos,wedetectedtheexpressionofmiR146aindifferent exosomesespeciallytheengineeredexosomestransfectedwith pac-miR146a-pacand/orSGMprotein.Inadditiontoabasic expressionofmiR146ainPMSC-derivedexosomes,overexpression ofpac-miR146a-pacnotablyboostedthemiR146alevelin

MiR146a-loadedengineeredexosomesreleasedfromsilk

broinpatch... Lietal.

Fig.3 SFPenhancedthebindingrate,releasesustainability,andstabilityofSGM-Exos. a ThebindingrateofSGM-ExoswithSFPwas signiﬁcantlyhigherthanthatofG-Exos(n = 6/group). b SFPenhancedthestabilityofSGM-ExosatRT(n = 8/group). c SFPenhancedthe stabilityofSGM-Exosat4°C(n = 8/group). d InternalizationofSGM-ExosreleasedfromSFPbyHaCaTcells(n = 8/group). e RetentionofSGMExoandSGM-Exo@SFPinvivomonitoredbyGaussialuciferase(Gluc)activity(n = 6/group).Thesignalactivitywasexpressedasphotons/s/ cm2/steradian(sr).Datarepresentthemean±S.D.ofthreedifferentexperiments(ns:nosigniﬁcance,**p <0.01,***p <0.001)

miR146a-Exos.Furthermore,pac-miR146a-pacandSGMcotransfectionsigniﬁcantlyenhancedaboutten-foldsloading efﬁciencyofmiR146ainSGM-miR146a-Exosviapac/MS2element interaction(Fig. 4a).Additionally,dose-dependentrelationship betweenmiR146aexpressionlevelsandtheamountofSGMmiR146a-Exos,usedtotreatreceptorcells,wasalsoobserved (SupplementaryFig.S5).

Next,wedetectedthebiologicalfunctionofmiR146ainSGMmiR146a-Exostosilencethetargetgenes.Interleukin-1receptorassociatedkinase1(IRAK1)wasapotentialtargetofmiR146a whichidentifiedinrecentstudies,26,27 andtheTargetscan (www.targetscan.org/vert_71/)alsosupporteditbaseonseveral putativemiR146abindingsiteinthe3’-untranslatedregions.To evaluatethesilencingeffectsofmiR146ainSGM-miR146a-Exoson IRAK1expression,HaCaTcellsweretreatedwithgradient concentrationofG-Exos,miR146a-Exos,SGM-Exos,andSGMmiR146a-Exos,respectively.TheresultsdemonstratedthatSGMmiR146a-ExoshadexcellenceinhibitingeffectonIRAK1compared withSGM-ExosandmiR146a-Exosintranscriptionandprotein levelswithadose-dependentmanner.(Fig. 4bandc).Atthe concentrationof1 μg/mL,IRAK1expressionwasslightly decreasedinmiR146a-Exosgroup,butsignificantlydownregulatedinSGM-miR146a-Exosgroup(Fig. 4d,e).Thedual-luciferase reportergenesFlucandRlucwereusedtoevaluatetheinhibitory effectsofmiR146aonthetargetgenes.Here,theexosomes withoutGlucfusionproteintags(SM-ExosandSM-miR146a-Exos) wereusedinthedoubleluciferaseassaytoavoidtheinfluenceon luciferaseobservationofFlucandRluc.TheresultshowedtheSMmiR146a-Exossignificantlydownregulatedthe fluorescenceintensityofIRAK1(Fig. 4f,SupplementaryFig.S6).

IRAK1iswellknowntobeanupstreamregulatorofNF-κB inflammationsignalingpathway.Consequently,wemonitored cellularsupernatantwithELISA,andtheinflammatorycytokines interleukin-1β (IL-1β),interleukin-6(IL-6),andtumornecrosis factor-α (TNF-α)weresignificantlydecreasedwithtreatmentof SGM-miR146a-Exosasexpected(Fig. 4g).Togetherthese

observationsdemonstratedthatwesuccessfullyencapsulated miR146ainSGM-miR146a-ExoswithhighefﬁciencyandmiR146a performedfavorablebiologicalfunction.

