TherapeuticsignificanceofexosomesasadrugdeliverysystemApplication:Parkinson’sdisease

1stexaminer:dr.MaartenH.K.Linskens2ndexaminer:prof.dr.ArjanKortholtStudent:IlyaIvanovStudyprogram:MScMolecularBiologyandBiotechnologyStudentnumber:S2961806Dateofsubmission:12.11.18

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Index

Abstract……………………………………………………………………………………………………………………..…..3

1.Introductiontoexosomes……………………………………………………………………………………………3

1.1Exosomebiogenesisandrelease……………………………………………………………..…………....3

1.2Compositionandloadingofexosomes…………………...………………………………………...…...4

1.3Exosomesandtheirbiologicalfunctionsandapplications……………………………………..5

1.3.1Cell-to-cellcommunication……………………….…………………………………………………..5

1.3.2Immunologicalresponse………………………………………………………………………………5

1.4Cellularrecognitionandinternalizationofexosomes…………………………………………….6

2.Exosomesfordrugdelivery…………………………………………………………………………………………6

2.1Advantagesandlimitationsofexosomesoverotherdrugdeliverysystems……………6

2.2Exogenousdrugloadingmethods…………………………………………………………………………6

2.3Thesignificanceofexosomesurfacemoleculesandtheirmodifications…………………8

3.Blood-brainbarrier(BBB)…………………………………………………………………………………………...9

3.1ChallengesofcrossingtheBBB……………………………………………………………………………..9

3.2ManipulatingexosomesforabetterBBBpermeability………………………………………...10

4.Parkinson’sdisease(PD)andtherapeuticapproachesforitstreatment……………………....11

4.1Catalase-loadedexosomes………………………………………………………………………………….11

4.2Deliveryofdopaminetothebrain………………………………………………………………………15

4.3Downregulationofα-synucleinexpressionwithRNAinterference………………………17

Conclusion……………………………………………………………………………………………………………………20

References……………………………………………………………………………………………………………………20

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AbstractExosomesarenanovesiclessecretedbyallcelltypesandcanbefoundinallbodyfluids.Theycarryacell-specificcompositionofmembranephospholipidsandbiomolecules.Exosomebiogenesisandcompositionismediatedbyanumberprecisemechanisms.InthisreviewexosomeswillbeaddressedasdrugdeliveryvesiclesfortreatmentofParkinson’sdisease.PDtargetingrequiresthecrossingoftheblood-brainbarrierandexosomesareabletodoso.Varioustherapeuticcargomoleculescanbeloadedintoexosomesexvivo,suchascurcumin,dopamineormRNA.Exosomesappeartobeaprominentdrugdeliverysystemduetotheirbiocompatability,versatility,andtheabilitytopreservecargocatalyticactivityandtocrosstheBBB.ThislastoneisessentialforallCNSdiseases,includingPD.DifferenttherapeuticstrategiesforPDtreatmentandtherapeuticcargoincorporationinexosomesfordrugdeliverytothebrainarethemaintopicsaddressedinthisreview.

1. IntroductiontoexosomesExtracellularvesicles(EVs)aresubcellularstructuressecretedbyallcells;theycanbefoundinallbodyfluids.TherearethreetypesofEVs:exosomes(30-100nm)[5],microvesicles(100-500nm)andapoptoticvesicles(500-1000nm)[1].Thisreviewwillfocusonexosomesaspotentialdrugdeliveryvehicles.Exosomeswereinitiallyconsideredcells’mechanismofwastedisposalbutitwasrecentlynoticedthatexosomescarryamixofbiomoleculesthatreflectintracellularmolecularcomposition.Thatmakesexosomesthekeytounderstandingtheintracellularenvironmentandthestateofacell.

1.1 ExosomebiogenesisandreleaseThebiogenesisofexosomesbeginswithendocytosis.Earlyendosomescombineandbecomebigger,transitioningintoalateendosome.Theinwardbuddingofthelateendosomalmembraneleadstotheformationofintraluminalvesicles(ILVs).EndosomescontainingILVsarecalledmultivesicularbodies(MVBs).AscanbeseenfromFig.1andthemechanismofILVformation,theILVshavelipidbilayerthatistopologicallyidenticaltothecellplasmamembrane.InsideofILVscloselyresemblescytosolicenvironment.TheprocessofILVformationismediatedbyendosomalsortingcomplexrequiredfortransport(ESCRT).Thiscomplexconsistsof4smallerproteincomplexes:ESCRT0,ESCRTI,ESCRTIIandESCRTIII.Thismachineryisinvolvedintheinvaginationoflateendosomalmembranewiththeaidofcurvature-inducingfactors[2].However,asecond,ESCRT-independent,mechanismwasfoundtobeinvolvedinMVBformation[27].Endosomalmembranecontainslipidrafts(moredetailsin1.3),thatareregionsofmembraneenrichedincholesterol,gangliosideGM3,andsphingomyelin.Neutralsphingomylinase2(nSMase2)isabletoconvertsphingolipidstoceramide.Ceramideinducescoalescenceanddomain-inducedbuddingoflateendosomalmembranethuspromotingILVformation[28].

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Figure1.BiogenesisofMVBsandreleaseofexosomesandotherEVs[2]AfterILVformationisfinishedMVBsarecarriedbymotorproteinstotheperipheryofacell,wheretheyfusewithplasmamembrane(Fig.1).ThefusionprocessismediatedbySNAREsandtetheringfactors.AfterbeingreleasedintotheextracellularspaceILVsbecomeexosomes[23].

