s41427-022-00419-y by Biogenomics

Microenvironmentalcue-regulatedexosomesas therapeuticstrategiesforimprovingchronic woundhealing

WeiDai1,YuchenDong1,TingHan2,JingWang2,BinGao3,HuiGuo4,FengXu 2,JingLi 1 andYufeiMa 2

Abstract

Exosomes,whicharenanoscalevesiclessecretedbyalmostallcelltypesviaparacrinepathways,playacrucialrolein intercellularcommunication.Fornearlyadecade,exosomeshavebeenwidelyexploitedtodevelopnovelbiologic therapeuticstrategiesforrecalcitrantclinicalproblemssuchaschronicwounds.Duetotheexcellentbiocompatibility andimmunestabilityofexosomes,exosome-basedtherapyhasshowngreaterpotentialforchronicwoundhealing thanotherbiotherapies(e.g.,transplantationofstemcells)andholdsgreatpromisefortheclinicaltreatmentof chronicwounds.Here,recentadvancesinregulatingthefeaturesandbiologicalfunctionsofcell-derivedexosomesby biochemicalandbiophysicalcuesinthecellmicroenvironmentaresystematicallysummarized.Subsequently, microenvironmentalcue-regulatedexosomesastherapeuticstrategiestoimprovechronicwoundhealingby regulatingtheinﬂammatoryresponse,promotingcellproliferationandmigration,facilitatingangiogenesis,and regulatingextracellularmatrix(ECM)remodelingarediscussed,andhydrogel-basedexosomedeliverysystemsusedin thetreatmentofchronicwoundsarehighlighted.Finally,ongoingchallengesandfutureopportunitiesinthisrapidly developing ﬁeldareproposed.

Introduction

Chronicwounds,suchasdiabeticfootulcers(DFUs), pressureulcers(PUs),andvenouslegulcers(VLUs),are skintissuedefectsthatfailtoproceedtothenormal woundhealingcascadeandpassthroughaninflammatory phase,resultinginchronicinflammationthatperpetuates thedegreeoftissuedamage.Clinically,woundsduetoany causethatfailtohealorshownohealingtendencyafter treatmentformorethanamonthareconsideredchronic wounds,whichareprevalentanddifficulttotreatamong elderlyindividuals.Chronicwoundsarecloselyrelatedto hightreatmentcostsandbecomealargeburdenforboth

Correspondence:JingLi(lijing02@fmmu.edu.cn)or YufeiMa(mayufei@xjtu.edu.cn)

1DepartmentofBurnsandPlasticSurgery,SecondAfﬁliatedHospitalofAir ForceMilitaryMedicalUniversity,Xi’an710038,P.R.China 2BioinspiredEngineeringandBiomechanicsCenter(BEBC),TheKeyLaboratory ofBiomedicalInformationEngineeringoftheMinistryofEducation,Xi’ an JiaotongUniversity,Xi’an710049,P.R.China

Fulllistofauthorinformationisavailableattheendofthearticle

©TheAuthor(s)2022

individualsandtheentirehealthcaresystem1.Ithasbeen estimatedthatindevelopedcountries,theprevalenceof chronicwoundsrangesfrom1.67per1000populationto 2.21per1000population2,andthetherapycostsare considerableandestimatedat~1–3%ofthetotalhealth careexpenditure3.Currenttherapeuticoptionsfor chronicwoundsincluderoutinedressingchanges,debridement,infectionmanagement,skintissuetransplantation,andnegativepressurewoundtherapy(NPWT)4, whichaimtorestoreskinintegrityratherthantarget woundhealingfromtheperspectiveofpathophysiological mechanisms.Althoughefﬁcient,thesestrategiesare associatedwithseverallimitations,suchascomplications, woundrecurrence,andvariabilityintheirhealingeffects5. Therefore,agreatneedremainsforadvancedtherapeutic optionsforchronicwoundhealing.

Itisnowwellknownthatthepathologicalcharacteristicsofchronicwoundsincludeprolongedexposureto proinﬂammatorycytokines,imbalancedexpressionof

OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionand reproduction inanymediumorformat,aslongasyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense, andindicateif changesweremade.Theimagesorotherthirdpartymaterialinthisarticleareincludedinthearticle’sCreativeCommonslicense,unlessindicated otherwiseinacreditlinetothematerial.If materialisnotincludedinthearticle’sCreativeCommonslicenseandyourintendeduseisnotpermittedbystatutoryregulationorexceedsthepermitteduse,youwillneedtoobtain permissiondirectlyfromthecopyrightholder.Toviewacopyofthislicense,visit http://creativecommons.org/licenses/by/4.0/

Fig.1Schematicillustrationofcell-derivedexosomesandtheirtherapeuticeffectsonchronicwounds. Theleftsideofthe figureshows sometypicalpathologicalcharacteristicsofchronicwounds,includingthepresenceofmanyinflammatorycells(e.g.,neutrophilsandM1 macrophages),overexpressionofinflammatoryfactors(e.g.,IL-1andTNF-α),insufficientexpressionofgrowthfactors(e.g.,VEGF,FGF,andTGF-β), overexpressionofMMPsandpoorangiogenesis.Therightsideshowstherolesofexosomesinchronicwoundhealingthrough,forinstance,M1to M2phenotypicswitchingofmacrophages,regulationofinflammation,promotionofcellproliferationandangiogenesis,andenhancedcollagen formation.

proteasesandtheirinhibitors,alteredavailabilityand activityofgrowthfactors,tissuehypoxia,andhighoxidativestresslevels6.Basedonsuchin-depthunderstanding,variousadvancedbiotherapieshavebeen recentlydeveloped,suchastheuseofacellulardermal matrix7,growthfactors8 orautologousplatelet-rich plasma(PRP)9,andtransplantationofstemcells10. Amongthesestrategies,stemcelltherapybasedon mesenchymalstemcells(MSCs)hasbeenthemostpromisingbecauseMSCssynthesizeasubstantialamountof growthfactorsandcytokines,whichpromotecellmigration,proliferation,andvascularization,playinganactive roleintissueregeneration11.However,consideringthe riskofuncontrolledproliferationandgraft-versus-host disease(GVHD)associatedwithMSCtransplantation,the directapplicationofMSC-basedtherapyisnotfeasible12. Recently,thetherapeuticeffectofMSCsinfacilitating tissuerepairwasfoundtooccurmainlythroughparacrine pathways.AsanessentialparacrinefactorofMSCs, exosomeshavebeendemonstratedtofacilitatetissue regenerationintissuesincludingthemyocardium13,

bone14,andskin15.Exosomes,whicharenanoscalevesiclessecretedbycellsintotheextracellularenvironment, playacrucialroleinintercellularcommunication16.The applicationofexosomesavoidstheriskofstemcell overproliferation.Inaddition,exosomesarenaturalproductsofthebody,leadingtoalowimmuneresponse17. Becausetheirsurfaceshaveintrinsicbiochemicalcharacteristicssimilartothoseofcells,exosomescanavoid phagocytosisandbypasslysosomalphagocytosis18. Therefore,exosome-basedtherapymayholdgreat potentialasaclinicalsolutionforchronicwoundhealing. Thetherapeuticeffectofexosomesisexertedwhenthey areinternalizedor,insomecases,attachedtothecell surface,andthiseffectusuallydependsonwhatthey carry,whichcanincludeDNA,proteins,mRNAs,lipids, andmiRNAs19.Bytransportingthesebioactivecomponents,exosomesparticipateinalmostallstepsofthe woundrepairprocess,includingregulatinginﬂammatory responses,promotingcellproliferationandmigration, boostingangiogenesis,andregulatingextracellularmatrix (ECM)remodeling(Fig. 1).Thecontentsofexosomes

mayvarybasedonthephysiologicalandpathological statesoftheoriginalcell20,21,andexosomesmayinherit theadaptivechangesofparentalcellsinresponseto biochemicalandbiophysicalcuesinthecellmicroenvironment,suchasECMmechanicsandtheoxygen level22,23.Therefore,itispossibletoregulatethebiologicalfunctionsofexosomesbychangingthecellmicroenvironmentandtothusimprovetheirtherapeuticeffects inchronicwoundhealing.

Manyoriginalarticlesandreviewshavediscussedthe applicationsofexosomesinwoundrepair,andmosthave focusedonthebiogenesisofexosomesandMSC-derived exosomesfortissueengineering24–26.Inaddition,some recentarticleshavediscussedexosome-loadedbiomaterials,suchashydrogelsandscaffolds,andtheirapplications inwoundhealing27–29.However,howbiochemicaland biophysicalcuesinthecellmicroenvironmentmediate thefeaturesandbiologicalfunctionsofcell-derivedexosomesandhowtheseexosomesexerttheirtherapeutic effectsoneachcrucialprocessduringwoundhealingare notyetclear.Inthisreview,wefullyprobethedevelopmentofmicroenvironmentalcue-regulatedexosomesas therapeuticstrategiestoimprovechronicwoundhealing. We firstsystematicallysummarizetheregulatoryeffects ofbiochemicalandbiophysicalcuesinthecellmicroenvironmentonthefeaturesandbiologicalfunctionsof exosomes.Then,cell-derivedexosomesarediscussedas therapeuticstrategiesforimprovingchronicwound healingthroughvariousmechanisms.Subsequently,we highlighthydrogel-baseddeliverysystemsformaintaining thebioactivityofexosomesandoptimizingexosome release.Finally,weconcludewithsomethoughtson relatedchallengesandfutureperspectives.Thisreview aimstoinspireandencourageresearchtofurther understandthis flourishing field,toadvancethedevelopmentofmicroenvironmentalcue-regulatedexosomes astherapeuticstrategiesinthe fieldofregenerative medicine,toacquireidealexosomedeliverysystemsfor efficientdeliveryandtoboostpotentialclinicalapplicationsofthesecell-derivedexosomes.

