Intracellular delivery of nanomaterials How to catch endosomal escape in the act

2021-04-11 来源：钮旅网

NanoToday(2014)9,344—364

Availableonlineatwww.sciencedirect.com

ScienceDirect

journalhomepage:www.elsevier.com/locate/nanotoday

REVIEW

Intracellulardeliveryofnanomaterials:Howtocatchendosomalescapeintheact

ThomasF.Martensa,b,∗,KatrienRemauta,JoDemeestera,StefaanC.DeSmedta,∗∗,KevinBraeckmansa,b

LaboratoryforGeneralBiochemistryandPhysicalPharmacy,FacultyofPharmaceuticalSciences,GhentUniversity,Harelbekestraat72,B-9000Ghent,Belgiumb

CenterforNano-andBiophotonics(NB-Photonics),GhentUniversity,Harelbekestraat72,9000Ghent,Belgium

Received31January2014;receivedinrevisedform11April2014;accepted21April2014Availableonline28May2014

KEYWORDS

Endosomalescape;Nanomaterials;Assays;Fusion;

Protonsponge;Poreformation

SummarySuccessfulcytosolicdeliveryofnanomaterialsisbecomingmoreandmoreimpor-tant,giventheincreaseinintracellularapplicationsofquantumdots,goldnanoparticles,liposomaldrugformulationsandpolymericgenedeliveryvectors.Mostnanomaterialsaretakenupbythecellviaendocytosis,yetendosomalescapehaslongbeenrecognizedasamajorbot-tleneckincytosolicdelivery.Althoughitisessentialtodetectandreliablyquantifyendosomalescape,noconsensushasbeenreachedsofaronthemethodstodoso.Thisreviewwillsum-marizeanddiscussfortheﬁrsttimethedifferentassaysusedtoinvestigatethiselusivesteptodate.

Introduction

Cytosolicdeliveryofnanomaterialshasgainedalotofinterest.Recentdevelopmentsinnanoscienceandnano-technologyhavecreatedalibraryofnanomaterialswith

Correspondingauthorat:Laboratoryforgeneralbiochemistryandphysicalpharmacy,FacultyofPharmaceuticalSciences,GhentUniversity,Harelbekestraat72,B-9000Ghent,Belgium.Tel.:+3292648049;fax:+3292648189.

∗∗Correspondingauthor.Tel.:+3292648076;fax:+3292648189.E-mailaddresses:Thomas.Martens@Ugent.be(T.F.Martens),Stefaan.Desmedt@Ugent.be(S.C.DeSmedt).

∗

potentialapplicationsinthevisualizationofsubcellularstructuresanddynamics,intracellulardeliveryofthera-peutics,genetherapyandthetreatmentordiagnosticsoforganelle-speciﬁcdiseases[1—4].Importantly,efﬁcientdeliverytotheintracellularenvironmentisnecessaryforexertingtheirintendedfunction.Thoughphysicaltech-niquesaresometimesusedtoforcethenanoparticlesacrosstheplasmamembrane(microinjection,electroporation,...)[4],uptakeofforeignnanomaterialsusuallyreliesonthecell’sinnateendocyticuptakemechanism.Thisresultsinthecargoresidinginendosomes(Fig.1),thusbeingphysi-callyseparatedfromthecytosolbytheendosomallimitingmembrane[5,6].Furthermore,maturationofmosttypesofendosomestomultivesicularlateendosomesiscoupled

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Figure1Mostnanoparticles,organicandinorganicones,fordrugdeliveryaswellasforimaging,areusuallytakenupbytargetcellsviaendocytosis,leadingtosequestrationofthecargointheendocyticvesicles(blueinteriordenotesneutralpH).Acidiﬁcationoftheendosomestypicallywilltriggeradestabilizationoftheendosomalmembranebythedeliveryvector(orangeinteriordenotesacidiﬁedpH).Afterdestabilizationoftheendosomalmembrane,dependingonthedeliveryvector,thecargowillbereleasedbyaburstingeffectifanosmoticgradientisestablishedbyendosomalbuffering,throughporesformedintheendosomalmembranebypersistentdestabilization,orbymembranefusionifthecargowaspackagedinanenvelopeddeliveryvector.Differentassaysareavailabletoinvestigatetheseendosomalescapemechanisms,asindicatedinred.Alsotheefﬁciencyofendosomalescapecanbequantiﬁedwithavarietyoftechniques,asindicatedinblue.IFP=intracellularﬂuorescenceproﬁle.

withadecreaseinintravesicularpHandfusionwithlyso-somes,potentiallyresultingindestructionofthefunctionalnanomaterialsbydegradativelysosomalenzymes[7].Thisendolysosomalsequestrationandhydrolyticdegradationofthenanoparticulatecargoimpliesthattheyshouldescapefromtheendosomesinatimelymannertoexertorpreservetheirintendedfunction.

Forinstance,semiconductornanocrystals,orquantumdots(Qdots),arebeingexploredtobeusedforlabelingintracellularstructuresandmoleculesbothinvitroandinvivo[8—10].Notonlydoessequestrationpreventtheiraccesstothecytosol,italsoresultsintoxicityasaresultfromionleachingbydegradationintheendolysosomalenvi-ronment,especiallyinthecaseofCd2+-containingQdots[11].Forcellularlabelingandinvivocelltracking,MRIcon-trastagentscanbedeliveredtothecellinteriorintheformofparamagneticGadolinium-nanoparticles(Gd3+)[12]orsuperparamagneticironoxide(SPIO)nanoparticles[13,14].IthasbeenshownthatsequestrationofsuchcontrastagentsinintracellularvesiclescouldresultinquenchingoftheMRIsignal[12,13].Therefore,itisoftenstronglypreferredthatnanoparticlesforimagingapplicationsshouldescapefrom

theendolysosomalpathwaytotheintracellularenvironment[12].

Intracellulardrugdelivery,forinstanceadaptiveimmunotherapyandgenetherapy,ismadepossiblebypackagingtherapeutic(macro)moleculesinnanomedicineparticlessuchaspolymermicellesordendrimers,nanogels,liposomes,mesoporoussilicaparticles,etc.,thatshouldprovideprotectiontoandmediateintracellulardeliveryofthesetherapeutics[2,15,16].However,therapeuticnucleicacidsexerttheireffectinthecytosolornucleusofthecell.MHC-IdependentantigenpresentationtoCD8+T-cellsalsoreliesonantigenicproteinorpeptidedeliverytothecytosolofantigenpresentingcells(APCs)suchasdendriticcellsormacrophages[17].Endolysosomalsequestrationandhydrolyticdegradationdrasticallydecreasestheefﬁciencyofdrugdeliverysystemsandshouldbecounteredbyefﬁcientendosomalescapeofthetherapeuticcargotothecytosol.