WoundhealingpromotingeffectsofSGM-miR146-Exos@SFP Subsequently,weinvestigatedwhethertheSGM-miR146a-Exo@SFP couldacceleratediabeticwoundhealingunderinvivoconditions. TheexperimentalprocedurehasbeendisplayedinFig. 5a.FullthicknesswoundsonthebackofBKS-DB(db/db)micewerelocally treatedwithSGM-miR146a-Exos,SFP,orSGM-miR146a-Exo@SFPand untreatedwoundswereusedascontrol.Nodeathorabnormality wasobservedinanyanimalduringthepostoperativeperiod. WoundstreatedwithSFPandSGM-miR146a-Exo@SFPweremuch fasterinhemostasis.Grossobservationofdorsalwoundsindicated thatthewoundhealingprocesswassigniﬁcantlyacceleratedby localapplicationofSGM-miR146a-Exos,SFP,orSGM-miR146aExo@SFPcomparedwithcontrolgroup(Fig. 5b).Thewoundareas weremeasuredatdays0,3,7,14,and21afterwounding.Asshown inFig. 5c,d,micetreatedwithSGM-miR146a-Exos,SFP,orSGMmiR146a-Exo@SFPdisplayedgreaterwoundclosurethanobserved inthecontrolgroupatdays3,7,14,and21post-wounding.The fastesthealinginSGM-miR146a-Exos,SFP,andSGM-miR146aExo@SFPgroupsoccurredbetween7and14days.Atday21,the SGM-miR146a-Exo@SFPgroupwoundswerecompletelyhealed,but thewoundareaincontrolgroupwasstillabout40%.Furthermore, thedegreeofwoundhealinginSGM-miR146a-Exo@SFPgroupwas signiﬁcantlybetterthanthatinSGM-miR146a-ExosorSFPgroups, indicatinganadditionalpro-healingfunctionbetweenSGMmiR146a-ExoandSFP.Consistentwiththewoundhealing,the narrowestscarwidthswereobservedinSGM-miR146a-Exo@SFP groupatdays3,7,and14post-wounding(Fig. 5e,f).

Granulationtissueformation,re-epithelialization,andcollagen depositionpromotedbySGM-miR146-Exo@SFP Histologicalstainingwasperformedtoobservegranulationtissue formationandre-epithelializationinrepairedtissues.Figure 6a

Fig.4 ExpressiondetectionofmiR146ainengineeredexosomesandevaluationofmiR146asilencingeffects. a ExpressionofmiR146ain engineeredexosomesassessedbyquantitativeRT-PCR(n = 6/group). b, c WesternblotanalysisofIRAK1expressionlevelsinHaCaTcells treatedwithgradientconcentrationsofengineeredexosomes. d, e Westernblotanalysisoftheinhibitoryeffectof1 μg/mLengineered exosomesonIRAK1expression. f Dual-luciferasereporterassaysofIRAK1expressionineachgroup. g ELISAanalysisofinﬂammatoryfactors IL-1β,IL-6,andTNF-α.(n = 6/group).Datarepresentthemean±S.D.ofthreedifferentexperiments(*p <0.05,***p <0.001)

showedrepresentativeH&Eimagesofthewoundstreatedaswell astheabovemethodsatday14andday21.Theformationof granulationtissueandepidermaltissuewasassessedbymeasuringthethicknessofgranulationtissueandepidermaltissue.The resultsshowedthatSGM-miR146a-Exo@SFPtreatedwounds displayedasigniﬁcantincreaseinthegranulationtissuethickness atday14incontrastwithcontrol,SGM-miR146a-Exos,andSFP groups.Butatday21,thegranulationtissuethicknessinSGMmiR146a-Exo@SFPgroupdecreasedcomparedwithday14,which wasalmostequivalenttothelevelofSGM-miR146a-ExosandSFP groups(Fig. 6aandb).

Rapidre-epithelializationisoneofthemostimportantstepsin woundhealing.Regardingthethicknessofneo-epidermis,atday 14,itwasthickerinSGM-miR146a-Exos,SFP,andSGM-miR146aExo@SFPgroups(89.3±17.25 μm,121.15±19.35 μm, 176.2±14.65 μm,respectively)thaninthecontrolgroup (10.54±9.75 μm),indicatinganacceleratedre-epithelialization progression.Atday21,thenewepitheliumoftheSGM-miR146aExo@SFPgroupexhibitedwell-arrangedandtherewereregularly distributedkeratinocytesthroughouttheepithelium,achieving there-epithelialization.Bycontrast,thetheneo-epidermis morphologyofthecontrolgroupwasirregularlyarrangedand hyperplastic(Fig. 6aandc).Moreencouragingly,matureskin structuressuchashairfolliclesandsebaceousglandswere observedinSGM-miR146a-Exo@SFPtreatedwoundsatday21 (Fig. 6a).