Regulationofthefeaturesandbiological functionsofcell-derivedexosomesby microenvironmentalcues

Cell-derivedexosomesareextracellularvesicles(EVs) withdiametersintherangeof40–160nm.Exosomesare importantmediatorsofintercellularcommunication throughtheparacrinepathway.Thetransportofexosomalcontents,forinstance,nucleicacidsandproteins,into recipientcellseffectivelychangesthebiologicalresponses oftherecipientcells30.Becauseexosomescanregulate complexintracellularpathways,theyshowgreatpotential inthetreatmentofmanydiseases,includingcentralnervoussystem-relateddiseases,cardiovasculardiseasesand

cancerprogression16.Indetail,designstrategiesforexosomeshavebeenrapidlydevelopedandwidelyappliedin thetreatmentoftumorsandneurologicaldiseases.Most ofthestrategiesaretoincreasethedurationofexosomes inthecirculationandtooptimizethetargetingofexosomesbymodifyingthesurfaceproteins19.However,in chronicwoundrepair,distanttargetedreleaseofexosomesisnotnecessary.Differentfromthestrategies mentionedabove,preconditioningormodifyingtheorigin cellstooverexpresssomecontentsofexosomestooptimizethespeciﬁcbiologicalfunctionsofexosomesinthe woundhealingprocessisconsideredanimportantdesign strategy.Forinstance,Taoandcolleaguesoverexpressed microRNA-126-3pinsynovialmesenchymalstemcellderivedexosomes(SMSC-126-Exos).Suchadesign strategycouldpromotethestimulatingeffectofSMSC126-Exosontheproliferationofepidermal ﬁbroblastsand vascularendothelialcells27.Inaddition,exosomes releasedfromnuclearfactorerythroid2-relatedfactor2 (Nrf2)-overexpressingadiposemesenchymalstemcells acceleratedwoundhealingbypromotingvascularization inadiabeticfootulcermousemodel31.Therefore,cellderivedexosomesloadedwiththerapeuticagentscanbe designedandthentransportedtothedesiredtarget. Consideringthecoreroleoftheloadedcomponentsin exertingthebiologicalfunctionsofexosomes,manystudieshavefoundthatdiversebiochemicalandbiophysical cuesintheextracellularmicroenvironmenthave remarkableregulatoryeffectsontheproduction,transport andespeciallycargoselectionofexosomes(Table 1). Theseregulatoryeffectscanbeconsideredtheresponses ofcellstomicroenvironmentalcuesmediatedbyexosomesthroughaparacrinepathway32,33.Inthefollowing section,wewillfocusontheregulatoryeffectsofbiochemicalandbiophysicalcuesinthecellmicroenvironmentonthefeaturesofcell-derivedexosomes(Fig. 2).

Biochemicalcues

Theoxygenconcentrationhasalwaysbeenregardedas oneofthemostimportantbiochemicalfactorsinthecell microenvironment.Alteredoxygenlevels,includingthose inhypoxia34 andunderoxidativestress35,arethepathophysiologicalbasisofmanydiseases.Cardiacprogenitor cells(CPCs)werefoundtosecreteproangiogenicexosomesinwhichthemiRNAsecretomeisalteredin responsetohypoxicconditions(Fig. 3A)34.Dougherty etal.conﬁrmedtheregulatoryeffectofhypoxiaonCPCEVs,whereCPCssecretedmoreEVsandhadabetter effectinischemicheartrepairunderphysoxicconditions (5%O2)thanundernormoxicorhypoxic(1%O2)conditions36.Alternatively,hypoxicadiposemesenchymal stemcell-derivedexosomes(ADSC-Exos)havealsobeen proventopromotetheangiogeniccapacityofhuman umbilicalveinendothelialcells(HUVECs)through

Table1Featuresofcell-derivedexosomesregulatedbybiochemicalandbiophysicalcuesincellmicroenvironment.

Refs.

ExosomefeaturesMechanismsExperimentalprotocols/ methods

GeneexpressionqPCRarray

Proteinarray

OrigincellsBiochemical/ biophysicalcues

CardiacprogenitorcellsHypoxiaCargoselection:increasedmiR-292,miR- 103,miR-210,etc.

AdiposeMSCsHypoxiaCargoselection:increasedVEGF,FGF,EGF, VEGF-R,MCP

α -overexpressingMSCsHypoxiaCargoselection:increasedJagged1Proteinarray; westernblotting

HIF-1

MastcellsOxidativestressCargoselection:mRNAalterationRNAmicroarrayanalysis

Humanplacenta-derivedMSCsNitricoxideCargoselection:increasedVEGF,miR-126qRT-PCR; Elisa; westernblotting

HumanumbilicalcordMSCs3,3 ′ -diindolylmethaneCargoselection:increasedWnt-11Wnt/ β -cateninsignalingactivationElisa; westernblotting

NanoSightanalysis; Elisa 42

Semi-quantitative ﬂ uorescent bead-basedassay; westernblotting

NanoSightanalysis; proteinarray; 46

Nanoparticletrackinganalysis; ﬂ uorescentstaining 51

Nanoparticletrackinganalysis; miRNAmicroarrayanalysis

Partiallyrelatedtocholesterol reduction

HeparanaseProduction:increasedexosomesecretion; Cargoselection:increasedsyndecan-1, VEGF,andHGF

Tumorcells (e.g.,myeloma,lymphoblastoid,andbreast cancer)

EBV-infectedcellsAspirinCargoselection:reducedlatentmembrane protein1andincreasedmiR-203

EpithelialcellsandmonocytesSimvastatinProduction:reducedexosomessynthesis, localization,andsecretion

AdiposeMSCsPlatelet-derivedgrowthfactorProduction:increasedexosomessecretion Cargoselection:carriedc-kitandSCF

Nanoparticletrackinganalysis; ﬂ owcytometry; qPCRarray

StimulatingnSMAse2-catalyzedand GW4869-sensitiveceramide formation

Production:enhancedexosomesyields Transport:moreef ﬁ cientsiRNAtransfer

3Dcultureandtangential ﬂ ow ﬁ ltration

Umbilicalcord derivedMSCs

MLE-12lungepithelialcellsMechanicalstretchProduction:increasedEVsrelease Cargoselection:miR ‐690,miR-711and miR ‐let ‐7c ‐5p,etc.

HUVECsShockwaveProduction:increasedexosomesrelease Cargoselection:miR-7i,miR-19a,miR-19b

Refs.

ExosomefeaturesMechanismsExperimentalprotocols/ methods

Nanoparticletrackinganalysis; Elisa

qRT-PCR; westernblotting

OrigincellsBiochemical/ biophysicalcues

TemperatureProduction:storagetemperaturerose,EVs decreased

Transport:lessC5b-9at 20°Cthan4°C and 70°C,theleastC5b-9at37°C

Cargoselection:enrichedmiRNA-16and miRNA-21

EndothelialcellsinfectedwithKaposi ’ s sarcoma-associatedherpesvirus(KSHV)

57 U87MGgliomacellsIonizingradiationProduction:increasedexosomesrelease

Activationofp53Nanoparticletrackinganalysis; RNAmicroarrayanalysis; westernblotting 58

Cargoselection:increasedIGFBP2 andCTGF

BonemarrowdendriticcellsLow-intensitypulsed ultrasound

VEGF/VEGF-R23.HIF-1α iswidelyknownasakey communicatorincellularadaptationtolow-oxygenconditionsandinactivatingangiogenicfactors.HIF-1αoverexpressingMSCshadenhancedsecretionofexosomesandchangedexosomalproteinandmiRNAlevels, whichpromotedangiogeniccapacity37.Inadditionto hypoxicconditions,exposuretooxidativestresshasregulatoryeffectsonexosomes.Forexample,mastcells exposedtooxidativestressproducedexosomesinwhich expressionoftheirmRNAtranscriptswasextensively differentfromexosomesproducedbycellsculturedunder normalconditions,andsuchexosomesstrengthenedthe toleranceofrecipientcellstooxidativestress35.Inaddition,nitricoxide(NO)wasfoundtobeinvolvedin manipulatingexosomebiofunctions,whereNOstimulatedMSCssecretedexosomeswithincreased miR-126andVEGFlevels,promotingtheangiogenic efﬁcacyofMSC-Exosbothinvitroandinvivo38.In summary,oxygenlevelsandthelevelsofothergaseous componentsinthecellmicroenvironmentmaybe importantchemicalfactorsthatregulatethebiological functionsofexosomes.

Inaddition,manyresearchershaveinvestigatedthe regulatoryeffectsofchemicalorbiologicalmoleculeson exosomefeatures.Forinstance,3,3′-diindolylmethane (DIM),anaturalsmall-moleculecompoundwithpotential intissueinjuryrepair39,40,facilitatedthestemnessand therapeuticeffectsofhumanumbilicalcordMSCs (hucMSCs)inwoundhealingbyupregulatingWnt11 expressioninhucMSC-derivedexosomes(Fig. 3B)41. Heparanasewasfoundtofacilitatetheproductionof exosomesderivedfromtumorcells(e.g.,myeloma,lymphoblastoidandbreastcancer)andtoupregulateexosomalproteincargoinvolvedintumorprogression,suchas syndecan-1,HGF,andVEGF42.Inaddition,some researchershaveexaminedtheregulatoryeffectsof pharmaceuticalmoleculesonexosomes.Transferofthe Epstein‒Barrvirus(EBV)oncoproteinlatentmembrane protein1(LMP1)viaexosomesisassociatedwithNPC metastasis.ThetumorsuppressormiRNA-203wasfound tobedownregulatedbyLMP143.Aspirinwasdemonstratedtoreversethisprocessbyinhibitingexosomal LMP1secretionbyEBV-positivecellsandbyboosting miR-203expression44.Inaddition,the3-hydroxy-3methylglutaryl-CoA(HMGCoA)inhibitorsimvastatin wasfoundtoreducethesynthesis,localizationand secretionofexosomesfromdifferentdonorcellsowingto itscholesterol-loweringeffect.Forexample,simvastatin showedaprotectiveeffectagainstatherosclerosisbecause itreducedexosomeproductionfrommonocytesand attenuatedendothelialcellmigrationmediatedby exosome-enclosedmiR-15045.Inaddition,theabilityof biomacromolecules,suchascytokinesorgrowthfactors, toregulatethefeaturesofexosomeshasbeeninvestigated.

Fig.2Regulatoryeffectsofbiochemicalandbiophysicalcuesintheextracellularmicroenvironmentonthefeaturesofcell-derived exosomes. Thefeaturesofexosomes,includingproduction,transport,andcargoselection,canbealteredwhencellsexperiencedifferent biochemical(e.g.,oxygen23 34 35,nitricoxide38,andchemical41 44 45 orbiologicalmolecules38 42 46)andbiophysical(e.g.,mechanicalstimulation22, environmentaldimensionality51,temperature56,ionizingradiation58,andlow-intensitypulsedultrasound(LIPUS)57)cuesintheirmicroenvironment.

Platelet-derivedgrowthfactor(PDGF)wasfoundtostimulateASC-EVsecretionandtoaltertheproteincompositiontofacilitateangiogenesis.PDGF-stimulatedEVs couldtrafﬁcc-kitanditsligand,stemcellfactor(SCF), whicharerelatedtoproangiogenicactivity(Fig. 3C)46. TGF-β1hasalsobeenfoundtoenhancetheexpressionof miR-135binMSC-derivedexosomesandtothuspromote chondrocyteproliferation47.Alltheseresultsindicatethat variousmoleculescanbeusedtoinﬂuenceexosome regulation.