Awidevarietyofapproacheshavebeendevelopedtofacilitatereleaseofnanoparticlesandmoleculesfromendo-somesbeforelysosomaldegradation,suchasfusogenicpep-tidesandphotochemicalinternalization(PCI),whichhave

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beenthetopicofnumerousreviews[1,2,4,6,8,16,18,19].Despitedecadesofresearch,however,endosomalescapeisstillrecognizedasaveryinefﬁcientprocessandamajorbottleneckincytosolicdeliveryofnanomaterials[2,16,18].Inordertoimproveonthosemethods,itisquiteessen-tialbeingabletodetectandreliablyquantifyendosomalescape[18,20].Althoughdifferentassayshavebeenpro-posedtodate,thereisnoconsensusyetonhowtoevaluatethiscriticalstepintheintracellulardeliveryprocess.

Inthisreview,weaimtoprovideanexhaustiveoverviewofmethodologiesusedtoevaluateendosomalescape,andtodiscusstheusefulnessandlimitationsofeach.First,abriefoverviewispresentedofthedifferentmechanismsandcom-poundstoenhanceendosomalescape.Next,anoverviewofreportedendosomalescapeassaysisgiven,withexemplaryapplicationsanddiscussionontheunderlyingassumptionsandlimitationsinherenttoeachtechnique.Speciﬁcally,adistinctionismadebetweenassaysinvestigatingthemech-anismofendosomalescape(relatingtomembranefusionormembraneintegrity),andthosethataimtovisualizeorquantifycargoreleaseinthecytosol,regardlessofthemechanismofendosomalescape.

Enhancingendosomalescape

Mostman-madematerialstoenhanceendosomalescapehavebeeninspiredbyviralandbacterialinfectionpathwaysinthecell,whichrelyonendocytosisfollowedbycleverlyevolvedwaysofescapingtowardthecytosoliccompart-ment[21,22].Typically,theendosomalmembraneisinitiallydestabilized,afterwhichendosomalescapecanoccurthrougheitherporeformation,ruptureormembranefusion,dependingonthenanoparticle’scharacteristics(Fig.2).Wewillbrieﬂydiscusstheseendosomalescapemechanisms,thoughthereaderisreferredtoseveralrecentreviewsonthistopicforamoredetailedoverview[1,2,4,18].

Endosomaldestabilizationandporeformation

Endosomalescapeischaracterizedbyaninitialmembranedestabilization(Fig.2B),whichisconﬁnedtotheendosomesasaresultoftheinherentacidiﬁcationduringendosomalmaturation(Fig.2A).Themostfrequentlyproposedmech-anismsforcausingendosomalmembranedestabilization,cationicchargeandmembrane-destabilizingpeptides,arediscussedbelow.Furthermore,persistentmembranedesta-bilizationcanleadtoporeformationintheendosomalmembrane,resultinginleakageofmoleculesandsmallerparticlesfromtheendosomalcompartmenttothecytosol(Fig.2C).

Sincetheouterlayersofendosomalmembranesaretypicallythoughttobecomposedofphospholipidswithanoverallnegativecharge,theinteractionofendoso-mallytrappedcationicnanoparticleswiththeendosomalmembraneisthoughttoinducea‘‘ﬂip-ﬂop’’mecha-nism,whereanionicphospholipidsfromthecytosolicleaﬂetwillﬂiptotheintraluminalsideoftheendo-some[23—25].Thischarge-neutralizedionpairwillresultinnon-lamellarphasechangesandsubsequentmembranedestabilization[25].Apermanentcationicchargecanresultfromquaternaryaminegroups,asisthecase

T.F.Martensetal.

withcationiclipidssuchasN[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride(DOTMA)or1,2-dioleyl-3-trimethylammonium-propane(DOTAP).Alternatively,pro-tonableaminegroupsonthenanoparticlesurfacecanalsoprovideacationiccharge,i.e.formaterialssuchaspoly(ethyleneimine)(PEI),poly(L-lysine)(PLL)andchitosan.Importantly,sincethecationicchargecansometimesbeenhancedalongwiththeacidiﬁcationoftheendosomalcompartment,membranedestabilizationwillthenprimarilyoccurinacidicendosomes.

Membrane-destabilizingpeptides,inspiredbynaturalviralentrypeptidessuchastheHA-2subunitoftheinﬂuenzavirushemagglutinin,areusuallycalledfusogenicpeptidessincetheirconformationalchangeexposeshydrophobic(␣-helical)domains,allowingthemtointeractwiththelipidmembraneoftheendosome.Theycantypicallybedividedinthreedifferentclassesbasedontheaminoacidresiduespresentinthepeptideandaslightlydifferentmechanismofaction[21,26]:(1)anionicamphiphilicpeptides,suchasINF7andE5WYG,whichcontainglutamateresiduesandundergoaconformationalchangefromarandomcoiltoan␣-helixunderacidicconditions(pH5—5.5);(2)histidinerichpeptidessuchasH5WYGthatprotonateundermildlyacidicconditionsanddestabilizemembranesduetocationicinteractionsandanosmoticbufferingeffect(alsoseesection‘Endosomalrupture’);(3)cationicamphiphilicpeptideswithlysineaminoacids,suchasK5andKALAthatcanbindanionicnucleicacidsandinduceapH-independentmembranedestabilizationbycationicinteractionswiththeendosomalmembrane.GALAisalsowidelyknownandcontainsbothglutamateresiduesforpHdependenceandhistidineaminoacidsforcationicchargeandbuffering[27].Adifferentapproachtoachievemembranedestabilization,withoutrelyingonacidiﬁcation,entailstheinteractionoftheendosomalmembranewiththelysogenicpeptideL-leucyl-L-leucinemethylester(Leu-Leu-OMe).Thisdipep-tideisconvertedintoamembrane-lysingcompound,notbyanacidicenvironment,butbythelysosomalenzymedipeptidylpeptidaseI[25,28].