1.2 CompositionandloadingofexosomesExosomesarecomposedofmanydifferentkindsofenzymes,proteincomplexes,nucleicacids.Themainphospholipidsfoundintheexosomalmembraneincludephosphatidylcholines,phosphatidylamines,andphosphatidylserines.Additionally,cholesterol,gangliosideGM3,andsphingomyelinshavebeenfoundinexosomalmembranesofmostcelltypes.Thislipidcompositionresembleslipidrafts,membraneregionspresentincellmembrane[30].Lipidrafts,previouslymentionedin1.1,areafeatureofcellmembraneand,therefore,arealsopresentinexosomalmembrane.However,exosomalmembraneseemstohavehigheramountofproteinthanthecellmembrane.So,exosomallipidraftsareonlyresembingthelipidraftsofcellmembrane[5].Thereisalistofproteinsthatarealwayspresentinexosomesastheyareessentialforthemaintananceofexosomalmicroenvironment.HSPs,orheat-shockproteins,arefoundinalltypesofexosomesandareimportantforwhenexosomeexperiencesstressconditions.Othercommontypesofproteinspresentinexosomesincludetetraspanins(CD9,CD63etc.),histones,integrins,smallGTPases,annexinsandactins[6].

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It’snotfullyunderstoodyethowproteinsaresortedintoILVs.Thereareseveralmethodsforproteinsorting,ofwhichthemostimportantoneistheubiquitin-dependentsortingbyESCRTmachinery[23].However,notallexosomalproteinswerefoundtobeubiquitinated,whichsuggestsanothermechanismofproteinuptakeintoexosomes[5].Plasmamembraneanchortags,suchasmyristoylation,prenylationandpalmitoylationcantargetproteinstowardsthesiteofmembranebuddingforILVformation.Therefore,notonlyubiquitinationbutalsoposttranslationallipidmodificationscanserveasasortingsignalforproteinloadingintoexosomes[2].ExosomesareknowntocontainnucleicacidssuchasmRNAandmiRNA.ThisfactplacesthemasapotentialRNAdeliveryvehicles.EssentialdifferencesbetweentotalRNAprofileofparentalcellsandofexosomeswerenoticed.ThismeansthatsomemRNAmaybeenrichedinexosomesbutnotintheparentalcells.Aconclusionisthatexosomalnucleicacidcompositionisalsoregulatedbyasortingmechanism[6].Thissortingmechanism,incontrasttosortingofproteins,isESCRT-independent,anddependsonceramide.WehaveseenalreadythatnSMase2isabletoconvertsphingolipidspresentinlipidraft-likeregionsintoceramide.SomeRNAscanbedeliveredtoraft-likeregionsbyRNA-bindingproteins(RBP).A2B1bindstomiRNAandhasanaffinityforceramide.Therefore,RNAloadingisdependentontheinteractionofRNAorRNA-RBPcomplexwithlipidraft-likeregionofMVBmembrane.Inthisway,RNAispresentatthesiteofILVformation[23].

1.3 Exosomesandtheirbiologicalfunctionsandapplications

1.3.1 Cell-to-cellcommunicationThehypothesisthatexosomespropagateintercellularcommunicationisbasedoncell-specificcontentofexosomesanditstargeteddeliverytorecipientcells.Moreover,studieshaveshownthatthedeliveryoffunctionalbiomoleculestocellsresultsinaphenotypicalresponse[7].Ithasbeenshownthattumor-derivedexosomescanpromoteangiogenesis[29].Thesameexosomescanalsotargetepithelialcellsandresultinaberrantmitosisandincreasedcellproliferationleadingtotheprogressionoftumor[8].Exosomesintumormicroenvironment(TME)canhaveanadverseeffectonCD8+T-cells.Exosomesofdevelopedtumorcellsareabletocarryimmunosuppressivemoleculesandmaketumorcellsescapeimmunesystemsurveillance.ThisresultsininhibitionofT-cellproliferation,suppressedactivityofCD8+T-cellsandinhibitionofkillercellsanti-tumorfunction[8].Thesefindingsindicatethattumorcellscanworktowardstheircommongoal(suchastumorproliferationandescapingimmuneresponse)bysecretionofexosomesexpressingspecificbiomolecules.

1.3.2 Immunologicalresponse

Exosomesinitiallywerebelievedtobeusedfor“wastedisposal”.Infact,exosomescanhaveaveryimportantroleinimmuneregulation.Forexample,B-cell(andlaterondendriticcell(DC))derivedexosomesareknowntoexpressMHCclassIIontheirsurface[6].B-cellsareabletouptakeantigens,whichthenbindtoMHCclassII.B-cellsthensecreteexosomesthatexpressantigenloadedMHCclassIIontheirsurface.AntigenisdeliveredtoCD4+T-cellsandinducesantigen-specificimmuneresponse[5].

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1.4 CellularrecognitionandinternalizationofexosomesExosometargetingiscell-specific,thereforecellsshouldhavewaystorecognizeanduptakeexosomes.Recognitionofexosomescanhappeninthreeways,asproposedbyMcKelveyetal.Recognitionbyfree-floatingleadstotheopsonization,i.e.thedefensiveactionofimmunesystem.Opsonizationmeansthatexosomesareinternalizedbymacrophages.ExosomesexpresslargearrayofcytokinesthatmayattractnearbyT-cellsandotherimmunecells.Thisresultsininternalizationbyphagocytosis[13].Recognitionbyadhesionisgovernedbyintegrinsthatundergoatransitionfromlowtohighaffinitystate.Integrinsarethenabletooligomerizecausinglymphocytes,thathaveaffinityforoligomerizedintegrins,tobindtotheintegrin-boundexosomes.ThisapproachcouldbepartiallyinvolvedinT-cellrecognitionofexosomes[13].Agoodexampleofantigen-basedrecognitionwouldbeactivationofT-cells.DC-derivedorBlymphocyte-derivedexosomeshaveMHCclassIImoleculeboundtospecificantigen.Suchexosomesareabletoactivateantigen-specificT-cellsandevokeanimmuneresponse[13].Thefactthatallthreeexamplesofexosomerecognitionincludeinteractionswithimmunecellsfurthersupportsthenotionthatexosomesplayveryimportantimmunologicalrole.Internalizationofexosomesbyrecipientcellshappenseitherbydirectfusionofmembraneandexosomecontentsreleaseintothecytosolorbyendocytosis.Endocytosiscanbemediatedbyclathrin,dynaminorcaveolin.Exosomescanalsobeinternalizedbyphagocytosisormacropinocytosis[24].