ToobservetheeffectofSGM-miR146a-Exo@SFPoncollagen depositionandremodeling,Masson’strichromestainingwasused tostaincollagen ﬁbers.AsshowninSupplementaryFig.S7,the increasedcollagendepositionwasobservedandthecollagen ﬁberswerealreadynormallyarrangedintheSGM-miR146a-

Exo@SFPgroupatday14.Atday21,theincreasedcollagen depositionandtypicalbundle-shapedcollagen fiberswerestill observedinSGM-miR146a-Exo,SFPandSGM-miR146a-Exo@SFP groups,whereasthecontrolgroupspresentedachaoticoutlook withinflammatorycellinfiltration.

Together,theseresultsindicatedthatSGM-miR146a-Exo@SFP enhancedgranulationtissueformation,re-epithelializationprocess,collagendeposition,andeventheregenerationofhair folliclesandsebaceousglands.

Anti-inﬂammationandangiogenesisrolesofSGM-miR146aExo@SFP

Theinflammationphaseisacriticalprocessofthewoundhealing, whiletheprolongedorexaggeratedinflammatoryresponsehas beensuggestedtobethepathogenicmechanismforchronic wounds.Next,wefurtherconfirmedtheanti-inflammationroleof SGM-miR146a-Exo@SFPinwoundtreatment.IRAK1andIL-6were chosenastheindicatorsforevaluatinginflammatoryactivityand anti-inflammatoryefficacy.Consistentwiththesilencingeffectsof miR146ainSGM-miR146a-ExosonIRAK1expressioninvitro,IRAK1 expressioninvivowasobviouslyinhibitedinSGM-miR146a-Exoor SGM-miR146a-Exo@SFPtreatedwounds(Fig. 7a,d).IL-6,as downstreamgeneregulatedbyIRAK1,isoneofrepresentative pro-inflammatorycytokines.28,29 Asexpected,IL-6expressionin thewoundswaseffectivelyattenuatedinSGM-miR146a-Exo@SFP groupcomparedwithcontrol,SGM-miR146a-Exos,andSFP groups.Furthermore,SGM-miR146a-Exogroupalsohada decreasedexpressionofIL-6incontrastwithcontrolgroup(Fig. 7b,e).Theanti-inflammatoryeffectwasverifiedattheprotein level.TheresultsshowedthatIRAK1andIL-6expressionswere apparentlydecreasedinSGM-miR146a-Exo@SFPgroup(Fig. 7g,h).

MiR146a-loadedengineeredexosomesreleasedfromsilk ﬁbroinpatch... Lietal.

Fig.5 SGM-miR146a-Exo@SFPacceleratedwoundclosureindiabeticmice. a Schematicillustrationofwoundtreatmentprocess. b Grossview ofwoundsatdays0,3,7,14,and21.Thewhitecirclesrepresenttheoriginalwoundareas. c Statisticalanalysisoftheresidualwoundareain eachgroup(n = 6/group). d Statisticalanalysisofwoundclosurearea(n = 6/group). e RepresentativeimagesofscarwidthsstainedbyH&E. f Statisticalanalysisofthescarwidthsineachgroup(n = 6/group).Datarepresentthemean±S.D.(ns:nosigniﬁcance,**p <0.01,***p <0.001)

Duringwoundhealing,newbloodvesselformationiscrucialfor transportoftrophicfactorstowoundsites.Wenextinvestigated whethertreatmentwithSGM-miR146a-Exo@SFPcouldpromote angiogenesisinwoundsites,therebyenhancingdiabeticwound healing.AsshowninFig. 7c,f,isolectinB4(IB4),aspeciﬁcmarker ofangiogenesis,30,31 showedincreasedexpressioninSGMmiR146a-Exos,SFP,andSGM-miR146a-Exo@SFPgroups,indicating enhancedneovascularizationcomparedwithcontrolgroup. Besides,morematurevesselsmarkedwithIB4wereobservedin SGM-miR146a-Exo@SFPgroup.CD31andVEGFexpressions detectedbywesternblottingwerealsoincreasedinSGM-

miR146a-Exo@SFPgroup(Fig. 7i,j).Furthermore,thenewblood vesselsaroundthewoundswerenotablyobservedinSGMmiR146a-Exo@SFPgrouponday14bythestereomicroscope (SupplementaryFig.S8).Collectively,these ﬁndingssuggested thatSGM-miR146a-Exo@SFPcouldpromoteangiogenesisand inhibitinﬂammatoryresponseatwoundsitesindiabeticmice. MechanisminvestigationofwoundhealingpromotedbySGMmiR146-Exos@SFP Togetinsightintothemechanismofthewoundhealingprocess, weperformedtranscriptomicsanalysisofwoundstreatedwithor