Endosomalrupture

Whereaspersistentmembranedestabilizationcanresultingradualleakageofsmallnanomaterialsthroughporeformation,burstingofendosomeshasalsobeenproposedasamechanismforendosomalescape(Fig.2D).Likelythebest-knownmechanismtoenhanceendosomalescapeisthe‘‘protonspongeeffect’’[29],whichisbasedoncationicpolymersorlipidswithexcessunchargedproton-ableaminegroupsthatcanbufferendosomalacidiﬁcationbyabsorbingprotonsintheendolysosomalcompartment.AslongasATPispresentinthecytosol,V-typeATPaseswillkeeppumpingprotonsagainsttheirelectrochemicalgradi-entacrosstheendolysosomalmembrane,withanassociatedinﬂuxofcounter-ionstobalancethetransmembranevoltagedifference(Fig.2A)[7,30].Endolysosomalruptureiscur-rentlybelievedtoresultfromacombinationofthreeeffects(Fig.2D).First,thebufferingcompoundswillinduceanini-tialmembranedestabilizationbytheﬂip-ﬂopeffectinducedbythecationicchargeoftheprotonatedaminegroups.Sec-ond,electrostaticrepulsionoftheprotonatedaminegroupsresultsinswellingofthebufferingagent,alsoreferred

Intracellulardeliveryofnanomaterials

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Figure2Differentproposedmechanismsforendosomalescape.(A)Normalacidiﬁcationoftheendosomesduringmaturationtolateendosomes.ATP-driventransportofH+-ionsacrosstheendosomalmembranebyV-typeATPasesproducesanelectricalgradient,whichisbalancedinpartbytheinﬂuxofcounter-ions,presumablyCl−-ions.(B)Uponacidiﬁcation,cationicparticlesinducenegativelychargedphospholipidsontheouterendosomalleaﬂettoﬂiptotheluminalsideoftheendosomebyaﬂip-ﬂopeffect,resultinginacharge-neutralpairandcausingmembranedestabilization.Alternatively,fusogenicorlysogenicpeptideswillundergoconformationalchangesinacidicenvironments,resultinginatriggereddestabilizationoftheendosomalmembrane.

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T.F.Martensetal.

toasthe‘‘umbrellaeffect’’[16],furthercontributingtomembranedestabilization[10].Third,theconstantinﬂuxofcounter-ionstobalancetheelectricpotentialcreatesanosmoticgradientbetweenvesicleandcytosol,leadingtoaninﬂuxofH2Otorestoretheosmoticbalance.Withanalreadydestabilizedendolysosomalmembrane,theswellingoftheintracellularvesiclesresultsinburstingoftheendosomeandreleaseofthecargointhecytosol.Typicalexamplesofpro-tonspongecompoundsarePEI[31],poly-amidoamines[32]andimidazole-containingpolymers(e.g.histidine)[2,18].Theyaretypicallyincorporatedintothenanoparticlesasthedeliveryvector,althoughinsomecasesthebufferingcom-poundsareeitherco-incubatedwiththenanoparticles(e.g.monensin)oraddedafterwards(e.g.chloroquine).Similartochloroquine,ammoniumchlorideandmethyl-aminearelipophilicintheunprotonatedformandwillpenetratecellu-larandvesicularmembranes.Uponprotonationinacidifyingendosomes,thesecompoundsbecometrappedandcanactasprotonsponges[18].

Incontrasttomembranedestabilizationtriggeredbytheendolysosomalcompartmentonly,certainphysicaltech-niquesallow(spatio)-temporallycontrolleddisruptionofintracellularvesicles.Awell-knownstrategytoinducerup-turewithouttheneedforacidiﬁcationisPCI[33,34],whichinvolvestheuseofamphiphilicphotosensitizers,e.g.ethyleosin[35]orTPPS2a(mesotetraphenylporphinecarryingtwosulfonategroupsonadjacentphenylrings)[33].Afterpulse-chasedadministrationofthephotosensitizertothecellsinvitro,theywillaccumulateinintracellularmembranes,amongstwhichtheendosomalmembranes.Uponillumina-tionwithaspeciﬁclightsource,excitationofthephoto-sensitizersinducestheformationofreactiveoxygenspecies(ROS),primarilysingletoxygen.Duetoashortlifetime,thedamagecausedbythishighlyreactiveintermediatewillbemainlyconﬁnedtotheproximatemembranes[33].Lighthasalsobeenusedasatriggerforheat-inducedendosomaldestabilizationorrupture,forexamplebyNIRirradiationofreducedgrapheneoxide[36]orbythegenerationofvapornanobubblesthroughpulsed-laserirradiationofgoldnanoparticles[37].Analternativemethodtoinduceburstingoftheendosomalvesiclesinatemporallycontrolledman-neristheso-called‘‘osmolyticshock’’.Thisisattainedbyloadingtheintracellularvesicleswithahypertonicsolutionviaendocytosisandafterwardsincubatingthecellswithahypotonicsolution,causingtheintracellularvesiclestoswellandburst[28,38,39].Similarly,carriermaterialshavebeendevelopedwhichexhibitswellinguponadecreaseintem-perature[40,41],therebydisruptingintracellularvesiclescontainingtheparticlesaftera‘‘coldshock’’treatment.

cytosol(Fig.2E).Endosomalescapethroughfusiononlyoccurswhenthenanoparticleitselfisenvelopedbyamembrane.Itisbeneﬁcialiftheendosomalmembraneisalreadydestabilized.Thisisthecaseforcationiclipo-somes,whosechargeensurescloseinteractionwithanddestabilizationoftheendosomalmembrane,resultinginfusionandreleaseoftheencapsulatedcargo.Incorporationoffusogenic‘‘helper’’lipids(e.g.1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamineorDOPE)furtherenhancesendosomalfusionandescape,byundergoingacon-formationalchangeuponacidiﬁcationandpromotinganon-lamellarlipidphasechange[42].Cholesterolhasalsobeenintegratedinliposomalparticlestoenhancefusogenic-ityinapH-independentway,bothattheplasmamembraneandafterendocytosis[43].

Studyingendosomalescapemechanisms

Aspreviouslyhighlighted,endosomalescapeinvolvesanini-tialmembranedestabilization,followedbyporeformation,endosomalruptureormembranefusion.Typically,themech-anismsforendosomalescapeareassayedbyinvestigatingtheintegrityoftheendosomalmembrane.Thoughtheseassayscanbeperformedincells,amorecontrolledenviron-mentcanbecreatedexcellulobyusingartiﬁcialendosomes.Theseartiﬁcialendosomesarefrequentlymodeledbycre-atingliposomeswithmembranesofaknownphospholipidcomposition,whichtheninteractwiththecompoundunderinvestigation.