2. Exosomesasdrugdeliveryvehicles

2.1 AdvantagesandlimitationsofexosomesoverotherdrugdeliverysystemsSeveralpropertiesmakeEVssuchgreatdrugdeliverysystem.Whilebeingprotectedbylipidbilayer,theircargoisstableinthecytosolycenvironment.Exosomesprovetobeespeciallygoodfordeliveringsmallhydrophilicmolecules,likemRNAsordopamine[6].Theirstructureallowsthemtobypassmanybiologicalbarriersandeventhetoughestonetopass,theblood-brainbarrier.Byexpressingligandsontheirsurfaceexosomescanbeutilizedfortargeteddeliverybymakinguseofligand-receptorspecificinteractions.However,exosome-baseddrugdeliveryhasitslimitations.First,notmuchisknownaboutthemechanismofBBBcrossing.Thislimitsknowledge-baseddiscoveryofmodificationsforincreasedBBBpermeability.Second,thereislackofunderstandingofpotentialside-effectscausedbytherapeuticcargo[6].Inaddition,currentEVisolationandpurificationtechniquesarestillfarawayfromthequalityrequiredforconductionofhumanclinicaltrials[6].Also,cargoloadingismuchmoreefficientwithsmallmoleculesandnucleicacidswhichsignificallynarrowsthescopeofpharmaceuticalapplicationofexosomes.

2.2 ExogenousdrugloadingmethodsIfexosomesareutilizedfordrugdeliverythentheyshouldcontainsometherapeuticcargoincorporatedinsidethem.Clearly,thechancesofsuchcargobeingloadedbythecell’sendogenoussortingmachinery(e.g.ESCRT)areverylow.Thiscallsforthedevelopmentofmethodsfortheloadingofanexternalcargo.Someofthesemethodsarepresentedinthissection.Theycanbedividedintotwomaingroups:loadinginvivoandinvitro.Thereare

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threegeneralapproachesforexosomaldrugloadingdescribedbyElenaV.BatrakovaandMyungSooKim(Fig.2).Thefirstapproachisbasedonisolationandpurificationofnativeexosomeswithsubsequentinvitroloadingofthedrugmolecules.Thereareactuallymanypossiblestrategiesforcargoloadinginvitro.Thestrategyshouldbechosenbasedontypeofcargomolecules.Moleculeswithlowmolecularweightlikecurcuminordopaminecanbeloadedintoexosomesbyincubationatroomtemperatureorbyfreeze/thawcycles.Theloadingcapacityvariesfrom7.2%to11.7%.Themildloadingcapacitycanbeexplainedbyalargearrayofmoleculesalreadybeingpresentinsideexosomes[1].Higherloadingcapacitycanbeachievedbyincreasingthecargoconcentrationtomaximizetheconcentrationgradientandenhancethediffusionacrossthelipidbilayer[21].LoadingefficiencycanbeincreasedtwicebytheadditionofsaponinwhenloadedbyincubationatRT[16].Largeandhydrophobicmoleculescanbemoreefficientlyloadedintoexosomesbysonicationorextrusion.Thesemethodsresultintheformationoftemporaryporesandpossiblelipidbilayerreformation.Theformationofsuchporesallowslargemoleculestoenterexosomes[16].ExogenousRNAcanbeloadedintoexosomesbyelectroporation[Alvarezetal].HighertemperatureandprecomplexationofRNAwithcationicliposomescanimproveRNAloading[1].Allinvitroloadingstrategiesaredifferentfromhowexosomesarenaturallyloaded.Theycanthereforehaveanindirectimpactonthefinalstructureofexosomes.Itisnotclearwhybutdifferentdifferentstrategiesofcargoloadingintoexosomescanhaveaneffectontherateofcargoreleasefromexosomes(Fig.4C)[16].Anotherwaytoloadcargoistomakeanorganismproducethecargoproteinandloaditinvivo(Fig.2C).Ndfip1isanenzymethatbindstoWWdomainandperformsubiquitination.BymakinguseofthisreactionSterzenbachetal.managedtoloadCrerecombinaseinvivo.CoexpressionofWW-CreandNdfip1inhostcellsresultedinasuccessfulubiquitinationofCrerecombinaseanditsloadingintoexosomes.WWtagdoesn’tinterferewithCrelocalizationoractivity[12].ByusingthisNdfip1reactivityitcouldbepossibletoloadotherproteinsintoexosomesinasimilarway.

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Figure2.Cargoloadingapproaches.(A)Throughexternalloadingofcargointopuriofiedexosomes.(B)Throughloadingofdrugintoparentalcellsandsubsequentincorporation.(C)Throughengineeringofrecombinantcellsthatproducethedrugandloaditintoexosomes.[1]