Fig.6 Granulationtissueformationandre-epithelializationpromotedbySGM-miR146-Exo@SFP. a Representativeimagesofskintissue sectionfromthewoundsonday14andday21stainedbyH&E(left,scalebar:2mm).High-magni ﬁcationimagesshowedgranulationtissue formationandepithelialtissueclosure(right,scalebar:200 μm).HairfolliclesandsebaceousglandscouldbeobservedinSGM-miR146aExo@SFP-treatedwoundsatday21(bottomright).Theextentofepidermisandgranulationtissueweremarkedwithgreenarrowsandred arrowsrespectively. b, c Statisticalanalysisofgranulationtissuethicknessandepidermalthicknessineachgroup(n = 6/group).Data representthemean±S.D.(*p <0.05,**p <0.01,***p <0.001)

withoutSGM-miR146a-Exo@SFPatday3andday7post woundingusinghigh-throughputmRNAsequencing(SupplementaryFig.S9).Thedifferentiallyexpressedgenes(DEGs)were deﬁnedashavingaminimumoftwo-foldchangeinexpression.

Figure 8ashowedthattheexpressionprofilesofSGM-miR146aExo@SFPgroupweredistinctfromtheprofilesofcontrol.We found482and133geneswithsignificantlydecreasedtranscript levelsinSGM-miR146a-Exo@SFPtreatedwoundscomparedtothe woundsincontrolgroupatday3and7respectively.Therewere 281genesthatexhibitedsignificantlyincreasedtranscriptlevelsin SGM-miR146a-Exo@SFPtreatedwoundsatday3and739atday7 (Fig. 8b).TheseresultsdemonstratethatdownregulatedDEGs predominatedinSGM-miR146a-Exo@SFPgroupatday3probably duetomiR146asilencingeffectsandupregulatedDEGspredominatedatday7probablyduetotheactivationofrepaircells inSGM-miR146a-Exo@SFPtreatedwounds.Top20DEGsassociatedwithinflammationandre-epithelializationdemonstrating thesignificantincreaseordecreaseintranscriptlevelsfromeach timepointwererespectivelypresentedinTablesS18andS19. AmongtheDEGs,wenotedthattheinflammation-relatedgenes, includingIL-1b,IL-1r2,IL-11,IL-19,IL-23a,Fasl,TNF,NF-κB,Tlr4, Tlr6,CXCR2,andCXCR5,weresignificantlyreducedinSGMmiR146a-Exo@SFPgroupatday3(SupplementaryTableS17).At day7,weobservedthatthegenesrelatedtothedevelopment anddifferentiationofepidermis,includingSprr1a,Sprr1b,Sprr2a1,

Sprr2a2,Sprr2d,Sprr2e,Sprr2f,Sprr2g,Sprr2h,Sprr2i,Krt10,Krt6a, Cnfn,Pou3f1,Trp63,Egf,Epgn,andEreg,weregreatlyincreasedin SGM-miR146a-Exo@SFPtreatedwounds(SupplementaryTable S18).ThroughGeneOntology(GO)enrichmentanalysisofDEGs, wefoundthatthedownregulatedDEGsinSGM-miR146a-Exo@SFP groupatday3weredistinctlyenrichedin “IFNresponse” , “immuneresponse” , “inflammatoryresponse”,and “innateimmunity” (Fig. 8c)andupregulatedDEGsatday7wereenrichedin epidermaldevelopmentandkeratinocytesdifferentiation(Fig. 8d). ThestatisticsofKyotoEncyclopediaofGenesandGenomes (KEGG)pathwayenrichmentanalysisofDEGsindicatedthatSGMmiR146a-Exo@SFPdownregulatedinterleukin,TNF,NF-κBsignalingpathwaysatday3(Fig. 8e)andupregulatedinsulinpathway andcelladhesionmolecule(CAM)pathwaywhichcouldbe involvedincelldevelopmentanddifferentiation32 atday7(Fig. 8f).Theconfirmationexperimentoftheinflammationsignaling pathwayswasperformedontheexpressionsofp-NFκB-p65,NFκBp65,p-IκBα,andIκBα.TheresultsindicatedtheexpressionsofpNFκB-p65wereobviouslydecreasedandtheactivationofNFκBp65wereinhibitedinSGM-miR146a-Exo@SFPgroup.Theexpressionlevelsofp-IκBα weredownregulatedandIκBα werereactively upregulatedinSGM-miR146a-Exo,SFP,andSGM-miR146aExo@SFPgroupcomparedwithcontrol.(Fig. 8g).Finally,we performedtheassociationanalysisof52DEGsinvolvedin inflammatoryreactionandcellularproliferationanddifferentiation