VerifyingpH-inducedmembranedestabilization

Endosomalmembranefusion

Fusionofananocarrierwiththeendosomalmembranecanresultinescapeofthenanocarrier’scargointothe

TheinitialmembranedestabilizationofendosomalescapeisusuallytriggeredbyanacidicpH,leadingtoconformationalchangesoffusogeniccompoundsoraﬂip-ﬂopeffectbycationicparticles.TheeffectofpHonthismembranedesta-bilizationisinvestigatedbycomparingendosomalescapescenarioswherepHeitherdoesordoesnotaffectthecationicorfusogeniccompounds.Thedifferenceinendo-somalescapebetweenthenormalandthepH-irrelevantscenarioisthenevaluatedusingthedifferentendosomalescapeassayswhichwillbediscussedfurther,andhavebeensummarizedinFig.1.RemovingtheinﬂuenceofpHonendosomalescapeisdonebyeitheralteringthecom-poundunderinvestigationsoitisnolongerpH-reactive[44],orthepHoftheendosomalcompartmentitselfisaltered.Inacontrolledexcelluloenvironment,thepHcanbemodeledbyusingbufferswithdifferentpH[45,46].Incellularassays,acidiﬁcationcanbeblockedbytheuseofinhibitors.Severalstudiesreporttheinhibitionofendo-somalacidiﬁcationbyusingahigherbufferingcapacityintheextracellularmedium[47]orco-incubationwithbuffer-ingagentsuchasammoniumchloride[48,49],chloroquine

(C)Persistentmembranedestabilizationbycationicnanoparticlesorbyfusogenicpeptidescanresultinporeformation.(D)Whenbufferingcompoundsarefoundintheendolysosomallumen,acidiﬁcationwillbebufferedbytheirproton-absorbingcharacteristics.Theincreasedcationicchargeandswellingofthecompound,asaresultoftheongoingprotonationoffreeprotonableamines,willresultinmembranedestabilization.Acontinuedinﬂuxofchlorideionswillcreateanosmoticgradientandinternalpressure,leadingtoruptureoftheendosomesandburstingofthecontentsintothecytosol.(E)Whenanenvelopednanoparticlecomesinclosecontactwithanalreadydestabilizedmembrane,e.g.bycationiccharge,fusionbetweennanoparticleandendosomalmembranecanresultincargoreleaseinthecytosol.

Intracellulardeliveryofnanomaterials

[50]andmonensin[48].However,itisimportanttotakeintoaccountthatthesebufferingagentsmightenhanceendosomalescapeduetotheproton-spongeeffect,ratherthanblockitthroughinhibitingacidiﬁcation.Therefore,itwouldmakemoresensetoblocktheacidiﬁcationprocessaltogether,whichisdonebytheadditionofionchannelinhibitors(ionophores)[51]ormoreimportantly,V-typeproton-pumpingATPaseinhibitorssuchasbaﬁlomycinsorconcanamycins[52].

Assaysforstudyingporeformation

Poreformationcanbeinvestigatedbymeasuringtheleakageoftracercompoundsintotheextravesicularenvironment(seeTable1).Typically,thoughnotalways,thesetrac-ersareﬂuorescentmoleculesthatarequenchedinsidethevesiclesandbecome(more)ﬂuorescentupontheirescape.Thisincreaseinﬂuorescenceintensitycanbemeasuredwithspectroﬂuorimetryinexcelluloassays,orwithﬂuo-rescencemicroscopyorﬂowcytometryincellularassays.Flowcytometryisconvenientinthatitcanprovideahigh-throughputquantiﬁcationoftheﬂuorescenceintensitypercell.Fluorescencemicroscopyhaslowerthroughputbuthastheadvantageofprovidingadditionalinformationontheintracellularﬂuorescenceproﬁle(IFP).Apunctateﬂuores-cencepatternisoftenconsideredanindicationofthetracercompoundbeingentrappedinendosomes,whileadiffusecytosolicstainingimpliesleakagefromtheendosomalvesi-cles(Fig.3).Itmustbenotedhowever,thatsuchatracerleakageassaycannotdistinguishbetweenendosomalescapebyporeformation,orbybursting(seesection‘Assaysforstudyingmembranerupture’).

8-Aminonaphthalene-1,3,6-TrisulfonicAcid(ANTS)and8-Hydroxypyrene-1,3,6-TrisulfonicAcid(HPTS)arepolyan-ionicﬂuorescentmoleculesthatarequenchedbythecationicquencherp-Xylene-Bis-PyridiniumBromide(DPX).BothANTS/DPX[55]andHPTS/DPX[55,56]havebeenusedinexcelluloassaystoinvestigatethemembraneintegrityofartiﬁcialendosomes,whereleakageanddilutionofbothcompoundsresultsindequenchingoftheﬂuorescentsignal.Fluorescein-labeledcell-penetratingpeptides(CPPs)havebeenusedinasimilarmannerwithpotassiumiodideasquencher[57].Otherwise,ratherthanusingaquenchermolecule,self-quenchingofcertainﬂuorophorescanbeachievedwhenusedatasufﬁcientlyhighconcentration,whichisrelieveduponleakageresultinginincreasedﬂuo-rescence.Forinstance,calcein[58,59],carboxyﬂuorescein(CF)[45]andsulforhodamineB(SulfoB)[61]havebeenusedinexcelluloleakageassaystoinvestigatemembraneintegrityofartiﬁcialendosomesbyspectroﬂuorimetry.Incellularassays,thesetracermoleculesareloadedintheendosomesbyconstitutiveendocytosis.Especiallycalceinhasbeenusedforthis,sincetheacidicpHinendolysosomeswillfurtherquenchitsﬂuorescencesothattheincreaseinﬂuorescenceintensityafterleakageisevenmorepro-nounced.Scoringporeformationincellularassaysusingcalceinhasbeenachievedbothbymicroscopy[47,60]andﬂowcytometry[53].

Whereasanincreasedcalceinﬂuorescencedoesnotdisclosefromwhichintracellularvesicleleakageoccurred,theuseofothertracermoleculessuchasDQ-ovalbumin[38]andacridineorange(AO)[38,47,51]relatespeciﬁcally

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toendolysosomalmembraneintegrity.DQ-ovalbuminisa45kDaproteintowhichahighnumberofBODIPY-FL(8-chloromethyl-4,4-diﬂuoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacene)isconjugated,quenchingitsﬂuorescence.Beforethisproteinisproteolyticallyprocessedintopeptidesintheendolysosomalcompart-ment,poreformationwillleadtoreleaseofthequenchedDQ-ovalbuminprotein.However,afterproteolyticaldegra-dation,poreformationwillleadtoreleaseanddilutionofthequenchedBODIPY-FL-labeledpeptidesandanincreaseinﬂuorescence.

Anotherinterestingapproachtospeciﬁcallyinvestigatelysosomalintegritythatdoesnotinvolveadequenchingofhighlyconcentrateddye,reliesonacidotropicdyeslikeAOthataccumulateatahighconcentrationinthelysosomallumen,formingﬂuorescentdimersinacidicenvironments.Uponlysosomalmembranedamageitsﬂu-orescencedecreasesbecausethelysosomalacidicpHcannolongerbemaintainedincombinationwithAOleakingoutinthecytosol.Quantiﬁcationofthisdecreasedﬂuores-cenceisthenrelatedtodamagetothelysosomalmembrane[38,47,51].