Biophysicalcues

Physicalchangessuchaschangesindimensionality, mechanicalstimulation,temperature,andradiationinthe extracellularenvironmentalsoinﬂuencecellsecretion behavior22,48,49.Forexample,thedimensionalityofthe environmentinwhichdonorcellsarelocatedhasbeen

confirmedtoimpactexosomeregulation50–52.Exosomes releasedfroma3Dtissue-engineeredEwing’ ssarcoma typeItumormodelweresmallerthanthoseinmonolayer culturesbuthadasizedistributionsimilartothatof exosomesinpatientplasma.Inaddition,3DbioengineeredtumorexosomescontainedhighermRNAlevelsof thepolycombhistonemethyltransferaseEZH2,apotential tumorbiomarker,thanmonolayercultures50.Haraszti andcolleaguesfoundimprovedproductionofMSC-Exos through3Dculturecombinedwithamethodofexosome isolationtermedtangential flow filtration(TFF).Moreover,theyusedexosomesproducedwiththiscombined strategytodeliver Huntingtin (Htt)siRNAtoneuronsand foundthatthese3D-TFF-derivedexosomesweremore efficientatsiRNAtransferandHttsilencinginneurons thancontrols,demonstratingthat3D-TFF-derived exosomesmoreefficientlytransferredexosomalcargo

Fig.3Regulationofexosomesbybiochemicalcues.A HypoxicCPC-ExosalteredthemiRNAsecretomeandpromotedangiogenesis.(a)Matrigel assayofcardiacendothelialcellstreatedwithCPC-Exosunderhypoxicornormoxicconditions.Scalebar,1mm.(b)qRT‒PCRanalysisofmiRNAs regulatedinhypoxicCPCexosomes(lightbars)anddonorCPCs(darkbars).ThreemiRNAswerefoundtobeincreasedinrecipientendothelialcells. Thedataareexpressedasthemean±SEM.*p <0.05,**p <0.01,***p <0.001.Reproducedwithpermission34.Copyright2014,WoltersKluwerHealth. B 3,3′-Diindolylmethane(DIM)improvedthetherapeuticeffectofhucMSC-ExosbyupregulatingWnt11expression.(a)H&Estainingofwoundtissue treatedwithshGFP-hucMSCs,shGFP-DIM-hucMSCs,shWnt11-hucMSCs,andshWnt11-DIM-hucMCs.(b)Westernblotanalysisoftheexpressionof Wnt11inhucMSCsandDIM-hucMSC-Exos.(c)ELISAwasusedtodetecttheexpressionlevelofWnt11inhucMSCs,DIM-hucMSC-derivedexosomes, andexosome-freeCdM(n = 6;***p <0.001).Reproducedwithpermission41.Copyright2017,IvyspringInternational. C Platelet-derivedgrowthfactor (PDGF)promotesexosome-mediatedangiogenesisbystimulatingEVsecretionandalteringtheproteincompositionofEVs.(a)Quantitativeanalysis ofvessel-likestructureformationinresponsetoEVsinthepresenceofantibodiesagainstc-kitorSCF(mean±SEM, #p <0.05vs. “control”,*p <0.05vs. “PDGF-EVs” , n = 7).(b)WesternblotanalysisofEVsderivedfromASCsunderbasalconditions(b-EVs),EVsderivedfromASCsafterPDGFstimulation (PDGF-EVs)andhumanmicrovascularendothelialcells(HMECs)afterstimulationwiththeseEVs.Reproducedwithpermission46.Copyright2014, BioMedCentral.

Fig.4Regulationofexosomesbybiophysicalcues.A 3DcultureandTFFtreatmentfacilitatedtheMSCsecretionofmoreexosomeswithhigher efficiencyintransportcapacity.(a)Yieldofexosomesisolatedby2D-UC,2D-TFF,3D-UC,or3D-TFF(n = 12).(b)Huntingtin(Htt)mRNAlevelsin neuronstreatedwithexosomescontainingHttsiRNA.(c)Timecourseof fluorescenceinprimaryneuronstreatedwithexosomescontainingCy3labeledsiRNA.Reproducedwithpermission51.Copyright2018,Elsevier. B MechanicalstretchregulatedEVreleaseandvesicularmiRNAexpressionin MLE-12cells.(a)Theeffectsof10%cyclicstretch(left)and5%continuousstretch(right)onthenumberofEVs.*p <0.05(b)Volcanographofthe miRNAlevelsinEVsafter5%continuousstretch(left)and10%cyclicstretch(right).Reproducedwithpermission22.Copyright2020,JohnWileyand Sons. C Ionizingradiationinfluencedexosomeabundanceandmolecularcompositiontopromotetumorcellmigration.(a)Exosomeabundanceat 24hposttreatmentwith2to8Gyofradiation(n = 3).(b)EffectofexosomesonthemigrationofU87MGcellswhenusedasachemoattractantor uponpreincubationofexosomeswiththecells(n = 3).(c)CTGFgeneexpressionlevelsinU87MGcell-derivedexosomesandU87MGcellsbyqRT‒PCR.Dataareshownasthemean±SEM, n = 3. #p <0.01,*p <0.05(d)ImmunoblotanalysisofIGFBP2proteinlevelsinU87MGcell-derivedexosomes andU87MGcells.Reproducedwithpermission58.Copyright2013,Elsevier.

(Fig. 4A)51.Inaddition,3DspheroidculturedhBM-MSCs generatedmoreexosomesthanthosepreparedby2D culture.Inadditiontoenvironmentaldimensionality, cellularadhesionitselfmightalsoplayacrucialrolein exosomeproduction.Thenumberofexosomesdecreased withincreasingcelldensityin2DculturedhBM-MSCs, demonstratingthatanonadherentstatemightfacilitate theefﬁciencyofexosomeoutput52.Thus,cellswithdifferentenvironmentaldimensionalityhavedifferentregulatoryeffectsonthesecretionandpropertiesof exosomes.

Inadditiontodimensionalit y,mechanicalstimulation mayaffecttheproductionandfunctionofexosomes.

WhenmechanicalstretchforceswereappliedtoMLE12lungepithelialcells,10%cyclicstretchingwasfound toaugmentEVrelease,while5%continuousstretching didnot.Ontheotherhand,bothcyclicandcontinuous mechanicalstimulationchangedtheexpressionofspeci ﬁ cmiRNAsinsidethevesicles(Fig. 4 B) 22 .Moreover, exosomesreleasedfromHUVECsimprovedangiogenic capacityintheischemicmyocardiumaftershockwave therapycomparedwiththatintheabsenceoftreatment. RNAinterferenceexperimentsconﬁ rmedthatmiR-19a3pisthevesicularcomponentresponsibleforthe mechanicalsimulationeffect 53 .Furthermore,Pironti etal.establisheddifferentmechanicalstressmodels

includingexposuretoosmoticstretchinvitroandthe seraofmiceundergoingcardiacpressureoverload invivotostudythein ﬂ uenceofmechanicalstresson exosomes 54 .Mechanicalstresswasfoundtoinducethe releaseofexosomescontainingangiotensinIItypeI receptor(AT1R).Furthermore,AT1R-enrichedexosomeswerepredominantlyreleasedbycardiomyocytes underpressureoverloadandtargetedcardiomyocytes, skeletalmyocytes,andmesentericresistancevesselsto elevatesystolicbloodpressureinresponseto angiotensinII.

Manyotherphysicalcuesinthecellmicroenvironment playaroleintheregulationofexosomes.Forinstance, temperaturehasbeenproventobeacriticalfactorin maintainingtheactivityofEVs.EVsfromhuman endothelialcellsinfectedwithKaposi’ ssarcomaassociatedherpesvirus(KSHV)wereshowntohavethe potentialtoactivatethecomplementsystem55 .Different storagetemperatureswerediscoveredtoimpactthe propertiesofEVsisolatedfromKSHV-infectedhuman endothelialcells.ThenumberofEVsdecreasedasthe storagetemperaturerose,andproteinmarkersofEVs andthebiologicalactivityoftheactivatedcomplement systemwerealteredunderdifferenttemperatureconditions 56.Inaddition,low-intensitypulsedultrasound (LIPUS)couldpromotethebioactivityofexosomes derivedfrombonemarrowdendriticcells(BMDC-Exos) andcouldthusinduceanti-infl ammatoryresponses 57 HUVECsincubatedwithBMDC-Exosexhibited decreasedTNF-α -inducedexpressionofintercellular adhesionmolecule-1(ICAM-1)andvascularcelladhesionmolecule-1(VCAM-1)atboththegeneandprotein levels.AfterLIPUStreatment,theBMDC-Exoswere enrichedinmiRNA-16andmiRNA-21,limitingthe activationofNF- κB,asre fl ectedbythedownregulation ofIκ Bkinase(IKK)-α andphosphorylatedp65.By transportingthesemiRNAsintoHUVECs,LIPUStreatedBMDC-ExosimpededTNF-α -inducedin fl ammationbyinhibitingtheNF- κBsignalingpathway.In addition,radiation,anothermajorphysicalfactorinthe environment,hasbeencon firmedtosignifi cantlyimpact intercellularcommunicationthroughvarioussignal transductionsystems.Forexample,ionizingradiation increasedthereleaseofexosomesfromU87MGglioma cellsandpromotedthebiologicalfunctionsofexosomes tofacilitatethemigrationofrecipientcells(Fig. 4 C)58 Furthermore,proteinsassociatedwithsignalingpathwayscrucialforcellmigration,suchasinsulin-like growthfactorbindingprotein2(IGFBP2)andconnective tissuegrowthfactor(CTGF),wereincreasedinirradiated cellsandtheexosomesreleasedbythesecells.Inaddition,coculturewithexosomesderivedfromirradiated cellsenhancedtheproteinexpressionofCTGFin recipientcells.

Mechanismsofexosome-basedtherapeutic strategiesfortreatingchronicwounds

Thebiologicalfunctionsofexosomesinregulating complexintracellularpathwayshavemadeexosomes promisingtherapeuticstrategiesfortreatingmanydiseases.SinceValadietal.discoveredthatexosomescould implementintercellularcommunicationthroughtransportationofRNAin2007,exosome-relatedstudieshave flourished59.Exosomeswereinitiallystudiedintherepair ofischemictissueinjuries,suchasacutekidneyinjuryand myocardialischemia‒reperfusioninjury60–62.Ringhino etal.in2012foundthatendothelialprogenitorcellderivedmicrovesiclesimprovedneovascularizationin hindlimbischemiamice63.Since2015,manystudieshave focusedontheapplicationofexosomesinwoundrepair, mostofwhichhavebeenconductedonMSC-derived exosomes.Withthedeepeningofresearch,studieson exosomesinchronicwoundrepairhavebeenlimitedto theobservationofthehealingprocess,andexploringthe mechanismsofexosomefunctionsintheprocessof woundhealinghasbegun.Specifically,thesestudieshave revealedhowexosomesactindifferentstagesofwound healing.Ascarriersofparacrinesignalsfromcells,exosomesdeliverproteins,mRNAs,microRNAs,andother signalingmoleculestotargetcellsandpromotewound healingthroughdifferentmechanisms,showinggreater advantagesthanotherbiotherapiesforchronicwound treatment(TableS1).Inthissection,wedescribethe specificmechanismsofcell-derivedexosomesastherapeuticstrategiesforimprovingchronicwoundhealing (Fig. 5).