MiR146a-loadedengineeredexosomesreleasedfromsilk ﬁbroinpatch... Lietal.

Fig.7 SGM-miR146a-Exo@SFPattenuatedinflammationandincreasedneovascularizationofdiabeticwounds. a–c Immunofluorescenceof IRAK1,IL-6,andIB4ofthewoundtissue(scalebar:200 μm). d–f StatisticalanalysesofIRAK1,IL-6,andIB4immunofluorescence. g TheIRAK1 expressionwasdecreasedinSGM-miR146a-Exo@SFPgroup. h TheIL-6expressionwasdecreasedinSGM-miR146a-Exo@SFPgroup. i The increasedCD31expressioninSGM-miR146a-Exo@SFPgroup. j TheincreasedVEGFexpressioninSGM-miR146a-Exo@SFPgroup.Data representthemean±S.D.ofthreedifferentexperiments(ns:nosignificance,*p <0.05,**p <0.01,***p <0.001)

MiR146a-loadedengineeredexosomesreleasedfromsilk

Fig.8 Transcriptomicsanalysisofthewoundtissueandconfirmationexperimentoftheinflammationsignalingpathways. a Volcanoplotof differentiallyexpressedgenes(DEGs)atday3andday7. b Venndiagramoftwooverlappingcirclesrepresentingtheoverlappeddownand upgenesatday3andday7. c GeneOntology(GO)enrichmentanalysisofDEGs. d DEGsatday7wereenrichedinskindevelopmentand differentiation. e KEGGpathwayenrichmentanalysisofDEGs. f SGM-miR146a-Exo@SFPupregulatedinsulinpathwayandcelladhesion molecule(CAM)pathwayatday7. g Theexpressionsofp-NFκB-p65,NFκB-p65,p-IκBα,andIκBα inwoundtissue.Datarepresentthemean± S.D.ofthreedifferentexperiments(ns,nosignificance,*p <0.05,**p <0.01,***p <0.001)

duringwoundhealing.SupplementaryFig.S10revealedthe interactionrelationshipamongtheseDEGs,withIL-1b,IL-1r2,NFκB,Tlr4,andTlr6themostcommon,whichcouldbeusedasthe potentialtherapeutictargetstopromotewoundhealing.

DISCUSSION

EVsorexosomes,asnaturalnano-scaledrugcarriers,providenovel promisingdeliverysystemsforthetherapyofdiseasesbyloading effectiveagents.Recently,variousbiomaterialsareusedtoextend theretentiontimeofexosomesandcontrolthereleaseof therapeuticagentsinvivo,whichareexpectedtobecomea practicallypowerfultoolforchronicwoundtreatment.33 Inthe treatmentstrategy,theloadingefficiencyofpro-healingagentsinto exosomesandexosomesontobiomaterialsiscrucial.Inthisstudy, wesucceededinengineeringtheexosomesforefficientmiR146a loadingandforthespecialattachmenttothesurfaceofSFPby constructingafusionproteininwhichtheexosomalmembrane proteinLactadherinwasfusedwithRNAbindingproteinandSFBP. Invitro,SGM-miR146a-Exosexhibitedexcellentinhibitingeffecton NF-κBsignalingpathwaybymiR146atargetingIRAK1,thereby downregulatingtheexpressionsofalargenumberofinflammatory cytokines.Invivo,SGM-miR146a-Exo@SFPsignificantlyenhanced thewoundrepairassociatedwithanti-inflammatory,collagen deposition,andneovascularization.SupplementaryFig.S11showed theschematicillustrationofSGM-miR146a-ExosreleasedfromSFP promotingdiabeticwoundhealing.