Endosomalmembraneintegritycanalsobeevaluatedincellswithouttheuseofﬂuorescenttracers.Forinstance,theleakageofcertaintoxinsfromendosomesisknowntoinhibitproteinsynthesis.Insuchaway,Pseudomonasexo-toxin[62],ribotoxin␣-sarcin[63]andsaporin[64]wereusedastracermoleculestoinvestigatemembranedestabilizationbydifferentviruses.Theinﬂuenceonproteinsynthesiswasevaluatedeitherwithradio-activelylabeled[3H]-leucine[62]or[35S]-methionine[63],orbyusinganMTTassay[64].Thoughtheseassayswereusedinaviralsetting,theycouldeasilybeappliedforman-madematerialsaswell.

Asalreadyhighlighted,theleakageassayisfrequentlyusedtoevaluatetheeffectivenessofcertaincompoundstoinduceporeformation,suchasviralnanoparticles[61—64]andCPPs[53,55,57—59].Nonetheless,theseassayswerealsousedtoverifytheeffectivenessofpolymericgenedeliv-erycomplexes,composedofcationicpolymerssuchasPEI[51,60]orpoly(2-alkylacrylicacid)[47],andpDNA.Alimi-tationofdetectingleakageofsmallmolecules,however,isthatitmaynotdirectlyrelatetothereleaseoflargermacro-moleculesornanoparticles.Therefore,radioactivelabeledproteinswereusedinsteadofsmallmoleculetracersinanexcelluloassay,wheretheradioactivityofleakedproteinsintheextravesicularenvironmentwasvisualizedafteragarosegelelectrophoresis[58].Fluorescentdextransofvariousmolecularweightsareinterestingforuseincellularassaysaswellsincetheyareefﬁcientlytakenupbycellswithoutspontaneousleakagefromtheendocyticvesicles,andtheycanbelabeledwithdifferentﬂuorophores[38,53,65].How-ever,asleakageofdextransintothecytosolwillnotleadtoﬂuorescencedequenching,itrequiresvisualizationoftheIFPwithﬂuorescencemicroscopy.Ofcourse,todistinguishpunctatefromdiffusestaining,itisessentialthattheselargetracercompoundsarestillabletodiffusethroughoutthecytosol,whichcanbecheckede.g.bymicroinjection.

Assaysforstudyingmembranerupture

Asidefromporeformation,endosomalescapecanalsooccurthroughtheruptureorburstingoftheendosomal

Table1Overviewofthedifferentassaysusedtoinvestigateporeformationbyleakageoftracercompounds,withtheiruseincelluloorexcellulo.Mostassaysarebasedonﬂuorescence,thoughalternativesarealsolisted.

Tracercompound

Molecularweight

Excellulo/inQuenchingMeasurementtechniqueReferenceComments

cellulo

andresponse

Tracer+quencher

ANTSDPXSpectroﬂuorimetry[55]

2molecules427Da

Excellulo

necessary

Fluorescence↑HPTS

524Da

Excellulo

DPX

Spectroﬂuorimetry[55,56]2moleculesnecessary

Fluorescence↑Fluorescein-CPP

1.500—3.000DaExcellulo

KI(100mM)

Spectroﬂuorimetry[57]

2moleculesnecessary

Fluorescence↑

Self-quenchingCalcein

622Da

Excellulo

±100mM

Spectroﬂuorimetry[58,59]

Quenchingconcentrationtracer

Fluorescence↑

calcein

Incellulo

25␮M/3.2mM

IFP:diffuse<>punctate

[47,60]

Quenchingconcentrationcalcein

QuantiﬁcationpossibleIncellulo

250␮M

Flowcytometry

[53]Quenchingconcentrationcalcein

SulforhodamineB

559Da

Excellulo

100mM

Spectroﬂuorimetry[61]QuenchingconcentrationFluorescence↑SulfoB

Carboxyﬂuorescein

376Da

Excellulo

±50mM

Spectroﬂuorimetry[45]

QuenchingconcentrationFluorescence↑

Lysosome-speciﬁcAcridineorange

302Da

Incellulo

Acidotropic

Flowcytometry[38,47,51]

Measuringdecreaseintracer

Fluorescence↓

ﬂuorescence

ComparetonormalcellsDQ-ovalbumin

45.000Da

Incellulo

BeforeproteolyticalIFP:diffuse<>punctate

[38]Onlyﬂuorescenceafterprocessing

lysosomaldegradation

Reporterassays

Pseudomonas71.000Da

Incellulo

Onlyactiveincytosol

Proteinsynthesis↓[62]

Nohydrophobicexotoxin[3H]-leucine

ﬂuorophores

Importanttonormalize␣-Sarcin

18.000Da

Incellulo

Onlyactiveincytosol

Proteinsynthesis↓[63]

Nohydrophobic[35S]-methionine

ﬂuorophores

ImportanttonormalizeSaporin

30.000Da

Incellulo

Onlyactiveincytosol

Proteinsynthesis↓[64]

NohydrophobicMTTassay

ﬂuorophores

Importanttonormalize

LargetracerProtein

20.000Da

Excellulo

Radio-activelabeling

Gelelectrophoresisand[58]

Nohydrophobiccompounds

radio-activityﬂuorophores

measurement

Relevantforcells?FluorescentDifferentsizes

Incellulo

—

IFP:diffuse<>punctate

[38,53,65]

Onlysmallerdextrans

dextrans

350T.F.Martensetal.Intracellulardeliveryofnanomaterials

351

Figure3(A)Theintracellularﬂuorescenceproﬁle(IFP)of3kDadextrans(greenﬂuorescence),illustratingthedifferencebetweenapunctatepattern(sequesteredcargo;left)anddiffusestaining(cytosoliccargo;right).Scalebar10␮m.Reprintedfrom[53],©2012,withpermissionfromElsevier.(B)IFPofQdots(greenﬂuorescence),visuallyclassiﬁedasapunctatepattern,moderatereleaseandahighamountofrelease.Bluecolorindicatesthenucleus.Scalebar50␮m.Reprintedwithpermissionfrom[54].©2013AmericanChemicalSociety.

vesicleswhenanalreadydestabilizedmembraneisper-turbedbyanoutwardforce,e.g.osmoticpressure(seesection‘‘Endosomalrupture’’).Theleakageassaysdis-cussedinsection‘Assaysforstudyingporeformation’canalsobeappliedheresinceruptureisalsoassociatedwithmembranedamage.However,theycannotdistinguishbetweenporeformationandrupture,seeingasthelatterisatransientevent.Instead,directvisualizationoftheburstingeventcanbeaccomplishedwithlive-cellvideomicroscopy.Forinstance,calceinreleasefromlight-responsivepoly-mersomesandendosomeswasimagedbyVasdekisandcolleaguesasaburstofﬂuorescencetowardthecytosol(Fig.4A),whichisindicativeofendosomalruptureratherthangradualleakageafterporeformation[35].LeakageofﬂuorescentdextransafterPCItreatmentwasvisual-izedinreal-timebyDeBruinandcoworkers,whonotedadisappearanceoftheamountoflabeledendosomes,indicatingfastreleaseofthecompounds(Fig.4B)[34].High-speedvideoacquisitionwasabletodocumentanasym-metricreleaseoftracermoleculesfromtheendosomes,representativeforaburst-likemechanism(Fig.4C).Fur-thermore,theauthorsinvestigatedreleasekineticsafterPCIofdifferentpDNA-polyplexescomposedofPEI,PLLandpoly-D-lysine(PDL),andnoticedadistinctinﬂuence