2.3 ThesignificanceofexosomesurfacemoleculesandtheirmodificationsExosomespossessanintrinsicabilitytocrossvariousbiologicalbarriersandthuscandelivertheircontentstomostcellsinanorganism.Exosomescontainmanydifferentmoleculesontheirsurface.Exosomesurfacecanbemodifiedforbetterrecipientcelltargeting,stabilityorasafluorescentbiomarkertomeasureitsdistribution.Theroleofexosomesurfacecompositionanditsmodificationsareexplainedinthissection.Exosomalsurfacemoleculescanbeusedfortherapeuticpurposes.ApplicationofexosomenativesurfaceglycosphingolipidglycanfortreatmentofAlzheimer’sdiseasewassuggestedbyYuyamaetal.Intheirstudytheytookadvantageoftheaffinityofamyloid-βforglycosphingolipidglycangroupsexpressedonthesurfaceofneuroblastomaexosomes.Amyloid-βbuildupinthebraindrivesthepathogenesisofAlzheimer’sdisease.Amyloid-βboundNBexosomesdeliveredamyloid-βtomicroglialimmunecellsinthebrain,whichdegradedamyloid-βtogetherwithexosomes.TakingadvantageofexosomalsurfacephospholipidscandevelopintoastrategyfortargetingADsymptomsinthefuture[32].Targetingtocertainregionsinabodycanbeachievedbyexpressingcertainpeptidesonthesurfaceofexosomes.Alvarez-Ervitietal.wasthefirstgrouptocomeupwithastrategyofexosomemodificationtotargetthebrain.ItwasachievedbyhostcellsexpressingLamp2bproteinfusedtorabiesvirusglycoprotein(RVG).Lamp2bisanexosomalmembraneproteinthatusuallygetsincorporatedintoexosomalmembrane.RVGpeptidecauseshomingofexosomestothebrain.TheyobtainedexosomesthatexpressLamp2bontheirsurfacefusedtoRVGandachievedbettertargetingtothebrain[23].Agroupofresearchersin2014noticedthatpeptidesattachedtoN-terminusofLAMP2baresusceptibletoproteolyticdegradationinacidicconditions.TheyrealizedthatglycosylationmotifGNSTMcanprotectpeptidesfromsuchdegradation.ThispotentiallyleadstomoreRVGexpressedonexosomalsurfaceandabetterdrugdelivery[25].

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3. Blood-brainbarrier(BBB)BBBgovernsahighlyrestrictedpassageofbiomoleculesfrombloodstreamtothebrain.StructureofBBBcanbeseeninFig.3.BigpartofBBBisbasallamina,whichislocatedonthebrainsideandseparatesendothelialcellsfromastrocytes.Itiscomprisedoflaminin,heparinsulfate,andcollagen.AstrocytesprovidesupportforendothelialcellsandalsocontributetoBBBoverallstructure.Inbetweentheendothelialcellsisthespacecalledtightjunctions.Thesetightjunctionsareformedbyclaudin,occludinandZO-1[15].

Figure3.Structureoftheblood-brainbarrier.Acquiredfrom:https://www.emf.ethz.ch/en/knowledge/topics/health/blood-brain-barrier/

3.1 ChallengesofcrossingtheBBBEndothelialcellliningisthemainbarrierthatpreventstheflowofpathogenmoleculestothebrain.Thisbarrierismaintainedduetothepresenceoftightjunctionsbetweenendothelialcells.Theirfunctionistokeependothelialcellliningintactandtopreventfreepassageofmoleculestothebrain.TightjunctionsarealsoresponsibleforexceptionallyhighelectricalresistanceacrossBBBandprevententryoftoxicsubstancesandpathogens.BBBnotonlyrestrictspassagetothebrainbutalsotakesaroleinexcretionbyexpressingABCtransportersontheendothelialcells.Thesetransportersprovideasecondlineofdefenseagainstunwantedmoleculespassingtothebrainandremovemoleculesthatmanagedtopassthroughthetightjunctions[15].

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BBBissopreventivebecausebrainisaverypreservedmicroenvironmentandmanymoleculescanhaveanadverseeffectonbrainfunction.ThetimespentinthebloodflowbeforecrossingtheBBBiscalledcirculationtimeandisoftenmaximizedwhenitcomestotherapeuticapplications.Thereasonisthattherearemacrophagespresentinbloodthatinternalizenanoparticles(aprocesscalledopsonization)thatappeartobepathogenic,meaningtheyexpressspecificantigenpeptidesthattriggeranimmuneresponse.Thebiggerthecirculationtimethelongerthenanoparticlescanresideinthebloodwithoutundergoingopsonization.Thisislikelythemaindisadvantageofusingliposomesorexosome-likeengineerednanoparticles–theyarecytotoxicandundergorapiddrugclearancebymononuclearphagocytesystem(MPS)[16].That’swhytheresearchnowisfocusedontheapplicationofnaturallyproducedexosomesinordertoavoidopsonizationandincreasebiocompatability.

3.2 ManipulatingexosomesforabetterBBBpermeabilityIt’spossibleforexosomestocrossBBBanditwasprovenbydetectingtumor-derivedexosomesandmicrovesiclesofglioma-bearingmicecirculatinginblood.ThisfindingsuggeststhatexosomeshaveanintrinsicabilityforcrossingtheBBB[16].However,it’sstillunclearwhattheprecisemechanismofBBBcrossingis.ItwasproposedthatexosomescouldbeinternalizedintoMVBsofthefirstlayerofendothelialcellstothenbereleasedandinternalizedbythesecondlayerofcellsandsoonuntilreachingthebrain[31].OneofthepossibilitiestolocallyincreaseBBBpermeabilityistodisruptthetightjunctionsbysonication.Theproblemisthatthisapproachraisestheriskofunwantedsubstancesenteringthebrain.Amuchmorefriendlyapproachisbytakingadvantageoftranscytoticpathwayofnanoparticlesbindingtoendothelialcells[15].Transcytosiscanbeoftwotypes:adsorptiveandreceptor-mediated.Incaseofadsorptivetranscytosisexosomeswouldcomeincontactandbindtotheplasmamembraneofendothelialcells.Nanoparticleswithpositivelychargedsurfacearemorelikelytoadsorbratherthannanoparticleswithnegativelychargedorneutralsurface[15].Receptor-mediatedapproachisbasedonligand-receptorspecificinteractions.Endocytosisthatfollowscanbemediatedbyclathrinorcaveolin.Withclathrin-mediatedendocytosis200nmendosomesarebuddingoffinclathrinenrichedmembraneregions.pHoftheseendosomeslowersastheyprogresstothelatestageandthecontentsaresubjectedtolysosomaldegradation[15].Caveolin-mediatedendocytosisoccursinlipidraftsandresultsinvesiclesofmuchsmallerdiameter(80nm).Caveolin-coatedvesiclesavoiddegradationandtheirfatedependsonthecelltype[15].BetterBBBpermeabilitycanbeachievedbyoptimizationofseveralpropertiesofdrugcarryingvesicles.First,nanoparticlessmallerthan200nmaremorelikelytocrossBBB[15].Asitwasmentionedpreviously,positivelychargedsurfacemoleculesincreasethechanceofadsorptivetranscytosis.NanoparticlescanbecoatedwithPEGcorona,whichleadstoincreasedcirculationtimeanddecreaseduptakebyimmunesystem[15].However,itwasreportedthatPEGylatedliposomeslosttheirlong-circuationpropertyonthesecondweekofsystemicadministrationtomice.Thiscreatesaproblemassociatedwithpossiblelong-termtreatmentandincreaseedopsonizationofPEGylatednanocarriervesicles[11].Surfacemodificationstoenhancereceptor-mediatedtranscytosishavebeenused,includingligandsforLf,LRP1andTfreceptors[9].Animportantconcepttonoteisavidity.