MiRNAs,aspossibletherapeuticagents,needaneffective deliverysystemtotransferintotargetcells.MS2isasinglestrandedRNAbacteriophageandtheRNAiscomposedof3569 nucleotidesencodingfourgenesincludingthematuration protein,thecapsidprotein,thelysisprotein,andthereplicase protein.TheRNAgenomepackagebycapsidproteinstartsfrom thebindingbetweenthecapsidproteindimerandaspecific19nucleotidestem-loopregion(pacsite).Recently,thebinding specificityofMS2capsidproteinforRNArecognitionhavebeen appliedfordevelopingvirus-likeparticlesfordrugdelivery.Inour experiments,wefusedLactadherin(amembrane-associated proteinenrichedinexosomes)withMS2capsidproteinattheC terminaltorecognizethedesignedmiR146awhichhavetwopac sitesat flanks.Duringtheprocessofexosomebiogenesis,the fusedLactadherin-MS2successfullyenrichedmiR146aintoexosomeswhenmiR146awasexcessivelyexpressedinPMSCs. ComparedtomiR146a-ExoswithoutthehelpofMS2pac/capsid proteinelementinteraction,aten-foldincreasedloadingefficiencyofmiR146awasobservedinMS2-miR146a-Exos.

EventhoughtherearesomanyresearchesabouteffectsofEVs onwoundtreatment,theadministrationandretentionofEVsin woundsstillfacechallenges.Applicationofbiomaterialsprovides sustainablereleaseandevendistributionofEVsonwound surface.34,35 Silkisanaturalhighpolymer fiberprotein,containing 70–80%silk fibroinwithgoodmechanical,physical,andchemical properties.Inourexperiment,SFPwasservedasexosome-laden scaffoldsafterevaluationofbiologicalsafetyinvivo.Toenhance theloadingefficiencyofexosomesontoSFP,weinsertedSFBP betweenSPandC1C2domainsofexosomalmembraneprotein Lactadherin.WiththehelpofSFBPpresentedontheexosomal membrane,theSF-affinityexosomesdistributedmoredenselyon SFPcomparedwiththenaturalexosomeswithoutthecapacityof SF-affinity.Additionally,wealsofusedLactadherinwithGluc followingSFBPasagoodagentforexosomelabeling.

Chronicdiabeticwoundsoccurfrequentlyindiabeticpatients suchasdiabeticfootanddiabeticulcer.Persistentinflammation andvasculardysfunctionarethemaincausesofdiabeticwound unhealing,sosuppressinginflammationandpromotingangiogenesiscanhelptackletherootofchronicwoundhealing problem.36,37 Inthisresearch,miRNA-146aasananti-inflammatory regulatorwasencapsulatedinexosomesandthentheexosomes

MiR146a-loadedengineeredexosomesreleasedfromsilk ﬁbroinpatch... Lietal.

wereloadedontoSFP.WithaboveefficientmiRNAdelivery system,wecreatedanewwounddressingnamedSGM-miR146aExo@SFP.InvivoexperimentsshowedthatSGM-miR146Exos@SFPaccelerateddiabeticwoundhealingbyinhibiting inflammatoryresponseandpromotingangiogenesiscompanied withenhancedgranulationtissueformation,re-epithelialization, andcollagendeposition.Consistentwithourresults,Zengetal.38 andLietal.39 alsoreportedthefunctionofmiR146torepress inflammatoryresponseinacutelunginjuryandinduceangiogenesisinosteoarthritiscartilage.