ofbufferingontheburstingeffect.Similarly,ﬂuores-centlylabeledoligodeoxynucleotides(ODNs)wereusedasself-quenchingtracermoleculesforthevisualizationofPEI-inducedendosomalrupture[66].Withaspinningdiskconfocalmicroscope,thereleaseofODNswasclearlyvisibleasaburstofﬂuorescenceﬁllingthecytosol,followedbyanaccumulationofﬂuorescentODNsinthenucleus(Fig.4D).ThesemeasurementsareaparticularlyniceconﬁrmationoftheprotonspongehypothesisofbufferingcationicpolymerslikePEI.

Thoughithasnotbeenprovenexplicitly,onecouldarguethatwhenvisualizingendosomalruptureofsmalltracermolecules,theco-incubationofsmalltracermoleculesmightaltertheactualosmoticpressurewithinendosomes,leadingtoaburstingeventwheretherenormallyisnotone.AnalternativeruptureassaynottroubledbythislimitationisproposedbyMaierandcolleagues[67],makinguseofacelllinestablyexpressingthelectingalectin-3(Gal-3)fusedtoanmCherryﬂuorophore.Gal-3bindsgalactoseresidues,whichunderphysiologicalconditionsarepresentexclusivelyontheextracellularorintraluminaldomainsofmembraneglycoproteins.Usinglive-cellvideomicroscopytheycoulddetectmembranerupturebyviralnanoparticlesinreal-timebasedontheaccumulationofcytosolicmCherry-Gal-3on

352

T.F.Martensetal.

Figure4Examplesofthevisualizationofendosomalrupturebyvideomicroscopy.(A)Light-inducedreleaseofcalceininmacrophagesbyburstingoftheendosomes,whichisnoticeableasaburstofcalceinﬂuorescenceinthecytosol.Reprintedwithpermissionfrom[35],©2012AmericanChemicalSociety.(B)Afastdecreaseintheamountofdextran-ﬁlledendosomesisseen,togetherwithanincreaseincytoplasmicﬂuorescence,indicativeofendosomalbursting.Scalebar5␮m.Reprintedwithpermissionfrom[34],©2008,withpermissionfromElsevier.(C)High-speedvideomicroscopyshowstheasymmetricreleaseofdextransfromanendosome,indicativeofbursting.Scalebar2␮m.Reprintedwithpermissionfrom[34],©2008,withpermissionfromElsevier.(D)Real-timevisualizationofendosomalescapeofﬂuorescentlylabeledoligodeoxynucleotides(FITC-ODNs).AsuddenreleaseofquenchedFITC-ODNscanbeseenasaburstofgreenﬂuorescence,accumulatingquicklyinthenucleusafterendosomalrelease.Reprintedwithpermissionfrom[66],©2013AmericanChemicalSociety.

intraluminalgalactoseresiduesinrupturedendosomalstruc-tures.Eventhoughthisassaywascoinedinaviralsetting,theauthorsconﬁrmedthatthiswasonlypossibleincaseoffullydisruptedmembranes,andnotincaseofporeforma-tion,extendingtheusefulnessofthisassaytoallkindsofman-madenanomaterials.

Assaysforstudyingmembranefusion

Whennanomaterialsaredeliveredbyenvelopednanopar-ticles,e.g.liposomalformulations,endosomalescapeishypothesizedtooccurviafusionwiththeendosomalmem-brane.Thefusionoflipidbilayersisusuallyassayedbyadyedilutionassay,whereﬂuorescentmarkersaredilutedoveranincreasedsurfacearea(Table2).Thiswillresultinachangeinﬂuorescenceintensity,whichcanbemonitoredwithﬂuorescencetechniquessuchasspectroﬂuorimetryorﬂuorescencemicroscopy.Asacontrol,lipidmembranesaretypicallylysedbyadetergentforcompletedilutionofthe

ﬂuorophores.Theﬂuorophoresusedareeitherlipophilicinnatureorcoupledtolipidcompoundssuchasphos-phatidylethanolamine(PE),sothattheycanbeefﬁcientlyincorporatedinthelipidbilayer.

Forexample,whenpyreneisloadedintoalipidmem-braneinasufﬁcientlyhighconcentration,excitablepyrenedimers,or‘‘excimers’’willbeformed.However,upondyedilutionoveralargersurfacearea,thesedimerswillbreakapart,leadingtoalossinﬂuorescence[59].AmorefrequentlyusedtechniquetomonitorﬂuorophoredilutionisFörsterresonanceenergytransfer(FRET).FRETisadistance-dependentinteractionbetweenapairofﬂuorophores,adonor(D)andacceptor(A),iftheemis-sionspectrumofthedonorﬂuorophoreoverlapswiththeexcitationspectrumoftheacceptorﬂuorophore.Ifbothﬂuorophoresareincloseproximity,typically1—10nm,theexciteddonorﬂuorophorecan(non-radiatively)transferitsenergytotheacceptorﬂuorophore.Thisresultsinanincreaseofacceptorﬂuorescenceattheexpenseof

Intracellulardeliveryofnanomaterials

353

Table2Successfullipid—lipidfusionleadstoadilutionoftheincorporatedﬂuorophoresoveralargersurfacearea.Dyedilutioncanbedetectedinanumberofways:dimerquenching,resultinginlossofﬂuorescence;FRET,resultinginashiftinﬂuorescenceratiooftwoﬂuorophores;dyedequenching,resultinginanincreaseinﬂuorescenceduetodequenchingofaself-quenchingﬂuorophore.