Regulationoftheinﬂammatoryresponse

Aprolongedinflammatoryresponseisanimportant factorassociatedwithrefractorychronicwounds.Previousstudieshaveshownthatthehealingofchronic woundsisimprovedbycorrectingthisextendedinflammatoryresponse64.Recentworkshaveshownthatcellderivedexosomesmediatetheinflammatoryresponse. Thus,theseexosomesshowgreatpotentialforchronic woundtreatment.Forexample,macrophage-derived exosomes(Mø-exos)wereproventohaveantiinflammatoryeffectsbyreducingthesecretionofproinflammatoryenzymesandcytokines,contributingtothe repairofdiabeticwoundsbysignificantlyaccelerating angiogenesis(Fig.S1A)65.Histologicalexamination showedthatMø-exossignificantlyreducedinfiltrating inflammatorycells,includingneutrophilsandmacrophages,andpromotedtheformationofgranulationtissue characterizedbymorenewbloodvesselsthanthose formedinPBS-treateddiabeticwoundsafter7daysof treatment.Moreover,Mø-exossuppressedtheexpression ofinflammatorycytokinesinvivo.Immunohistochemistry stainingrevealeddecreasedlevelsofTNF-α andIL-6in

Fig.5Schematicillustrationofthesignalingpathwaysofcell-derivedexosomesforimprovingchronicwoundhealing. huMSC-exos:human umbilicalcordmesenchymalstemcell-derivedexosomes;EPC-exos:endothelialprogenitorcell-derivedexosomes.

Mø-exo-treatedwoundsitescomparedtothoseinthe controls.Notably,thecombinationoflipopolysaccharides (LPS)andMø-exosenhancedinflammatorycellinfiltration,elevatedtheexpressionofIL-6andTNF-a,and decreasedtheformationofneovessels,demonstratingthat LPS-inducedlocalinflammationoffsetexosome-mediated pro-healingeffects65.Inaddition,furtherstudiesbyKim andcoworkersreportedthatexosomesderivedfromM2 macrophages(M2-Exos)couldpromotecutaneouswound repairbyinducingtheconversionofmacrophageswitha proinflammatoryM1phenotypeinwoundedtissuesinto macrophageswithareprogrammedM2-likephenotype (Fig.S1B)66.Within24haftertreatmentwithM2-Exos, M1-andM2-specificmarkers(e.g.,iNOSandarginase) hadclearlybeenswitchedoffandswitchedon,respectively,suggestingsuccessfulcompletionofmacrophage phenotypeswitching66.Alternatively,MSC-derivedexosomespreconditionedwithLPS(LPSpre-Exos)had similarinhibitoryeffectsonchronicinflammatory responsesbyregulatingmacrophagepolarization.In detail,inflammatoryTHP-1cellswereconvertedtoM2 macrophagesbyLPSpre-Exos.TheRT‒PCRresults showedthattheexpressionofanti-inflammatorycytokines(e.g.,TGF-β andIL-10)andM2macrophagesurface markers(e.g.,CD163)inTHP-1cellswasincreased,while theexpressionoftheproinflammatorycytokinesTNF-α, IL-1,andIL-6wasreducedafter48hofLPSpre-Exo treatmentunderhigh-glucoseconditions(Fig.S1C)67. Theseexosomalfunctionswerefurtherproventobe achievedviatheTLR4,NF-κB,STAT3,andAKT

signalingpathwaysbythetransportationofmiR-let-7b.In addition,miR-181cinhuc-MSC-derivedexosomes (hUMSC-Exos)suppressedtheinflammatoryresponseby inhibitingtheToll-likereceptor4(TLR4)signaling pathwayinaburnedmousemodel(Fig.S1D)68.InmacrophageswithLPS-stimulatedinflammation,theprotein expressionofNF-κB/P65,p-P65,andTLR4wasobviously higherthanthatinmacrophagestreatedwithhUMSCExos.Moreover,theexpressionlevelofmiR-181cwas higherinLPS-stimulatedmacrophagescoculturedwith hUCMSC-ExosthaninLPS-stimulatedmacrophages withoutexosometreatment.ELISAsdemonstratedthat hUCMSC-ExossuccessfullyinhibitedIL-1β andTNF-α levelsafterLPSstimulationbutnotablyincreasedIL-10 levels.Alltheseresultsindicatedthatcell-derivedexosomes(e.g.,exosomesfrommacrophagesandMSCs)can regulateinflammatoryresponses,protectinjuredtissues andacceleratewoundhealing.Inparticular,exosomesare richinmiRNAs,whichplayacrucialroleintheregulation ofinflammation.

Promotionofcellproliferationandmigration

Cellproliferationandmigrationplayfundamentalroles inwoundrepair.Specifically,epithelialcells, fibroblasts, andendothelialcellsthatmigratetothewoundsiteperformspecificfunctionsduringwoundhealing.Indetail, fibroblasts filltissuedefectsandsynthesizeECM,endothelialcellspromoteangiogenesis,andepithelialcells graduallycoverthewound69.MSC-derivedexosomescan regulatetheproliferationandmigrationoftheabovecells

bytransportingtheirencapsulatedcomponents.For example,exosomesderivedfromadiposemesenchymal stemcells(ASCs-Exos)couldstimulatetheproliferation andmigrationof fibroblastsinadose-dependentmanner tofacilitatecutaneouswoundhealing(Fig.S2A)26.After treatmentwithASC-Exos, fibroblastmigrationat12hand 24hwasincreasedcomparedtothatofthecontrolgroup, asdeterminedbyscratchandTranswellmigrationassays. Inaddition,theexpressionofgenesassociatedwithcell migrationandproliferation,suchasN-cadherin,cyclin-1 andproliferatingcellnuclearantigen(PCNA),wasupregulatedin fibroblastsbyASC-Exos.Furthermore,thehigh levelsofmicroRNA-21inASC-Exosenhancedthe migrationandproliferationofHaCaTcellsthroughthe PI3K/AKTpathway,whichindicatedthatmicroRNA-21 mightbeakeycargoinASC-Exosthatpromotescell migrationandproliferation70.Similarly,exosomesisolatedfromhumanbonemarrowMSCs(BMSC-Exos) couldinducethegrowthandpromotethemigrationof fibroblastsfrombothnormalanddiabeticwounds.The MTTassayrevealedthatthegrowthofthesetwo fibroblastlineswassignificantlyincreasedinadose-dependent mannercomparedtothatincontrols.Furthermore,the resultsofthescratchassayindicatedtheincreased migrationof fibroblastsfrombothnormalanddiabetic woundsintheBMSC-Exogroup71.Inaddition,Zhang’ s groupfoundthathUMSC-Exoscouldreverseacute thermalinjury‐inducedcellapoptosisviaactivationofthe β‐cateninsignalingpathwaytoacceleratewoundhealing (Fig.S2B)72.IncomparisontoPBS,hUMSC-Exossignificantlysuppressedheatstress‐inducedapoptosisin HaCaTcellsandprimarydermal fibroblasts(DFLs)and promotedtheirproliferation.Westernblotanalyses showedlowerBaxlevelsinHaCaTcellsandDFLstreated withhUMSC-ExosthanincellswithouthUMSC-Exo treatment,butBcl‐2expressionwashigherinthe hUMSC-Exosgroupthaninthecontrolgroups.Inaddition,theenhancedPCNAexpressionbyhUMSC-Exos indicatedanincreasedlevelofcellproliferation.The Wnt4proteininhUMSC-ExosactivatedtheWnt/ β-cateninsignalingpathwayandenhancedtheexpression ofproliferativeproteins(e.g.,PCNA,N-cadherinand cyclinD3)inHaCaTcells,thuspromotingcellproliferationandmigration.Thiseffectwasabrogatedbythe β-catenininhibitorICG001.Thedecreasedexpressionof PCNA,N-cadherin,andcyclinD3andsuppressedcell proliferationandmigrationobservedinthepresenceof ICG001con fi rmedtheroleofthe β -cateninpathway.In addition,underhigh-cell-densityconditions,the hUMSC-Exo-derivedprotein14-3-3ζ couldactivatethe HippopathwaytoreversetheWnt/β -cateninpathway andsuppresscellproliferationandmigration,preventing excessiverepairandscarformation73.Inadditionto stemcell-derivedexosomes,exosomesfromother

sourcesareinvolvedinpromotingcellproliferationand migrationduringthewoundhealingprocess.For example,exosomesisolatedfromPRPcouldpromote ﬁ broblast-relatedfunctionsandfacilitatewoundhealing 74 .Thesestudieshaveshownthatexosomesderived fromdiversesourcescanenhancetheproliferationand migrationofcells,especially ﬁ broblasts,therebyacceleratingwoundhealing.

Promotionofangiogenesis

Angiogenesisisconsideredtobeessentialforwound healingsinceitiscrucialf orthemaintenanceofoxygen andnutrientdelivery.However,thisimportantprocess isusuallycompromisedinchronicwounds,especiallyin DUs 75 .Manyrecentworkshaveinvestigatedtheroleof exosomesintheprocessofangiogenesisduringchronic woundrepairduetotheirdeliveryofproteinsand miRNAs.Forinstance,Chenandcolleaguesfoundthat urine-derivedstemcellexosomes(USC-Exos)could facilitatediabeticwoundhealingbyenhancingangiogenesisbytransferringaproangiogenicproteinnamed deletedinmalignantbraintumors1(DMBT1) 76 .ASCExoswerefoundtoupregulatetheexpressionofthe proangiogenicgenesAng1andFlk1,downregulatethe expressionoftheantiangiogenicgenesVash1andTSP1 inHUVECsandenhanceangiogenesisbothinvitroand invivo(Fig.S3A) 77 .Speci ﬁ cally,ASC-Exosmayfacilitatetheformationofendothelialtipcellstomodulate endothelialcellangiogenesisbyinhibitingtheexpressionoftheangiogenicinhibitordelta-like4(DLL4) throughthetransportationofmiRNA-125a.Moreover, aseriesofmiRNAs,suchasmiR-30b,miR-30c,miR424,andlet-7f,arethoughttobetransferredbyMSCExosandtoplayrolesinregulatingangiogenesis 78 . Exosomesderivedfromhumanumbilicalcordbloodderivedendothelialprogenitorcells(EPC-Exos)were alsodemonstratedtohaveproangiogenicandprohealingeffectsindiabeticratmodels.EPC-Exoscouldbe internalizedintoendothel ialcellsandpromotetheir proliferationandmigrationandenhancetubeformation(Fig.S3B) 25 .Erk1/2signalingexertsitsimportant effectinpromotingangiogenesisresponsesbyEPCExos.Inaddition,MSC-Exosarethoughttoactivatethe AKT,STAT3,andNF- κ Bsignalingpathwaystoinduce theupregulationofgrowthfactors(e.g.,HGF,IGF1, PDGF,EGF,andFGF),therebypromotingtheproliferationandmigrationof vascularendo thelialcells andenhancingangiogenesis 71 , 79 .Therefore,exosomes fromdifferentsourceseitherdirectlyaffectendothelial cellsorindirectlyactonendothelialcellsbyupregulatingtheexpressionofangiogenesis-relatedgrowth factors,thusenhancingendothelialcellproliferation andmigrationandpromotingtheformationofnew bloodvessels.