WenextinvestigatedthemechanismresponsiblefortheantiinflammatoryandangiogenesisfunctionofmiR146duringwound healing.MiR146ahasbeenpredictedtotargetTNFreceptorrelatedfactor6(TRAF6),IRAK1,andSmad4.Researchersfound thatmiR146ainhibitedtheexpressionofTRAF6andIRAK1and blockedtheactivationofTLRs/NF-κBsignalingpathway,thereby reducingthereleaseofpulmonaryinflammatoryfactors.38 Inour experiment,miR146ainhibitedtheexpressionofIRAK1,TNF-α,IL1β,andIL-6invitroandtheexpressionofIRAK1,IL-6,andp-NFκBp65invivo.Intermsofangiogenesisfunction,wefoundthat miR146enhancedtheexpressionofIB4,CD31,andVEGF. Consistentwithourresults,Lietal.39 reportedthatmiR146a upregulatedVEGFexpressionthroughtargetedinhibitionof Smad4,contributingtothepathogenesisofosteoarthritis. Collectively,wegeneratedengineeredexosomesSGMmiR146a-ExobyfusingLactadherinwithSFBPbetweenSPand C1C2domainstoattachthesurfaceofSFP,withGlucfollowing SFBPtomonitorexosomesinreal-time,andwithMS2attheC terminaltoloaddesignedmiR146awhichhavetwopacsitesat flanks.SGM-miR146a-Exo@SFPcreatedasanewwounddressing wasproventoinhibitinflammationandpromoteangiogenesis, therebyacceleratingwoundhealing.Consequently,thisstudynot onlyprovidesanefficientapproachtoenrichmiRNAsinto exosomes,butalsodevelopsapracticalexosome@biomaterialbaseddrugdeliveryplatformformiRNAstotreatdiabeticwounds.

MATERIALSANDMETHODS

Ethicsstatement

Theanimalexperimentswereapprovedbythemedicalethics committeeofChinesePLAGeneralHospital.

PhagedisplayforSFBP

TheSFBPwasselectedthroughscreeningthe12-phagepeptide libraryandvalidationbyELISA.ThebiopanningofSFBPschematic wasshowninSupplementaryFig.S1.Thedetailedscreeningand preparationmethodsofSFBPwereprovidedintheSupplementary Material.

Preparationandidentiﬁcationofexosomes

ExosomesderivedfromPMSC(Exo),G-PMSC(G-Exo),SGM-PMSC (SGM-Exo),miR146a-PMSC(miR146a-Exo),andSGM-miR146aPMSC(SGM-miR146a-Exo)werepreparedasthemethods describedintheSupplementaryMaterial.ExoandG-Exowere usedascontrol.AlltheexosomesweresummarizedinaTable (SupplementaryTableS17).Exosomemorphologieswere observedusingtransmissionelectronmicroscopy(TEM).Thesize distributionofexosomeswasmeasuredbyusingNanosizer technology(MalvernInstruments,Malvern,U.K.).Expressionof theexosomalcharacteristicmarkersCD9(1:500),CD63(1:1,000), TSG101(1:500),andCalnexin(1:500)(CST,MA,USA)wereanalyzed byWesternblotting.

PreparationofSFPencapsulatingengineeredexosomes

Thesilk ﬁbroinprotein-basedporousmaterialswerepreparedas themethodsintheSupplementaryMaterial.Thesilk ﬁbroin proteinporousmaterialswerepreparedintopatchesofthe woundsize.100 μgofexosomesolution(50wt%)wasaddedto

theSFP,followedbyincubatingat37°Cfor10minforexosome attachingtotheSFP.Theuncombinedexosomeswerewashed awayfromSFPwithdeionizedwater.Finally,SGM-ExoloadedSFP (SGM-Exos@SFP)wasobtainedthroughtheaboveprocess.The controlSFPwasinﬁltratedwithPBSfordetection.

ReleaseandinternalizationofGluc-labeledexosomes

ThetotalproteinofexosomeswasmonitoredbyGaussia luciferase(Gluc)activityusinganexposureapparatus.Therelease ratioofexosomeswastestedinthepresenceoftheGlucsubstrate water-solublecoelenterazine(GeneCopoeia).Brieﬂy,theGluclabeledexosomes(G-Exos)isolatedfromPMSCs,whichtransducedwithGlucfusionprotein,wereusedasthecontrolexosome inbindingrateanalysis.100 μgexosomeswereaddedinapatch of10-mmdiameter,followingcombinationwitheachotherin48wellplateat37°Cfor10min.Then,adding200 μLPBSineachwell tosubmergetheSFP.Afterincubatingina37°Cincubatorfor differentialtime,thesupernatantPBSwascollectedandmovedto another48wellsplateforBLIanalysis.Theamountsofleaching exosomesweremeasuredviaGlucsignalsaccordingtothetrend oflineardependentrelationshipbetweenthequantityof exosomesandGlucactivity.TheinternalizationofG-Exoswas analyzedinHaCaTcells.HaCaTcellswereseededat5×104 cells perwellina24-wellplateonedaypriortoaddingSGM-Exos (100 μg/mL)andSGM-Exo@SFP,thenincubatedfor1,3,6,9,12, 18,24,and36h.AfterwashingbyPBStwice,theinternalizationof exosomeswasanalyzedbyluminescenceimagingsystem.