Dimerquenching

PyrPC

󰀁exc=345nm;󰀁em=480nm

Excellulo

[59]

FRET

NBD(D)

󰀁exc=465nm;󰀁em=535nmRhodamine(A)󰀁exc=545nm;󰀁em=576nm

Excellulo

[17,44]

Incellulo

[68]

Dyedequenching

Rhodamine󰀁exc=545nm;󰀁em=576nmDiD

󰀁exc=633nm;󰀁em=650nmLPDiI

󰀁exc=549nm;󰀁em=565nm

Excellulo

[55]

Incellulo

[69,70]

Incellulo

[71]

FRET=Försterresonanceenergytransfer;F=ﬂuorophore;D=donor;A=acceptor;NBD=4-chloro-7-nitrobenz-2-oxa-1,3-diazole;PE=phoshpatidylethanolamine;DiD=1,1󰀅-dioctadecyl-3,3,3󰀅,3󰀅-tetramethylindodicarbocyanine;DiI=1,1󰀅-dioctadecyl-3,3,3󰀅,3󰀅-tetramethylindocarbocyanine;PyrPC=1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine.

donorﬂuorescence[44].AfrequentlyusedFRETpairisNBD(4-chloro-7-nitrobenz-2-oxa-1,3-diazole)asadonorandrhodamineasanacceptordye[17,44,68].Liposomalpar-ticlesaredouble-labeledinahighconcentrationsothatthedistancebetweendonorandacceptorallowFRET.Uponsuccessfullipidfusionhowever,bothﬂuorophoreswillbedilutedoveralargersurfaceareaandthedistancebetweendonorandacceptorwillincrease,resultinginadecreasedFRETefﬁciency.Inanexcellulosetting,thisdouble-labelingcanbeappliedtoeithertheliposomalvector[17]orthearti-ﬁcialendosome[44],andFRETefﬁciencycanbemonitoredbyspectroﬂuorimetry.FREThasbeenusedincellularexper-imentsaswell,wherethedonorandacceptorﬂuorophoresareincorporatedinthelipidenvelopofthenanoparticulatecargoandFRETefﬁciencyismonitoredbylivecellspectralimagingmicroscopy[68].Athirdalternativefordyedilu-tionreliesontheself-quenchingcharacteristicsofspeciﬁcﬂuorophoreswhentheyareloadedaboveacertainconcen-trationinalipidmembrane.Uponfusion,theﬂuorophores

aredilutedbelowtheirself-quenchingconcentrationandtheincreaseinﬂuorescenceintensitycanbemonitored[55,69—71].

Fluorophoredilutioninlipidmembranesisfrequentlyusedtoinvestigatemembranefusion,eitherinducedbyCPPs[55,59,68],viralnanoparticles[69—71]orliposomaldeliv-eryvectorsofproteins[17]orpDNA[44,68].Nevertheless,itshouldbeemphasizedthatthemajorlimitationofthisassayistheinabilitytodistinguishbetweenlipidfusionandlipidmixing(Fig.5A)[72].Whereasbothfusionandmixingdenotestheinteractionbetweenlipidbilayersandwillleadtodilutionoftheincorporatedﬂuorophoresoveralargersurfacearea,lipidmixingwillnotresultintheintendeddis-placementofthecargo.Therefore,complementaryassayshavetobeincludedtodistinguishlipidmixingfromlipidfusion.Asanadditionalconﬁrmationinanexcelluloassay,itwassuggestedtomeasurethehydrodynamicsizebydynamiclightscattering.Iffusionoflipidnanoparticlesandarti-ﬁcialendosomesoccurs,thisshouldleadtoanincrease

354

T.F.Martensetal.

Figure5Combiningmembranedyedilutionandcontenttransferassaystoverifylipidfusion.(A)Asinglecolormembranedyedilutionassay(1)cannotdistinguishbetweenlipidmixingandlipidfusion,astheincreaseinredﬂuorescenceresultingfromdequenchingafterdyedilutioncouldbeattributedtobothcases.Acontenttransferassay(2)canbeusedasacomplementaryassay,whereﬂuorescenceofthecontentmarkerwillbelostupondilutiononlyaftermembranefusion.Bothassayscanbecombinedwhenusingtwo-colorﬂuorescencemicroscopy(3),whereasub-resolutionparticlewillappeargreen,yelloworred,dependingonwhetherthereisnointeraction,lipidmixingorlipidmembranefusionrespectively.(B)Byusingtwocolorﬂuorescencemicroscopy,Miyauchiandcolleaguesproposedanassaytodistinguishlipidmixingfromlipidfusionattheplasmamembraneandinendosomes.Labelingboththeenvelopewithamembranemarker(redﬂuorescent)andthecorewithsolubleNC-GFP(greenﬂuorescent),theviralparticlesoutsideofthecellsandinsideoftheendosomeswillemitﬂuorescencefrombothﬂuorophores(appearingyellow).Lipidmixingattheplasmamembranewillresultinalmostinﬁnitedilutionoftheredlabelandlossoftheredsignal.Sincelipidmixingdoesnotresultincargodisplacement,thegreencontentsignalisstillvisible.Lipidmixingwiththeendosomalmembraneontheotherhandwillstillresultinthepresenceofyellowparticlesintheimage,astheredmembranemarkerwillstillemitﬂuorescenceafternegligibledilutionintheﬁniteendosomalmembrane,andthecontentsignalwillalsoremainthesame.Whenlipidfusionoccurshowever,eitherattheplasmaorendosomalmembrane,thecontentmarkersignalwillbelostduetoinﬁnitedilutioninthecellcytoplasm.Fusionattheplasmamembrane,therefore,willresultinlossofbothsignals,whereasfusionwiththeendosomalmembranecanbeascertainedbylossofgreenﬂuorescenceandemissionofredﬂuorescencefromtheendosomalmembrane.Adaptedfrom[69],©2009,withpermissionfromElsevier.

Intracellulardeliveryofnanomaterials

inaverageparticlesize[44].Alternatively,contenttrans-ferfromtheenvelopednanoparticlehasalsobeenusedtoprovefusioninsteadofmixing(Fig.5A).Forinstance,thetransferofﬂuorescentlylabeledODNstogiantunilamel-larvesicleswasvisualizedbyﬂuorescencemicroscopy[73],thoughthisassaylackedhigh-throughput.Contentdisplace-mentoftracercompoundshasalsobeenemployedtoverifylipidfusion,bothtowardartiﬁcialendosomesexcellulo[59],andtothecytosolincellulo[69].However,suchcon-tentdisplacementcouldbeanindicationofbothmembranefusionandporeformation[72].Therefore,bothcontentdis-placementanddyedilutionassaysshouldbecombinedtoascertainlipidfusion.Miyauchiandcoworkersproposedsuchacombinationofdyedilutionandcontenttransferassays,whereadual-colorﬂuorescencelabelingofanenvelopedviruscoulddistinguishlipidfusionfromlipidmixingattheplasmamembraneortheendosomes[69](Fig.5B).Thoughthisassaywasusedinaviralsetting,thesametechniquecouldbeappliedtoman-madeenvelopednanoparticles.Basically,boththecontentandtheenvelopearelabeledinﬂuorophoreswithdifferentspectra(greenandred,respec-tively)resultinginayellowsignalwhentheviralparticleisintactoutsideofthecellorinsideoftheendosome.Toascer-tainiftheparticleinteractswiththeplasmamembraneortheendosomalmembrane,thedilutionofthemembranedyewillleadtocompletelossoftheredsignalwheninﬁnitelydilutedovertheplasmamembrane,orthesignalwillstillbevisiblewhendilutedoverthesmaller,ﬁnitesurfaceoftheendosomes.Nevertheless,thisdyedilutionassay,thoughinformativeaboutwhereinteractionoccurs,doesnotdistin-guishlipidmixingfromlipidfusion.Theﬂuorescentcontentmarkerisincorporatedtothisend,asalossofitsﬂuores-cenceindicatesfusioninsteadofmixing,regardlessofthelocation.