RegulationofECMremodelingandinhibitionofscar formation

Theremodelingphase,whichischaracterizedbyECM remodelingandscarformation,isthelastkeyphaseinthe woundhealingprocess.Animbalancebetweenthe synthesisanddegradationofECMcanpreventhealingor leadtopathologicalscarformation80.Numerousexosomes,especiallythosereleasedfromMSCs,havebeen proventobeinvolvedintheprocessofECMremodeling. Forexample,hUMSC-Exoswerefoundtofacilitatethe synthesisofelastinandcollagenI81.Inaddition,exosomes derivedfromhuman-inducedpluripotentstemcellderivedMSCs(hiPSC-MSCs)weredemonstratedtopromotetheexpressionofcollagenI,collagenIIIandelastin (Fig.S4A)24.Furthermore,endothelialcell-derivedexosomescouldfacilitatethecrosslinkingofECMbyupregulatinglysyloxidase-like2(LOXL-2)underhypoxic conditions82.Alltheseresultsindicatethatexosomes exerttheireffectonECMremodelingduringwound healing.Ontheotherhand,exosomeshavealsobeen foundtosuppresstheexcessiveproductionofECM componentsandoverdifferentiationof fibroblastsinto myofibroblaststopreventscarformationandexcessive repair.ADSC-Exoswerefoundtoinhibitscarformation bypreventing fibroblastsfromdifferentiatingintomyofibroblastsandregulatingtheTGF β3/TGF β1,collagenIII/ collagenI,andMMP3/TIMP1ratios(Fig.S4B)83.In addition,hUMSC-ExosinhibitedtheformationofmyofibroblastsbytransportingspecificmicroRNAs(e.g.,miR145,miR-125b,miR-23a,andmiR-21)thattargetthe TGF-β/SMAD2signalingpathway(Fig.S4C)84.Therefore, exosomesmaycontrolcollagensynthesisandmyofibroblastdifferentiationatdifferentstagesofthewound healingprocesstoachieveidealwoundrepair.

Hydrogel-baseddeliverysystemsofexosomesin chronicwoundtreatment

Cell-derivedexosomeshaveshownoverwhelming potentialinthe ﬁeldofregenerativemedicine.However, manychallengesintheirusealoneexist.Forexample, deliveringexosomesatatherapeuticdosagetothetarget site,especiallyviasystemicinjection,isnotasimple process.Thedirectapplicationofexosomesthrough approachessuchasintravenous,subcutaneous,orintraperitonealinjectionoftenleadstorapidexosomeclearancefromthebloodcirculationandexosome accumulationintheliver,spleen,lung,andgastrointestinaltract85,86.Furthermore,macrophagesinthe reticuloendothelialsystemtakeupandreleasethe majorityofinjectedexosomes,regardlessofthedelivery methodorcellsource87.Therefore,maintainingexosome stabilityovertimeaftertransplantationisacentralchallengefortheirclinicalapplication,andsustaineddelivery ofexosomesthatincreasestheirlong-termefﬁciencyand

concentrationisneeded.Inadditiontocontrollablephysicochemicalproperties,hydrogelswith3Dnetworkshave propertiessimilartothoseofthenativeECM,suchas goodbiocompatibilityandbiodegradability88.Withthese advantages,hydrogelshavebeenwidelyappliedto encapsulateanddeliverdrugs89–91,growthfactors92–94 , andotherbioactivesubstances95,96,andhaveshown satisfactorydeliveryeffects.Recently,manystudiesonthe applicationofhydrogelsforthedeliveryofexosomesin promotingwoundrepairhavebeenreported(Table S2)27,29,74,97–101.Inthissection,wewillintroduce hydrogel-basedexosomedeliverysystems.

Directencapsulationofexosomesinhydrogels

Todate,mosthydrogel-baseddeliverysystemshave encapsulatedexosomesdirectlyinhydrogels.Exosomes aretypicallymixedwithapolymersolutionandcrosslinkingagentsbeforehydrogelgelationorareincorporatedintoporoushydrogelscaffoldsafterhydrogel crosslinking.Asthehydrogelsdegradeinvivo,the encapsulatedexosomesaregraduallyreleased.For example,alginate(Alg)-basedhydrogelswereusedto deliverADSC-Exosfortherepairoffull-thicknessskin defects28.ThereleaseofADSC-ExosfromanAlg-based hydrogelwassustainedfor172h,andmorethan50%of thetotalexosomereleasewascompletedinthe first72h, suggestingreliableandcontinuousdelivery.Loading ADSC-ExosintoanAlg-basedhydrogelaccelerated woundrepairbypromotingcellmigrationandproliferation,collagendepositionandangiogenesisincomparison withwoundrepairintheuntreatedcontrolgroupandthe grouptreatedwithAlgalone.Inaddition,specific degradation-sensitivehydrogelshavebeendevelopedto moreefficientlydeliverexosomesindifferentmicroenvironments.Loadingofathermosensitivechitosan(CS) hydrogelwithexosomesfromhumanplacenta-derived MSCs(hP-MSCs)improvedtheirretentionandstability invivo(Fig. 6A)102.Labeledexosomesgraduallyincreased bothinthesurroundingsafterincubationwithCS-Exosat 37°Cinvitroandinmouseischemichindlimbsafterthe injectionofCS-Exosinvivo,suggestingsustainedrelease ofthermosensitiveCS-Exos.Thisapproachenhancedthe stabilityofexosomesderivedfromhP-MSCsand improvedtheirtherapeuticeffects,thusfacilitatingtissue repairafterischemicinjury.Zhouetal.introducedan MMP2-sensitiveself-assemblingpeptide(KMP2)hydrogelcontaininganMMP2-cleavablemotifforthetreatmentofischemia‒reperfusion(I/R)injury(Fig. 6B)103. DuetoenzymaticdegradationbyMMP2,thenetwork structureoftheKMP2hydrogelwasdestroyed,andthe loadedEVswerethenreleased.InresponsetoMMP2, theKMP2hydrogelincreasedmasslossandaccelerated thereleaseofMSC-EVs,demonstratingcontrolledrelease ofEVsfromtheKMP2hydrogel.Moreover,thereleased

Fig.6Encapsulationofexosomesinhydrogelsbydirectmixingofexosomeswiththehydrogelprecursorsolutionandthereleaseof exosomesduringdegradation.A Thermosensitivechitosan(CS)hydrogelforthesustainedreleaseofexosomesfromhP-MSCs.(a)Opticalimages oftheCSsolution(I)andhydrogel(II).(b)ScanningelectronmicroscopyimageoftheCShydrogel.(c)TherheologicalpropertiesoftheCShydrogel withtemperaturechangeswereanalyzedbyrheologicalmeasurements.(d)SustainedreleaseofExosfromtheCShydrogelwastestedbytracking Glucsignalsusingbioluminescenceimaging(BLI)analysis.(e)InvivomonitoringoftheretentionoftransplantedCS-ExosorExosthroughthe trackingofGlucsignalsbyBLIanalysis.(f)ThestabilityofthemainfunctionalmoleculemiR-126inCS-ExosorExosat37°Cwasassessedbyreal-time qPCRanalysis.(g)QuantitativeanalysisofGlucsignalsindicatedtheretentionofExosin(e).*p <0.05,**p <0.01.Reproducedwithpermission102

Copyright2018,AmericanChemicalSociety. B MMP2-sensitiveKMP2hydrogelforthecontrolledreleaseofMSC-EVs.(a)Schematicillustrationofthe KMP2hydrogelforenhancedEVreleaseandtissuerepair.(b)DegradationoftheKMP2hydrogelwithorwithoutMMP2wasmeasuredbygelmass loss.(c)InvitroreleaseofEVsfromKMP2hydrogelsinPBSoranMMP2solution(0.4or4mg/ml, n = 3).*p <0.05,**p <0.01,PBSgroupvs.MMP2_0.4 group; #p <0.05, ##p <0.01,PBSgroupvs.MMP2_4group; &p <0.05, &&p <0.01,MMP2_0.4groupvs.MMP2_4group.(d)Real-timePCRanalysisofBax andFasmRNAlevelsinHK2cells.(e)WesternblotanalysisofICAM-1andIL-1β proteinlevelsinHK2cells.HK2cellswereexposedtohypoxiareoxygenation(H/R)andweretreatedwith10 μgprotein/mlfreshEVsorreleasedEVs.*p <0.05,**p <0.01,H/Rgroupvs.Congroup; #p <0.05, ##p <0.01,FreshEVsgroupvs.H/Rgroup.Reproducedwithpermission103.Copyright2019,Elsevier.

EVsmaintainedtheirproperties,suchastheirantiapoptotic(e.g.,FasandBax)andanti-inﬂammatory(e.g., MCP-1,IL-1β,andICAM-1)potencyinHK2cellsunder hypoxia-reoxygenation(H/R)conditionsinvivo.

Encapsulationofexosomesinhydrogelsthroughphysical/ chemicalinteractions

Inadditiontothedirectincorporationofexosomesinto hydrogels,someresearchershaveusedphysicalorchemicalinteractionstoimplementtheencapsulationof exosomesinhydrogelsandtheirrelease.Forinstance, PluronicF127graftingpolyethylenimine(PEI)andaldehydepullulan(APu)wereusedtosynthesizea polysaccharide-basedFEPhydrogelbyaSchiffbase reaction;ADSC-derivedexosomeswereencapsulatedinto thehydrogelviaelectrostaticinteractionsandwere releasedinapH-responsivemanner(Fig. 7A)104. AlthoughsustainedexosomereleasebyFEPhydrogelwas observedfor21days,fasterexosomereleaseoccurred atpH5.5thanatpH7.5duetothepresenceofa

Fig.7 (Seelegendonnextpage.)

(see ﬁgureonpreviouspage)

Fig.7Encapsulationofexosomesinhydrogelsandthereleaseofexosomesfromhydrogelsthroughphysical/chemicalinteractions. A ADSC-derivedexosomeswereloadedinanFEPhydrogelthroughelectrostaticinteractionsandreleasedinresponsetopHchanges.(a)Schematic illustrationofthesynthesisofamultifunctionalFEPhydrogelscaffold.(b)SchematicofexosomereleasefromtheFEPscaffolddressing.(c) RepresentativepH-dependentreleaseproﬁleofloadedexosomesintheFHEscaffolddressing.Reproducedwithpermission104.Copyright2019, AmericanChemicalSociety. B Light-triggerableHAhydrogelforthecontrolledreleaseofSEVs.(a)SchematicillustrationoftheformationofthelighttriggerableHAhydrogelcontainingSEVs(hydrogelistranslucid:a1,swollengelinPBS;a2,swollengelinDMEM).PhotocleavageoftheHAhydrogel overtimedemonstratingthatthereleaseofSEVswasdependenton(b)thenumberofirradiationsand(c)theexposuretime.*p <0.05,**p <0.01, ***p <0.001,UV-lightgroupvs.nonirradiatedgroup; # p <0.05, ## p <0.01, ### p <0.001,blue-lightgroupvs.nonirradiatedgroup.Reproducedwith permission105.Copyright2019,AmericanChemicalSociety.

pH-sensitiveC=NbondintheFEPhydrogel.Inaddition, Helenaetal.encapsulatedPCL-modifiedsmallextracellularvesicles(SEVs)incysteine-modi fiedhyaluronic acid(HA)throughchemicalinteractionsbetweenthe terminalacrylategroupsonPCLandthethiolgroupson HA,yieldingalight-triggerableHAhydrogelcontaining SEVs(Fig. 7B)105.Duetotheformationofaphotolabile orthonitrobenzylesterbond,controlledreleaseofthe SEVscouldbeachieveduponUV/bluelightirradiation.In addition,releaseoftheSEVswasdependentonthe numberofirradiationsandtheexposuretime.Asignificantdifferenceinfull-thicknesswoundrepairwas observedinanimalstreatedwiththeHAhydrogel+EVs +lightirradiationgroupcomparedwiththecontrol groups,indicatingimprovedwoundhealingwiththe controlledreleasesystem.Overall,theutilizationof hydrogelsasexosomedeliverysystemsmaybeapromisingwaytoachievesustainedtherapeuticeffectson chronicwounds.