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It’srecommendedtoavoidintroducingtoomanydifferentligandsontheexosomalsurfaceasinthatcasetheywillallhinderonanother.Goodendocytosiscanbeachievedbyabalancedexpressionofnecessaryligands.

4. Parkinson’sdisease(PD)andtherapeuticapproachesforitstreatmentPDisthesecondmostcommondiseaseinneurodegenerativegroupafterAlzheimer’sdisease.NeurodegenerationistheprocessofaccumulateddamagereceivedbyneuronsinCNS.It’ssymptomsincludedifficultywhilewalking,shakingaswellassleeping,thinking,andbehavioralproblems.DementiaisusuallyoneofthesymptomsofPDinanadvancedstage[17].Thecostofbraindiseasesin2014intheUSreachedUSD$789billion.Asneurodegenerativediseasesareknowntoprogressandworsenwithageandaveragelifeexpectancyinthedevelopedcountriesisonlyincreasingitisexpectedforthesecoststoriseevenhigher[15].AtthispointthereisnocureforPD.It’sstillunclearwhatthecauseofPDisbutitisconsideredtoinvolvebothgeneticandenvironmentalfactors.Neuronsinsubstantianigraaredyingandthiscausesmotordisorders(thusthename–neurodegeneration).Neurodegenerationleadstodecreaseddopaminelevels.Therefore,dopaminesupplyappearstobeanattractivestrategytodelaytheprogressionofPD.Alotofreactiveoxygenspecies(ROS)werefoundinthebrainlesionassociatedwithPDpathology.ROSaredangerousastheyaredamagingthetissuearoundthemandcanleadtolossofneuralcells.NormallytheeliminationofROSisdonebyantioxidantsorreductases.SupplyingastrongantioxidantcanresultinreducedROSlevelanddelayedPDprogression.OneofthekeyfeaturesofPDisanincreasedaccumulationofα-synucleininLewybodies.AnincreasedproductionofthisproteintogetherwithshapealteringS129phosphorylationcausetheformationofaccumulatedα-synucleinaggregatesinLewybodies.ThisprocessiscloselyassociatedwithPDatthemoment[18].GenetherapyorRNAinterferencestrategiesseemappealingasawaytoreducetheamountofα-synuclein.

4.1 Catalase-loadedexosomesCatalaseisanenzymethatcatalyzesdecompositionofhydrogenperoxidetooxygenandwater.Catalase’smainfunctionisthereductionofoxidativestressandeliminationofROS.CatalaseisoneofthestrongestantioxidantsandcanbeusedforreductionofROSinPDpatients.Injectingcatalaseintravenouslyispossiblebutit’salargehydrophobicproteinsoitwillbechallengingforbulkcatalasetocrossBBB.Therefore,catalasewillprobablyhavelongcirculationtimemeaningthatitwillmostlikelyundergoopsonization.Incorporatingcatalaseintoexosomeswillpreserveitscatalyticactivityandpreventdegradation.Haneyetal.showedintheirresearchthatcatalase-loadednanoparticleswereabletoreachvariouscelltypesinvitro[19].IntheiresearchtheymixedcatalasewithPEI-PEGblockcopolymer.Thisresultedinnanoparticleswithcatalase-polymercomplexcoreandPEGcorona,termednanozymes.Nanozymeswereloadedintomacrophagecellsbyincubationfor1h.[19].Theyinvestigatedtheabilityofmacrophage-derivednanozymestoreachthreetypesofcells:brainmicrovesselendothelialcells(BMVEC),neurons,andastrocytes.Ineachcasemacrophagenanozymesshowedbettertransfertotherecipientcellsthanfreenanozymes(Fig.4)[19].

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NanozymetransferfrommacrophagecellstoBMVEC,bovinebrainmicrovesselendothelialcells(BBMEC),andneuronswasmodelledwithandwithout(2mmdistance)thephysicalcontactofthetwocelllines.TotestwhethernanozymetransferwascontactdependentforBBMECandneuroncellsadhesioninhibitorswereadded,namelytrypsinandlocostatin.Nanozymetransfertothesecelltypeswasnotaffectedbyadhesioninhibitors.Nevertheless,closecontactseemstoimprovenanozymetransferfrommacrophagecellstoBMVEC[19].Nanoparticleaccumulationinrecipientcellswasmeasuredbyflowcytometry.Haneyetal.managedtoshoweffectivetransferofcatalasenanoparticlesfrommacrophagecellstoBMVEC,neurons,orastrocytes.Theyshowedthatcellularcontactfavorsthetransportbutadhesionisnotnecessary.Themainlimitationoftheirworkistheabsenceofinvivotrials.Nanoparticlesareknowntobesusceptibletoopsonization,henceit’snotclearwhetherthesenanoformulationswillreachbraincellsinmousePDmodel.