BLIanalysis

TheretentionofSGM-ExoandSGM-Exo@SFPweremonitoredby Gaussialuciferase(Gluc)activity.Inthevivoexperiments,100 μg SGM-Exosinavolumeof100 μLwereloadedinSFP,andthenthe SGM-Exo@SFPwassubcutaneouslyimplantedinthebackofnude mice.100 μgSGM-ExosolutionwithoutSFPwasusedascontrol. Attheindicatedtimepoints(0,12,24,36,48,and72h),watersolublecoelenterazine(5mg/kg,NanolightTechnology)was injectedsubcutaneouslyandtheanimalswereimagedimmediatelyusingIVISLuminaImagingSystem.ThesignalactivityofBLI wasexpressedasphotons/s/cm2/steradian(sr).

Dual-luciferasereporterassay

Renillaluciferase(Rluc)wasusedasaninternalcontrol.The ﬁreﬂy luciferin(Fluc)wasusedasthesubstratetodetecttheexpression ofIRAK1.Luciferasecatalyzedtheoxidationofluciferinto oxyluciferinwiththeparticipationofMg2+ andO2.Inthe oxidationprocessofluciferin,bioluminomescenceoccurred.Then, insubsequentexperiments,thebioluminescenceofFlucwas terminatedbytheadditionofcoelenterazine,thesubstrateofRluc thatinhibitedtheluciferasecatalysisofFluc.Bioluminescence occurredthroughthecatalyticoxidationofluciferasetocoelenteramide.After2min,thebioluminescencewasmeasuredbya chemiluminescenceinstrument.TheBLIsignalswereallmeasured byaverageradiancefromregionsofinterest(ROI).IntheBLI experimentsperformedinplates,theROIsweremergedwiththe wellsineachgroup.

Diabeticwoundmodeltreatmentandanalysis

Thedetailedmethodsofdiabeticwoundmodeltreatmentand analysiswereprovidedintheSupplementaryMaterialsand Methods.

DATAAVAILABILITY

Statisticalanalysis

Allresultspresentedarefromatleastthreeindependent experimentsforeachcondition.Dataareexpressedasmean± standarddeviation(S.D.).One-wayortwo-wayanalysisofvariance wasusedtodeterminethelevelofsigniﬁcanceusingGraphPad Prism9.2software.Differenceswereconsideredstatistically signiﬁcantat p <0.05.

TheRNA-seqdatageneratedduringthisstudyhavebeendepositedintheNCBI’s GeneExpressionOmnibus(GSE217981).Otherdatathatsupportthe ﬁndingsofthis studyareavailablefromthecorrespondingauthoruponreasonablerequest.

ACKNOWLEDGEMENTS

ThisworkwassupportedbytheNationalNatureScienceFoundationofChina (81901971,82172211,81830064,82172231),NaturalScienceFoundationofBeijing Municipal(7194316,7202197),NationalKeyResearchandDevelopmentProgramsof China(2022YFA1104303),theCAMSInnovationFundforMedicalSciences(CIFMS, 2019-I2M-5-059),theMilitaryMedicalResearchandDevelopmentProjects (AWS17J005,2019-126)andMilitaryMedicalScienceandTechnologyYouthTraining Program(21QNPY128).

AUTHORCONTRIBUTIONS

C.Z.,X.F.,andH.L.designedtheprojectsandsupervisedthisstudy.Q.L.andW.H. performedmostoftheexperimentsandanalyzedthedata.Q.H.,J.Y.,B.L.,K.M.,Q.W., Y.W.,J.S.,M.S.,andS.C.participatedintheexperimentalworkanddataanalysis.Q.L. andW.H.draftedthemanuscript.R.Y.andC.Z.editedthemanuscript.Allauthors havereadandapprovedthearticle.

ADDITIONALINFORMATION

Supplementaryinformation Theonlineversioncontainssupplementarymaterial availableat https://doi.org/10.1038/s41392-022-01263-w

Competinginterests: Theauthorsdeclarenocompetinginterests.

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