Conclusionandfutureperspectives

Fordecades,exosomeshavebeenwidelyexploitedto developnovelstrategiesfortissueregeneration,drug delivery,anddiseasediagnosisandtreatment106.Among exosomesderivedfrombiologicalsources,MSC-derived exosomeshaveshowngreatpromiseinthe fieldof regenerativemedicine,astheycaninheritmostofthe regenerativeeffectsofMSCs107.Asdrugcarriers,exosomesandliposomeshavecomplementaryadvantages anddisadvantages.Liposomesarecommonlyused nanoparticle-baseddrugdeliverysystemsduetotheir highbiocompatibilityandlowtoxicity.Liposomesare composedofphospholipidsandcholesterol,whicharethe maincomponentsofcellmembranes,makingthemthe firstartificialdrugcarrierstoreceiveclinicalapproval108 Todate,liposomeswiththeadvantagesofhighloading andgoodprotectionfordrugshavebeenabletomeetthe requirementsofcommercialmassproduction.However, problemswithimmunogenicityandpoortargetingstill restraintheapplicationofliposomesforidealdrug delivery109.Incomparison,exosomes,asnaturalvesicles releasedbycells,havegreatimmunecompatibility. Moreover,withavarietyofproteinsontheirmembranes

(e.g.,tetraspinins),exosomesareprovidedwithhighand specificorganotropism,whichisoneofthemajorgoalsof alltypesofnanoparticle-baseddrugdeliverysystems.Itis worthnotingthattherearealsosomedisadvantagesof exosomesasdrugcarriers,suchasinsufficientproductivity,lowdrugload,andhighclearancerateinthe circulation110.Therefore,thedevelopmentofvarious typesofdesignedexosomesorliposome-basedartificial exosomesfortargeteddrugdeliveryiscurrentlyunderindepthstudy111.Inthe fieldofwoundrepair,exosomes transportavarietyofbioactivecontentsandparticipatein almosttheentirewoundhealingprocess;forexample, theyregulateinflammatoryresponses,promotecellproliferationandmigration,boostangiogenesis,andregulate ECMremodeling.Allthese findingshaveproventhatcellderivedexosomeshavegreatpotentialastherapeutic strategiesforchronicwoundhealing.However,some challengesstillremain.Weconcludewithsomeopinions ontheextractionandpurificationofexosomes,thesafety andapplicationofexosomes,theregulatoryeffectsof microenvironmentalcuesoncell-derivedexosomes,and exosomedeliverysystems.

1.Theultimategoaloftheuseofexosomesisto achieveclinicaltherapeuticresults,butthereisstilla longwaytogo.First,theinsufficientproductionof exosomesisthemostvitalproblem.Traditional approachesfortheextractionandpurificationof exosomesarequitecomplex.Somerecently developedtechniques,suchasEXODUS112,can achieveultrafastisolationofexosomesfrombiofluids withhighpurityandhighyield.Second,standards relatedtovesiclesize,purity,andcontamination levelsandidentificationofspecificbiomarkersare lacking.Methodstodistinguishexosomesbasedon cellsourcehavenotbeendeveloped,anda proteomicanalysisofexosomeshasnotbeen carriedout.Finally,anyclinicalapplicationsmust bebasedonsafety.However,asofnow,studies assessingthesafetyofexosome-basedtherapiesare intheirinfancy,andmanyuncertaintiesregarding theirsafetyremain.Forexample,thepro-and anticancereffectsofexosomesindifferent applicationsarestillcontroversial.Exosomesmay

acceleratecancerprogressionthroughdirect interactionsorbysecretingvariousfactors113 Furtherstudiesarenecessarytodeterminethe effectivedoserangeforthesafeclinicalapplication ofexosomes.

2.Heterogeneityamongexosomesduetotheirdiverse originsisacrucialissuethatcannotbeignored.Even amongexosomesfromthesamekindofcellsor biospecimens,itisnotclearwhetherexosomesdiffer amongindividuals.Inaddition,theextracellular microenvironmentcaninfluencecellsecretion51 Cell-derivedexosomescaninherittheadaptive changesoftheirparentalcellsinresponseto environmentalfeaturesandmainlyshowchanges intheirproduction,qualityandthecomponentsthat theyencapsulate;suchchangescaninfluencethe therapeuticefficiencyofexosomes.Cell preconditioningmaybeawaytoresolvethisissue. Wehavesummarizedtheregulatoryeffectsofsome biochemicalandbiophysicalcuesintheextracellular microenvironmentonthefeaturesofexosomes. Hence,itispossibletoregulatethebiological functionsofcell-derivedexosomesbyalteringthe cellmicroenvironmenttopromotetherapeutic efficiencyorevenachievepersonalizedtreatments, whichisanemergingareathatmayserveasa researchfocusforfuturestudies.

3.Toenhancetherapeuticefficiency,hydrogel-based deliverysystemsforthesustainedandpreciserelease ofexosomesduringtreatmenthavebeendeveloped. Stimulus-responsivehydrogelsmayprovide customizablemethodsofexosomedelivery.However, theclinicalapplicationofhydrogel-baseddelivery systemsinvivostillfacesmanychallenges.For example,residualcrosslinkersorcomponentsin hydrogelsaretoxicandcarcinogenic.Uncontrollable crosslinkingofpH-ortemperature-sensitivehydrogels canoccur,whichcanblocktheneedleduringinjection. Therefore,itisnecessarytooptimizetheconditions underwhichthegelformstopreventprematuregel formation.Inaddition,thereleaseprofilesofhydrogelbaseddeliverysystemsdeterminedinvitromaynotbe applicabletoinvivoapplications.Theeffectsofthe invivomicroenvironmentondeliveryefficiencystill needtobeexplored.

Acknowledgements

ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina (11972279),theYoungEliteScientistSponsorshipProgrambyCAST (2018QNRC001),andtheNaturalScienceFoundationofShaanxiProvince (2020JQ-060).

Authordetails

1DepartmentofBurnsandPlasticSurgery,SecondAfﬁliatedHospitalofAir ForceMilitaryMedicalUniversity,Xi’an710038,P.R.China. 2Bioinspired EngineeringandBiomechanicsCenter(BEBC),TheKeyLaboratoryof

BiomedicalInformationEngineeringoftheMinistryofEducation,Xi’ an JiaotongUniversity,Xi’an710049,P.R.China. 3DepartmentofEndocrinology, SecondAfﬁliatedHospitalofAirForceMilitaryMedicalUniversity,Xi’an710038, P.R.China. 4DepartmentofMedicalOncology,FirstAfﬁliatedHospitalofXi’ an JiaotongUniversity,Xi’an710061,P.R.China

Authorcontributions

W.D.,J.L.,andY.M.contributedtothepaperdesign, ﬁgureandtable preparation,andpapereditingandrevision.Y.D.andJ.W.contributedto ﬁgure preparation.T.H.contributedtotheliteraturecollection.B.G.,H.G.,andF.X. contributedtothereviewofthepaper.

Conﬂictofinterest

Theauthorsdeclarenocompetinginterests.

Publisher’snote

SpringerNatureremainsneutralwithregardtojurisdictionalclaimsin publishedmapsandinstitutionalafﬁliations.

Supplementaryinformation Theonlineversioncontainssupplementary materialavailableat https://doi.org/10.1038/s41427-022-00419-y

Received:13August2021Revised:7July2022Accepted:12July2022. Publishedonline:9September2022

References

1.Rice,J.B.etal.Burdenofdiabeticfootulcersformedicareandprivate insurers. DiabetesCare 37,651–658(2014).

2.Martinengo,L.etal.Prevalenceofchronicwoundsinthegeneralpopulation: systematicreviewandmeta-analysisofobservationalstudies. Ann.Epidemiol. 29,8–15(2019).

3.Olsson,M.etal.Thehumanisticandeconomicburdenofchronicwounds:a systematicreview. WoundRepairRegen. 27,114–125(2019).

4.Frykberg,R.G.&Banks,J.Challengesinthetreatmentofchronicwounds. Adv.WoundCare. 4,560–582(2015).

5.Akopian,G.etal.Outcomesofconventionalwoundtreatmentinacomprehensivewoundcenter. Am.Surg. 72,314–317(2006).

6.Nunan,R.,Harding,K.G.&Martin,P.Clinicalchallengesofchronicwounds: searchingforanoptimalanimalmodeltorecapitulatetheircomplexity. Dis. ModelMech. 7,1205–1213(2014).

7.Chou,P.-R.etal.Supercriticalcarbondioxide-decellularizedporcineacellulardermalmatrixcombinedwithautologousadipose-derivedstemcells: itsroleinaccelerateddiabeticwoundhealing. Int.J.Med.Sci. 17,354–367 (2020).

8.Boeringer,T.,Gould,L.J.&Koria,P.Protease-resistantgrowthfactorformulationsforthehealingofchronicwounds. Adv.WoundCare. 9,612–622(2020).

9.Oneto,P.&Etulain,J.PRPin woundhealingapplications. Platelets 32, 189–199(2020).

10.Huang,Y.-Z.,Gou,M.,Da,L.-C.,Zhang,W.-Q.&Xie,H.-Q.Mesenchymalstem cellsforchronicwoundhealing:currentstatusofpreclinicalandclinical studies. TissueEng.PartBRev. 26,555–570(2020).

11.Shin,L.&Peterson,D.A.Humanmesenchymalstemcellgraftsenhance normalandimpairedwoundhealingbyrecruitingexistingendogenous tissuestem/progenitorcells. StemCellsTransl.Med. 2,33–42(2013).

12.Volarevic,V.etal.Ethicalandsafetyissuesofstemcell-basedtherapy. Int.J. Med.Sci. 15,36–45(2018).

13.Song,Y.etal.Humanumbilicalcordblood-derivedMSCsexosomeattenuate myocardialinjurybyinhibitingferroptosisinacutemyocardialinfarction mice. CellBiol.Toxicol. 37,51–64(2020).

14.Liu,W.etal.Hypoxicmesenchymalstemcell-derivedexosomespromote bonefracturehealingbythetransferofmiR-126. ActaBiomater. 103, 196–212(2020).