Figure4.LevelsofRITC-labelledcatalasenanozymeaccumulationin(A)BMVEC,(B)Neurons,(C)Astrocytesmeasuredbyflowcytometry[19].4yearslaterthesamegroupofresearchersdidasimilarexperimentbutwithseveralimportantchanges.Theywerealsoaimingatthedeliveryofcatalasetothebrainbutthistimedecidedtotakeadifferentapproach.Firstofall,insteadofPEG-coatednanoparticlestheyusedexosomes.Althoughintheir2011worktheyshowedlowcytotoxicityofnanozymesdrugdeliveryusingexosomeswouldprobablyprovetobemorebiocompatible.Macrophage-derivedexosomeswereloadedwithcatalaseinvitroviaincubationatRT(withandwithoutsaponin),freeze/thawcycles,sonication,andextrusion[16].Catalase-loadedexosomeswerecalledexoCATs.ExoCATsweretestedontheirloadingefficiency,catalasereleaserateandantioxidantactivity.

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TheresultsofloadingefficiencyareshowninFig.5A.Loadingwithsonicationandextrusionresultedinthehighestloadingefficiency,possiblybecausecatalaseisalargeprotein(≈240kDa)andsimplepermeationwillnotresultinagoodcatalaseloading.Extensivereformationofexosomesmembraneundersonicationandextrusionmethodsallowedcatalasetobypassthelipidbylayer[16].Fig.5BshowsenzymaticactivityofexoCATsthatwasdonetoevaluatetheabilityofexosomestopreservecatalaseactivity.Catalase,loadedbysonicationandextrusion,showsthehighestenzymaticactivity,followedbycatalase,loadedwithfreeze/thawcyclesandincubationatRT.ThesefindingsfurtherprovethedatafromFig.5A.Catalasereleasewasmeasuredwithdialysismembraneswith2000kDacutoff.CatalaseloadedatRTwithoutsaponinshowedthefastestreleasewhileexosomesloadedbysonicationonlyreleased40%catalasein24h(Fig.5C).Thus,sonicationofexosomesresultedinhighloadingefficiencyandsustained,prolongedcatalaserelease[16].

Figure5.Resultsofloadingefficiency(A),catalyticactivityofcatalase(B)andrateofcatalsereleasefromexosomes(C)[16]Theresearchersinthisstudydidnotdoanymodificationstotheexosomesurface,theyreliedontheintrinsicabilityofexosomestocrosstheBBB.Hence,itwasnotentirelyclearwhichinteractionstookplaceinexosomerecognition.RateofexosomaluptakebyPC12recipientcellswasmeasuredbymeasuringfluorescence.Thisexperimentrevealedthatexosomesloadedbysonicationshowedthehighestuptakerate.Freeze/thawloadedexosomesandexosomesloadedbyincubationatRTbothdidntresultinhighuptakebyPC12cells(Fig.6).Itispossiblethatduetomembranereformationofexosomesloadedbysonicationtheywereacceptedatahigherratethantheexosomesloadedbyothermethods.Membranereorganizationcouldrevealcertainhydrophobicregionsorintegralproteinsthatresultedinimprovedinteractionswithrecipientcellmembrane[16].

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Figure6.UptakeofexosomesbyPC12recipientcells.ExosomeswerestainedwithDILdyeandfluorescencewasmeasuredbyShimadzuRF5000spectrophotometer.Theamountofcatalasewasnormalizedforthetotalproteincontent[16]ExoCATneuroprotectiveabilitywasmeasuredbyMTTassayandtheresultsarepresentedinFig.6.Theextentofneuronprotectionisshownasthepercentageofsurvivedneuronsrelativetotheinitialnumberofneurons.NeuroprotectionisthehighestforexoCATsloadedbysonication(Fig.6).AnadditionofsaponinduringincubationatRTsignificantlyimprovesneuroprotectionbyexoCATs.AllotherloadingmethodsdidnotresultinexoCATswithhighneuroprotectiveactivity,includingcatalasealone[16].

Figure6.Neuroprotectionevaluatedoncellspre-incubatedwith6-OHDA(C)[16].

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Overall,exosomesloadedbysonication,extrusion,andincubationatRTwithsaponinshowedhighloadingefficiency,preservationofcatalaseenzymaticactivityaswellasprolonged,sustainedrelease.Itwashypothesizedthatsaponinmayselectivelyremovesomeofintegralmembranemolecules,likecholesterol,andthuscreateporesforsuccessfulcatalasediffusion[16].ExoCATsloadedbypermeationwithsaponinandbysonicationprovidedhighloadingandsustainedrelease.ThesetwotypesofexoCATsalsoshoweddecreasedbraininflammationandimprovedneuronalsurvivalinvivo(Fig.6).Potentially,exosomesloadedbypermeationwithsaponinaresuperiortoexosomesloadedbysonicationastheycouldhaveamoreuniformandintactsurfacemorphology.Suchmorphologycandecreasetheirrecognitionbyimmunesystemandincreasetheirbloodcirculationtimeforfutureclinicaltrials[16].