15.Ferreira,Ad.F.&Gomes,D.A.Stemcell extracellularvesiclesinskinrepair. Bioengineering 6,4(2018).

16.Kalluri,R.&LeBleu,V.S.Thebiology, function,andbiomedicalapplicationsof exosomes. Science 367,eaau6977(2020).

17.ElAndaloussi,S.,Mäger,I.,Breakeﬁeld,X.O.&Wood,M.J.A.Extracellular vesicles:biologyandemergingtherapeuticopportunities. Nat.Rev.Drug Discov. 12,347–357(2013).

18.Xu,J.,Camﬁeld,R.&Gorski,S.M.Theinterplaybetweenexosomesand autophagy partnersincrime. J.CellSci. 131,jcs215210(2018).

19.Hood,J.L.&Wickline,S.A.Asystematicapproachtoexosome-based translationalnanomedicine. WileyInterdiscip.Rev.Nanomed.Nanobiotechnol. 4,458–467(2012).

20.Colombo,M.,Raposo,G.&Théry,C.Biogenesis,secretion,andintercellular interactionsofexosomesandotherextracellularvesicles. Annu.Rev.CellDev. Biol. 30,255–289(2014).

21.Schorey,J.S.&Harding,C.V.Extracellularvesiclesandinfectiousdiseases: newcomplexitytoanoldstory. J.Clin.Investig. 126,1181–1189(2016).

22.Najrana,T.etal.Mechanicalstretchregulatestheexpressionofspeciﬁc miRNAinextracellularvesiclesreleasedfromlungepithelialcells. J.Cell. Physiol. 235,8210–8223(2020).

23.Han,Y.,Ren,J.,Bai,Y.,Pei,X.&Han,Y.Exosomesfromhypoxia-treatedhuman adipose-derivedmesenchymalstemcellsenhanceangiogenesisthrough VEGF/VEGF-R. Int.J.Biochem.CellBiol. 109,59–68(2019).

24.Zhang,J.etal.Exosomesreleasedfromhumaninducedpluripotentstem cells-derivedMSCsfacilitatecutaneouswoundhealingbypromotingcollagensynthesisandangiogenesis. J.Transl.Med. 13,49(2015).

25.Zhang,J.etal.Exosomesderivedfromhumanendothelialprogenitorcells acceleratecutaneouswoundhealingbypromotingangiogenesisthrough Erk1/2signaling. Int.J.Biol.Sci. 12,1472–1487(2016).

26.Hu,L.etal.Exosomesderivedfromhumanadiposemensenchymalstem cellsacceleratescutaneouswoundhealingviaoptimizingthecharacteristics of ﬁbroblasts. Sci.Rep. 6,32993(2016).

27.Tao,S.C.etal.Chitosanwounddressingsincorporatingexosomesderived frommicroRNA-126-overexpressingsynoviummesenchymalstemcells providesustainedreleaseofexosomesandhealfull-thicknessskindefectsin adiabeticratmodel. StemCellsTransl.Med. 6,736–747(2017).

28.Shafei,S.etal.Exosomeloadedalginatehydrogelpromotestissueregenerationinfull-thicknessskinwounds:aninvivostudy. J.Biomed.Mater.Res.A. 108,545–556(2020).

29.Wang,C.etal.Engineeringbioactiveself-healingantibacterialexosomes hydrogelforpromotingchronicdiabeticwoundhealingandcompleteskin regeneration. Theranostics 9,65–76(2019).

30.Schorey,J.S.&Bhatnagar,S.Exosomefunction:fromtumorimmunologyto pathogenbiology. Trafﬁc 9,871–881(2008).

31.Li,X.etal.Exosomesfromadipose-derivedstemcellsoverexpressingNrf2 acceleratecutaneouswoundhealingbypromotingvascularizationina diabeticfootulcerratmodel. Exp.Mol.Med. 50,29(2018).

32.Wang,X.etal.Cardiomyocytesmediateanti-angiogenesisintype2diabetic ratsthroughtheexosomaltransferofmiR-320intoendothelialcells. J.Mol. CellCardiol. 74,139–150(2014).

33.Haraszti,R.A.etal.Serumdeprivationofmesenchymalstemcellsimproves exosomeactivityandalterslipidandproteincomposition. iScience 16, 230–241(2019).

34.Gray,W.D.etal.IdentiﬁcationoftherapeuticcovariantmicroRNAclustersin hypoxia-treatedcardiacprogenitorcellexosomesusingsystemsbiology. Circ.Res. 116,255–263(2015).

35.Eldh,M.etal.Exosomescommunicateprotectivemessagesduringoxidative stress;possibleroleofexosomalshuttleRNA. PLoSOne 5,e15353(2010).

36.Dougherty,J.A.etal.Humancardiacprogenitorcellsenhanceexosome releaseandpromoteangiogenesisunderphysoxia. Front.CellDev.Biol. 8,130 (2020).

37.Gonzalez-King,H.etal.Hypoxiainduciblefactor-1α potentiatesjagged 1-mediatedangiogenesisbymesenchymalstemcell-derivedexosomes. StemCells 35,1747–1759(2017).

38.Du,W.etal.Enhancedproangiogenicpotentialofmesenchymalstemcellderivedexosomesstimulatedbya nitricoxidereleasingpolymer. Biomaterials 133,70–81(2017).

39.Busbee,P.B.,Nagarkatti,M.&Nagarkatti,P.S.Naturalindoles,indole-3-carbinol (I3C)and3,3’-diindolylmethane(DIM),attenuatestaphylococcalenterotoxin B-mediatedliverinjurybydownregulatingmiR-31expressionandpromoting caspase-2-mediatedapoptosis. PLoSONE 10,e0118506(2015).

40.Elliott,D.M.,Nagarkatti,M.&Nagarkatti,P.S.3,39-DiindolylmethaneamelioratesstaphylococcalenterotoxinB–inducedacutelunginjurythrough alterationsintheexpressionofmicroRNAthattargetapoptosisandcell-cycle arrestinactivatedTcells. J.Pharmacol.Exp.Ther. 357,177–187(2016).

41.Shi,H.etal.3,3’-DiindolylmethanestimulatesexosomalWnt11autocrine signalinginhumanumbilicalcordmesenchymalstemcellstoenhance woundhealing. Theranostics 7,1674–1688(2017).

42.Thompson,C.A.,Purushothaman,A.,Ramani,V.C.,Vlodavsky,I.&Sanderson, R.D.Heparanaseregulatessecretion, composition,andfunctionoftumor cell-derivedexosomes. J.Biol.Chem. 288,10093–10099(2013).

43.Zuo,L.L.etal.Cadherin6isactivatedbyEpstein-BarrvirusLMP1tomediate EMTandmetastasisasaninterplaynodeofmultiplepathwaysinnasopharyngealcarcinoma. Oncogenesis 6,402(2017).

44.Zuo,L.etal.TargetingexosomalEBV-LMP1transferandmiR-203expression viatheNF-κBpathway:thetherapeuticroleofaspirininNPC. Mol.Ther. NucleicAcids 17,175–184(2019).

45.Kulshreshtha,A.etal.Simvastatinmediatesinhibitionofexosomesynthesis, localizationandsecretionviamulticomponentinterventions. Sci.Rep. 9, 16373(2019).

46.Lopatina,T.etal.Platelet-derivedgrowthfactorregulatesthesecretionof extracellularvesiclesbyadiposemesenchymalstemcellsandenhancestheir angiogenicpotential. CellCommun.Signal. 12,26(2014).

47.Wang,R.,Xu,B.&Xu,H.TGF-β1promotedchondrocyteproliferationby regulatingSp1throughMSC-exosomesderivedmiR-135b. CellCycle 17, 2756–2765(2018).

48.Kao,J.-K.etal.Heat-shockpretreatmentreducesexpressionandreleaseof TSLPfromkeratinocytesunderTh2environment. Pediatr.AllergyImmunol. 27, 62–69(2016).

49.Jelonek,K.,Widlak,P.&Pietrowska,M.Theinﬂuenceofionizingradiationon exosomecomposition,secretionandintercellularcommunication. Protein Pept.Lett. 23,656–663(2016).

50.Villasante,A.etal.Recapitulatingthesizeandcargooftumorexosomesina tissue-engineeredmodel. Theranostics 6,1119–1130(2016).

51.Haraszti,R.A.etal.Exosomesproducedfrom3DculturesofMSCsbytangential ﬂow ﬁltrationshowhigheryieldandimprovedactivity. Mol.Ther. 26, 2838–2847(2018).

52.Kim,M.,Yun,H.-W.,Park,D.Y.,Choi,B.H.&Min,B.-H.Three-dimensional spheroidcultureincreasesexosomesecretionfrommesenchymalstemcells. TissueEng.Regen.Med. 15,427–436(2018).

53.Gollmann-Tepeköylü,C.etal.miR-19a-3pcontainingexosomesimprove functionofischaemicmyocardiumuponshockwavetherapy. Cardiovasc. Res. 116,1226–1236(2020).

54.Pironti,G.etal.Circulatingexosomesinducedbycardiacpressureoverload containfunctionalangiotensinIItype1receptors. Circulation 131,2120–2130 (2015).

55.Jeon,H.etal.ExtracellularvesiclesfromKSHV-infected endothelialcells activatethecomplementsystem. Oncotarget 8,99841–99860(2017).

56.Park,S.J.,Jeon,H.,Yoo,S.-M.&Lee,M.-S.Theeffectofstoragetemperature onthebiologicalactivityofextracellularvesiclesforthecomplementsystem. Vitr.Cell.Dev.Biol.Anim. 54,423–429(2018).

57.Li,X.,Li,X.,Lin,J.,Sun,X.&Ding,Q.Exosomesderivedfromlow-intensity pulsedultrasound-treateddendriticcellssuppresstumornecrosisfactorinducedendothelialinﬂammation. J.UltrasoundMed. 38,2081–2091(2019).

58.Arscott,W.T.etal.Ionizingradiationandglioblastoma exosomes:implicationsintumorbiologyandcellmigration. Transl.Oncol. 6,638–648(2013).

59.Valadi,H.etal.Exosome-mediatedtransferofmRNAsandmicroRNAsisa novelmechanismofgeneticexchangebetweencells. Nat.CellBiol. 9, 654–659(2007).

60.Bruno,S.etal.Mesenchymalstemcell-derivedmicrovesiclesprotectagainst acutetubularinjury. J.Am.Soc.Nephrol. 20,1053–1067(2009).

61.Gatti,S.etal.Microvesiclesderivedfromhumanadultmesenchymalstem cellsprotectagainstischaemia-reperfusion-inducedacuteandchronickidneyinjury. Nephrol.Dial.Transpl. 26,1474–1483(2011).

62.Lai,R.C.etal.ExosomesecretedbyMSCreducesmyocardialischemia/ reperfusioninjury. StemCellRes. 4,214–222(2010).

63.Ranghino,A.etal.Endothelialprogenitorcell-derivedmicrovesiclesimprove neovascularizationinamurinemodelofhindlimbischemia. Int.J.Immunopathol.Pharmacol. 25,75–85(2012).

64.Zhao,R.,Liang,H.,Clarke,E.,Jackson,C.&Xue,M.Inﬂammationinchronic wounds. Int.J.Mol.Sci. 17,2085(2016).

65.Li,M.etal.Macrophage-derivedexosomesacceleratewoundhealing throughtheiranti-inﬂammationeffectsinadiabeticratmodel. Artif.Cells Nanomed.Biotechnol. 47,3793–3803(2019).

66.Kim,H.etal.Exosome-guidedphenotypicswitchofM1toM2macrophages forcutaneouswoundhealing. Adv.Sci. 6,1900513(2019).

67.Ti,D.etal.LPS-preconditionedmesenchymalstromalcellsmodifymacrophagepolarizationforresolutionofchronicinﬂammationviaexosomeshuttledlet-7b. J.Transl.Med. 13,308(2015).

68.Li,X.etal.Exosomederivedfromhumanumbilicalcordmesenchymalstem cellmediatesMiR-181cattenuatingburn-inducedexcessiveinﬂammation. EBioMedicine 8,72–82(2016).

69.Singer,A.J.&Clark,R.A.Cutaneouswoundhealing. N.Engl.J.Med. 341, 738–746(1999).

70.Yang,C.etal.Highly-expressedmicoRNA-21inadiposederivedstemcell exosomescanenhancethemigrationandproliferationoftheHaCaTcellsby increasingtheMMP-9expressionthroughthePI3K/AKTpathway. Arch. Biochem.Biophys. 681,108259(2020).

71.Shabbir,A.,Cox,A.,Rodriguez-Menocal,L.,Salgado,M.&VanBadiavas,E. Mesenchymalstemcellexosomesinduceproliferationandmigrationof normalandchronicwound ﬁbroblasts,andenhanceangiogenesisinvitro. StemCellsDev. 24,1635–1647(2015).

72.Zhang,B.etal.HucMSC-exosomemediated-Wnt4signalingisrequiredfor cutaneouswoundhealing. StemCells 33,2158–2168(2015).

73.Zhang,B.etal.HucMSCexosome-delivered14-3-3ζ orchestratesself-control oftheWntresponseviamodulationofYAPduringcutaneousregeneration. StemCells 34,2485–2500(2016).

74.Guo,S.C.etal.Exosomesderivedfromplatelet-richplasmapromotethereepithelizationofchroniccutaneous woundsviaactivationofYAPinadiabeticratmodel. Theranostics 7,81–96(2017).

75.Kota,S.K.etal.Aberrantangiogenesis:thegatewaytodiabeticcomplications. IndianJ.Endocr.Metab. 16,918–930(2012).

76.Chen,C.Y.etal.ExosomalDMBT1fromhumanurine-derivedstemcells facilitatesdiabeticwoundrepairbypromotingangiogenesis. Theranostics 8, 1607–1623(2018).

77.Liang,X.,Zhang,L.,Wang,S.,Han,Q.&Zhao,R.C.Exosomessecretedby mesenchymalstemcellspromoteendothelialcellangiogenesisbytransferringmiR-125a. J.CellSci. 129,2182–2189(2016).

78.Gong,M.etal.Mesenchymalstemcellsreleaseexosomesthattransfer miRNAstoendothelialcells andpromoteangiogenesis. Oncotarget 8, 45200–45212(2017).

79.Anderson,J.D.etal.Comprehensiveproteomicanalysisofmesenchymal stemcellexosomesrevealsmodulationofangiogenesisvianuclearfactorkappaBsignaling. StemCells 34,601–613(2016).

80.Xue,M.&Jackson,C.J.Extracellularmatrixreorganizationduringwound healinganditsimpactonabnormalscarring. Adv.WoundCare. 4,119–136 (2015).

81.Kim,Y.-J.etal.Exosomesderivedfromhumanumbilicalcordblood mesenchymalstemcellsstimulatesrejuvenationofhumanskin. Biochem. Biophys.Res.Commun. 493,1102–1108(2017).

82.Jong,O.G.,Balkom,B.W.M.,Gremmels,H.&Verhaar,M.C.Exosomesfrom hypoxicendothelialcellshaveincreasedcollagencrosslinkingactivity throughup‐regulationoflysyloxidase‐like2. J.Cell.Mol.Med. 20,342–350 (2016).

83.Wang,L.etal.Exosomessecretedbyhumanadiposemesenchymalstem cellspromotescarlesscutaneousrepair byregulatingextracellularmatrix remodelling. Sci.Rep. 7,13321(2017).

84.Fang,S.etal.Umbilicalcord-derivedmesenchymalstemcell-derivedexosomalmicroRNAssuppressmyoﬁbroblastdifferentiationbyinhibitingthe transforminggrowthfactor-β/SMAD2pathwayduringwoundhealing. Stem CellsTransl.Med. 5,1425–1439(2016).

85.Takahashi,Y.etal.Visualizationandinvivotrackingoftheexosomesof murinemelanomaB16-BL6cellsinmiceafterintravenousinjection. J.Biotechnol. 165,77–84(2013).

86.Smyth,T.etal.Biodistributionanddeliveryefﬁciencyofunmodiﬁedtumorderivedexosomes. J.Control.Release 199,145–155(2015).

87.Wiklander,O.P.B.etal.Extracellularvesicleinvivobiodistributionisdeterminedbycellsource,routeofadministrationandtargeting. J.Extracell. Vesicles. 4,26316(2015).

88.Annabi,N.etal.25thanniversaryarticle:rationaldesignandapplicationsof hydrogelsinregenerativemedicine. Adv.Mater. 26,85–123(2014).

89.Chen,X.etal.Magneticandself-healingchitosan-alginatehydrogelencapsulatedgelatinmicrospheresviacovalentcross-linkingfordrugdelivery. Mat. Sci.Eng.C.-Mater. 101,619–629(2019).

90.Mahanta,A.K.etal.Nanoparticle-inducedcontrolleddrugdeliveryusing chitosan-basedhydrogelandscaffold:applicationtoboneregeneration. Mol. Pharm. 16,327–338(2019).

91.Wei,W.etal.Synthesisandcharacterizationofamulti-sensitivepolysaccharidehydrogelfordrugdelivery. Carbohyd.Polym. 177,275–283(2017).

92.Neuerburg,C.etal.Growthfactor-mediatedaugmentationoflongbones: evaluationofaBMP-7loadedthermoresponsivehydrogelinamurine femoralintramedullaryinjectionmodel. J.Orthop.Surg.Res. 14,297(2019).

93.Chan,S.J.etal.Promotingneuro-supportivepropertiesofastrocyteswith epidermalgrowthfactorhydrogels. StemCellsTransl.Med. 8,1242–1248(2019).

94.Zhang,X.etal.Stimulationofwoundhealingusingbioinspiredhydrogels withbasic ﬁbroblastgrowthfactor(bFGF). Int.J.Nanomed. 13,3897–3906 (2018).

95.Ding,L.etal.Aself-assembledRNA-triplehelixhydrogeldrugdeliverysystem targetingtriple-negativebreastcancer. J.Mater.Chem.B. 8,3527–3533 (2020).

96.Chen,Z.etal.TargeteddeliveryofCRISPR/Cas9-mediatedcancergene therapyvialiposome-templatedhydrogelnanoparticles. Adv.Funct.Mater. 27,1703036(2017).

97.Xu,N.etal.WoundhealingeffectsofaCurcumazedoariapolysaccharide withplatelet-richplasmaexosomesassembledonchitosan/silkhydrogel spongeinadiabeticratmodel. Int.J.Biol.Macromol. 117,102–107(2018).

98.Wang,C.etal.Thefabricationofahighlyefﬁcientself-healinghydrogelfrom naturalbiopolymersloadedwithexosomesforthesynergisticpromotionof severewoundhealing. Biomater.Sci. 8,313–324(2019).

99.Zhang,Y.etal.Preparationofexosomesencapsulatednanohydrogelfor acceleratingwoundhealingofdiabeticratsbypromotingangiogenesis. Mat. Sci.Eng.C.-Mater. 120,111671(2021).

100.Shi,Q.etal.GMSC-derivedexosomescombinedwithachitosan/silk hydrogelspongeaccelerateswoundhealinginadiabeticratskindefect model. Front.Physiol. 8,904(2017).

101.Zhao,D.etal.GelMAcombinedwithsustainedreleaseofHUVECsderived exosomesforpromotingcutaneouswoundhealingandfacilitatingskin regeneration. J.Mol.Histol. 51,251–263(2020).

102.Zhang,K.etal.Enhancedtherapeuticeffectsofmesenchymalstemcellderivedexosomeswithaninjectablehydrogelforhindlimbischemiatreatment. ACSAppl.Mater.Inter. 10,30081–30091(2018).

103.Zhou,Y.etal.Injectableextracellularvesicle-releasedself-assemblingpeptide nanoﬁberhydrogelasanenhancedcell-free therapyfortissueregeneration. J.Control.Release 316,93–104(2019).

104.Wang,M.etal.Efﬁcientangiogenesis-baseddiabeticwoundhealing/skin reconstructionthroughbioactiveantibacterialadhesiveultravioletshielding nanodressingwithexosomerelease. ACSNano 13,10279–10293(2019).

105.Henriques-Antunes,H.etal.Thekineticsofsmallextracellularvesicledelivery impactsskintissueregeneration. ACSNano 13,8694–8707(2019).

106.He,C.,Zheng,S.,Luo,Y.&Wang,B.Exosometheranostics:biologyand translationalmedicine. Theranostics 8,237–255(2018).

107.Rani,S.,Ryan,A.E.,Grifﬁn,M.D.&Ritter,T.Mesenchymalstemcell-derived extracellularvesicles:towardcell-freetherapeuticapplications. Mol.Ther. 23, 812–823(2015).

108.Almeida,B.,Nag,O.K.,Rogers,K.E.&Delehanty,J.B.Recentprogressin bioconjugationstrategiesfor liposome-mediateddrugdelivery. Molecules 25, 5672(2020).

109.Allen,T.M.&Cullis,P.R.Liposomal drugdeliverysystems:fromconceptto clinicalapplications. Adv.DrugDeliv.Rev. 65,36–48(2013).

110.Zhang,Y.etal.Exosome:areviewofitsclassiﬁcation,isolationtechniques, storage,diagnosticandtargetedtherapyapplications. Int.J.Nanomed. 15, 6917–6934(2020).

111.Antimisiaris,S.G.,Mourtas,S.&Marazioti,A.Exosomesandexosome-inspired vesiclesfortargeteddrugdelivery. Pharmaceutics 10,218(2018).

112.Chen,Y.etal.Exosomedetectionviatheultrafast-isolationsystem:EXODUS. Nat.Methods 18,212–218(2021).

113.Lin,R.,Wang,S.&Zhao,R.C.Exosomesfromhumanadipose-derived mesenchymalstemcellspromotemigrationthroughWntsignalingpathway inabreastcancercellmodel. Mol.Cell.Biochem. 383,13–20(2013).