4.2 DeliveryofdopaminetothebrainNeurodegenerationinsubstantianigraisfollowedbydopamindeficiencyinPDpatients.Dopaminesupplyservesasaneffectivestrategytoincreasedopaminelevelinthebrainandincreaseneuronalsurvival.Deliveryofbulkdopaminetothebrainseemshardlypossibleduetotherapidclearancebyimmunesystem.Itwasconfirmedin2015byPahujaetal.thatdopamine-loadednanoparticlesarelesscytotoxicthanbulkdopamine.Administrationofdopamine-loadednanoparticlessignificantlyreducesdopamineopsonizationandensuresdopaminedeliverytoSH-SY5Ycellsinducedwith6-OHDA[20].Althoughincorporationofdopamineintonanoparticlesreduceddopamineopsonization,NPsarestillusuallyrecognizedbymacrophagesandrisktobeinternalized.Tumor-derivedvesiclesoriginatinginmicebrainwerefoundinbloodcirculation.ThisindicatesthatexosomesareabletopenetratetheBBBwithoutanyadditionalenhancements[16].TheresearchgroupofQuetal.usedthisabilityofexosomesandisolatedbonemarrowderivedmacrophageexosomes.TheseexosomeswereloadedwithdopaminebyincubationatRTfor24h.Theyusedsaturateddopaminesolutionandachievedloadingof16%.Freedopaminewasremovedbyultracentrifugation.Dopamineisarelativelysmallandhydrophilicmolecule,thereforeincubationatRTseemslikeagoodstrategytoloaddopamineintoexosomes[21].BraintargetingbyexosomesandtheirdistributioninthebrainwasmodelledonbEnd.3andSH-SY5Ycells.InbEnd.3modelofBBBtheconcentrationofdopaminedeliveredbybloodexosomeswas1.02±0.15mmol/g,whiletheconcentrationoffreedopaminewasnon-detectible.Freedopamineaccumulatedinkidney,liver,andlungswhereasdopaminefrombloodexosomesachieved15-foldincreaseindsitributioncomparedtofreedopamine6hoursafterthestart.Freedopaminelevelsaremuchloweralsobecauseofopsonization[21].AnexperimentwithSH-SY5Ycellsmodelshowedthatexosomaluptakehappenedthroughenergy-dependentendocytosis,whichinvolvedclathrin-andcaveolin-dependentprocessesandmacropinocytosis[21].ExosomesappearedtofacilitatedopaminetransportacrossBBB.ThistransportcouldbeinhibitedbyanadditionofNaN3oratlowtemperature(Fig.7).

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Figure7.AccumulatedclearancevolumeobtainedinBBBinvitromodelwithtime[21]Oneofthegoalsoftheresearchwastoinvestigatetheinteractionsunderlyingexosomerecognitionbytargetcells.Transferrinisanabundantproteinpresentinbloodthatbindstoitsreceptor,TfR.ThereisTfRpresentonthesurfaceofblood-derivedexosomesaswellasonthesurfaceofendothelialcells.Quetal.exploredtheinteractionbetweenbEnd.3cellsandblood-derivedexosomesandelucidateditwasdependentontransferrin-TfRbinding.WiththehelpofZDOCKrigid-bodyproteindockingprogramtheydiscoveredthatthebindingoccursbetweentransferrindimerandtwoTfRsformingatetramer(Fig.8)[21].

Figure8.TransferrindimerboundtotwoTfRproteinspredictedbyZDOCKdoickingprogram.Transferrinshowninsurface(left)andinribbon(right)structure[21]Fortheassessmentoftherapeuticefficacyinvivoclinicalmiceweredividedinto5groups.Thegroupsare:shamcontrol(healthymice,treatedwith0.9%saline),micetreatedwithemptybloodexosomes,freelevodopa,freedopamine,anddopamine-loadedexosomes.Activitywasmeasuredasdopaminelevelrelativetohealthymice(shamcontrol).Fromfig.9wecanseethatDAexosomestreatmentwasthemostsuccessfulinelevatingdopaminelevel,resultinginover55%dopaminecomparedtoshamcontrol[21].

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Figure9.Relativedopaminelevelinmicecomparedtoshamcontrol[21]DA-loadedexosomeswerealsothebesttreatmenttoelevatetheamountofsuchimportantenzymesassuperoxidedismutaseandglutathionineperoxidase,importantantioxidantenzymes.Thissuggeststhatdopamine-loadedbloodexosomes“canhelpimprovedopaminergicneuronsandamelioratediseasephenotypeinamousemodelofPD”[21].ThisstudyconductedbyQuetal.presentsincreaseddopaminelevelinmousemodelofPDachievedbysystemicinjectionofdopamineloadedbloodexosomes.It’spossiblethatdopaminecouldbethecausefordopaminergicneurogenesis,meaningthattheneuralcellsstartedproducingendogenousdopamineafterthistreatment[21].Suchtreatmenthasotherindirectpositiveeffectslikeanincreaseinantioxidantenzymesresultinginloweredoxidativestress.Intheend,bloodexosomeswereabletotargetbEnd.3cellsduetotransferrin-TfRinteractionsandachieveddecreasedoxidativestressandincreasednumberofneuronsanddopamine.

4.3 Downregulationofα-synucleinexpressionwithRNAinterferenceItisknownthatPDischaracterizedbyaccumulationofmutatedα-synucleinaggregatesinLewybodies.ThefirstexternallyloadedcargosfordrugdeliverybyNPswereRNAmolecules.Theyaresmallmoleculesandhaveaverybroadtargetscope.DeliveryofRNAmoleculesinliposomesdatesbackto1978[22].IntheirresearchCooperetal.attemptedtoreducetheexpressionofα-synucleininthebrainbysystemicinjectionsofsiRNA.Anadvantageofthisstrategyisthatitisversatile.AnyproteinintheorycanbethetargetforRNAi,thereforeRNAihaswiderangeofapplications(cancer,cardiovasculardiseasesetc.).α-synucleinsiRNAwasloadedintoDC-derivedexosomespost-isolation.ThemosteffectivewaytoloadsmallhydrophilicmoleculesuchasRNAisbyelectroporation.150µgsiRNAwaselectroporatedinto150µgexosomes,centrifuged,andresuspendedin5%glucose[18].BraintargetingwasachievedbyloadingcellswithLamp2b-RVGcodingplasmid,a

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strategypreviouslymentionedin2.3.TheeffectofsiRNAdeliverywastestedonhealthymiceandtransgenicmicewithhumanα-synucleinmimic.InthehealthymicemodeltheresearchersassessedsiRNAabilitytoreduceendogenousα-synucleininmousebrain.Thebrainwasanalyzedfortheamountofα-synucleinandα-synucleinmRNA3and7daysafterinjection(Fig.10).mRNAlevelswereanalyzedbyqPCRrelativetoGAPDHandproteinlevelswereanalyzedbywesternblotrelativetoactin[18].Bothweremeasuredinmidbrain,striatum,andcortex.mRNAandα-synucleinlevelsaredecreasedinallthreebrainregionsbyapproximately50%.Theexceptionisα-synucleinlevelincortex,whereit’s75%ofthecontrol(Fig.10).Theexplanationforthatcouldbeslowerturnoverofcorticalα-synuclein[18].7daysaftertheinjectionα-synucleinandmRNAlevelsdidn’tchangemuchmeaningmostofRNAiactivityhappenedinthefirst3dayspost-injection(Fig.10).Thesefindingssuggestthatthemajortherapeuticeffectisachievedwithin3daysandfurtherwaitingforhigherefficiencyisnotverypractical.Ontheonehand,themajortherapeuticeffectisachievedwithin3days.Ontheotherhand,thereisnotmuchroomfortheimprovementoftherapeuticefficacy.

Figure10.Amountofα-synucleinandα-synucleinmRNAshownaspercentageofcontrol3(A,B)and7(C,D)daysafterinjectioninmidbrain,striatumandcortex[18]Intheexperimentwithhealthymicetheyusedmouseα-synucleinandnothumanα-synuclein.Themicewerehealthyandα-synucleindidn’tformaggregates.Itstillwasn’tconfirmedthatsiRNAwillbeaseffectiveinreducingaggregatedα-synucleinlevelsasitwaswithnormalmouseα-synuclein.Therefore,thenextexperimentwasdoneontransgenic(Tg13)micewithhumanα-synucleinmimic.InhumanPDpatientsα-synucleinisphosphorylatedatS129andformesaggregates[26].TheyusedmicemodelwithS129Dα-synuclein.ThismutationmimicsthephosphorylationandS129Dα-synucleinformsaggregatesinmousebrain.

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Figure11.S129Dα-synuclein(WB)andmRNA(PCR)levelsinTg13mice7daysaftertheinjectioncomparedtountreated(control)Tg13mice.Measuredinmidbrain,striatum,andcortex[18]mRNAlevelsarereducedsignificantlyinallthreebrainregionstoapproximately50%comparedtomRNAlevelinuntreatedmice.Proteinlevelsalsodecreasedtoabout70%ofthecontrol(Fig.11).Toensuretheseresultsweren’trandomCooperetal.didanotherexperimentinwhichtheydideverythingthesameexceptsiRNAhadmutationinonebasepair.TheresultsofCsiRNARVG-exosomestreatmentofTg13miceispresentedinFig.12.Ascanbeseen,CsiRNAdidn’tcauseanyreductioninmRNAandS129Dα-synucleinlevels,confirmingthatS129Dα-synucleindownregulationwasduetoRNAinterferenceofsiRNA[18].

Figure12.S129Dα-synuclein(WB)andmRNA(PCR)levelsinTg13mice7daysaftertheinjectionofcontrolsiRNAcomparedtountreatedTg13mice.Measuredinmidbrain,striatum,andcortex[18]ThesametherapeuticapproachwasappliedfortreatmentofADbythesamegroupofresearchers4yearspriortothisexperiment.BACE1istheproteinresponsibleforN-terminalcleavageofamyloidprecursorprotein.Inotherwords,BACE1producesβ-amyloidpeptidethatformsaggregatesandisinvolvedinADpathogenesis.In2011Alvarez-

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Ervitietal.deliveredBACE1siRNAinDC-derivedexosomestomousebrainandobtaineda60%reductioninbothBACE1andBACE1mRNAexpression[23].Itwasshownthatsystemicinjectionsofα-synucleinsiRNAcansignificantlyreducethelevelsofmouseα-synucleinaswellashumanS129Dα-synucleininaggregates.ThesuccesssiRNAsystemicinjectionsopensapossibilityforclinicaltrialsandthedevelopmentoftherapeuticstrategiesforotherapplications,suchasAlzheimer’sorHuntington’sdiseases.

ConclusionDrugdeliverynowadaysisatthepointwhenthereareplentyofapplicationsbuttherapeuticapproachesarestillindevelopment.Exosomesappeartobeaneffectivedrugdeliverymodelduetotheirlowcytotoxicity,prolongedcirculationtimeandtargeteddrugdelivery.Parkinson’sdiseaseisthe2ndmostcommonneurodegenerativediseasewithnocurefoundtoday.TherearecertainstrategiesthatcanhelptohalttheprogressionofthediseasebuttheirdeliveryisveryinefficientduetopoorBBBpermeabilitybyforeignmolecules.Exosomesofferagoodplatformforthedeliveryoftherapeuticagents,notonlybecausetheycancrossBBBbuttheyalsopreservetheactivityofcargodrug[19].Exosomescanbeisolatedfromthehostorganismandpurified,thoughpurificationstrategiesarenotveryefficient.Exosomescanbeloadedinvitrowithdifferentkindsofcargomolecules,e.g.proteins,mRNA.AgoodwaytoloadsmallhydrophilicmoleculesiswithincubationatRT,whereaslargeproteinsarebetterincorporatedbysonicationorextrusion[16].AdditionofsaponinduringincubationatRTcanincreaseexosomalloading.mRNAormiRNAarebestloadedwithelectroporation.ItwaspresentedthatthetherapeuticstrategiesofusingexosomesfordrugdeliverytorelievePDsymptomsweresuccessful.ExosomaldrugdeliveryshowsbetterresultindeliveringcatalasetotherelevantPDbrainregionsthanbulkcatalase.Widespreaddistributionofcargointhebrainleadstodiversepositiveeffects.Experimentsonmiceshownoabnormalitiescausedbytheadministrationofexosomedrugnanoformulations.Withmoreknowledgeofexosomalbiologyandblood-brainbarrierphysiologyexosomescanbeappliedinclinicaltrialforPDpatients.Exosomesareaneffectiveplatformfordrugdelivery,showingbetterresultsinrecentyearsthanliposomes,exosome-likenanoparticlesorbulktherapeuticagents.DuetotheirintrinsicabilitytocrossBBB,preservativefunctionandimmenserobustnessexosomespromisetobeasuccessinthefieldofdrugdeliveryinfuture.References

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