Biologicallyrelevantartiﬁcialmembranes

Artiﬁcialmembranesmimickingtheendosomalmembranearefrequentlyusedinexcelluloassaystoevaluateendo-somalescapeinacontrolledenvironment.ThemajorconstituentsoftheendosomalmembranearePC(phos-phatidylcholine),PEandPS(phosphatidylserine),thatarepresentataratioof55%,25%and10%oftotallipidcontent,respectively[73,74].AlthoughoftenartiﬁcialmembranesaresimpliﬁedversionsconsistingonlyofPCandsome-timescholesterol,moreandmorestudiestrytomimictheendosomalmembraneasaccuratelyaspossible[44].TheimportanceoflipidcompositiononmembraneinteractionswasprovenforexampleinastudybyBerezhnaandcowork-ers[73].Lipidfusionwasevaluatedbetweenlipoplexesandgiantunilamellarvesicles(GUVs)consistingofdifferentcompositionsofPC,PE,PSandsphingomyelin(SM).Remark-ably,theauthorsﬁndthatfusionofthecationiclipoplexeswiththeartiﬁcialmembraneandsubsequentreleaseofnucleicacidsispredominantlymediatedbythenegativelychargedPSandPE,whilePCandSMaresupposedlyinertinthisprocess.Similarly,astudybyYangetal.[55]showedthatTAT-mediatedfusionwasdependentontheanioniclipidbis(monoacylglycero)phosphate(BMP),highlyenrichedintheintraluminalvesiclesoflateendosomes.Thus,thesestudiesclearlyshowthatthelipidcompositionofthe

355

artiﬁcialmembraneusedintheexcelluloassayisofutmostimportance.

AnotherkeyaspectofendosomesistheshiftinpHcom-paredtotheextravesicularenvironment.Torecreatetheendosomalacidicenvironment,abufferwithsimilarpHcanusedtoresuspendtheartiﬁcialendosomes[26].AdifferentapproachbyMadanietal.involvespreparingartiﬁciallargeunilamellarvesicles(LUVs)withbacteriorhodopsin(BR)inte-gratedinthelipidlayer.Uponillumination,BRwillactasaproton-pumpingV-typeATPase,henceacidifyingtheLUV’sinteriorandmimickingthelateendosomalenvironmentinacontrolledmanner[57].

Nevertheless,liposomeswillalwaysbeasimpliﬁcationofactualendosomalmembranes,giventhelackofproteinsandlipidasymmetry[75].Inanattempttoinvestigateinter-actionswithanartiﬁcialendosomalmembranewithashighabiologicalrelevanceaspossible,redbloodcells(RBCs)havebeenfrequentlyusedasamodel.Adyedequench-ingassaywithRBCsasamodelmembranehasbeenusedbyLakadamyalietal.[70]tostudyfusioninaviralcon-text.Similartoleakageassays,RBCintegrityisfrequentlyemployedtoevaluateporeformationbyendosomalescapeenhancingcompounds,suchasCPPs[26],PEI[46]orotherdrugdeliveryvectors[47,56,76].DamagetotheRBCmem-branewillleadtoleakageofhemoglobin,whichcanbequantiﬁedbyabsorptionmeasurementsat450nmafterremovalofintacterythrocytes.Anevenmorerepresen-tativemodelmembranewasusedinaleakageassaybyPrchlaetal.[77].HeLacellswerepreviouslyloadedwithahighconcentrationofbiotin-dextrans,afterwhichendo-someswereisolatedfromthecells.Afterwards,leakageofbiotin-dextransexcellulocouldbedetectedintheextrav-esicularenvironmentwithanELISAassay.Interestingly,LeBlancandcoworkersadoptedasimilarmethod[71],thoughelaboratedbyincubatingthepre-loadedisolatedendosomesinacytosol-mimickingsolutioncontainingbuffersandATPforcontinuousacidiﬁcationofthepuriﬁedlateendosomes.Thoughthisassaywasusedinaviralsetting,themimickingofthecytosolicenvironmentcanbeextrapolatedforuseinanyexcelluloassay.

Studyingendosomalescapeefﬁciency

Ofevengreaterpracticalusethanelucidatingthemecha-nismofendosomalescape,isdeterminingifandtowhichextentendosomalescapeoccurs.Insteadofinvestigatingthemembraneintegrityorfusion,these(mostlycellu-lar)assaysactuallymonitortheamountofcytosoliccargo.Therefore,theseassaysarenotinﬂuencedasmuchbythetypeofdeliveryvectorastheyarebythetypeofcargo.Inthissectionanoverviewisprovidedofthedifferentmethodsthathavebeenreportedtodeterminesuccessfulcytosolicdeliveryofnanoparticulatecargo(Table3).

Biologicalactivity

Whentheaimistodeliverbiologicallyactivemolecules,e.g.therapeuticmoleculesindrugdelivery,successfulcytosolicdeliverycanbeeasilyassessedbythebiologicalactivity.Forinstanceincaseofgenetherapy,amodelmRNAorpDNAisusedencodingforareporterproteinsuchasluciferase

Table3

Overviewofdifferenttechniquesusedtomonitorcargodisplacementtothecytosol.

DistinguishcytosolicMeasuringtechniqueUse

Reference

Pro’s

Con’s

fractionfrom

sequesteredfraction

BiologicalactivityinReversetranscriptase-Knockdownofreporter[80]

-Invivomeasurements-Indirectmeasurecytosol

quantitativePCR

orhouse-keepinggene-Easyhigh-throughput-Fixedendtime-pointbysiRNA

quantiﬁcation

-LimitedtosiRNASpectro/luminometer

-Expressionor

[46,79]

-Applicabletodifferentcargo-Indirectmeasureknockdownofreporter-Easyread-out

-Populationaveragegene

-Easyhigh-throughput-Fixedendtime-point

-Enzymeactivity[81]quantiﬁcation

Flowcytometry

-Expressionor

[78]

-Applicabletodifferentcargo-Indirectmeasureknockdownofreporter-Fastandeasyread-out-Fixedendtime-point

gene

-Easyhigh-throughputquantiﬁcation

-Single-cellmeasurementsELISA

-DetectionofIL-2[17]

-Live-cellmeasurement-Indirectmeasure

secretionasantigen-Easyhigh-throughput-LimitedtoantigendeliverytoAPCs

presentationassayquantiﬁcation

Immunoﬂuorescence-Immunostainingof[35]

-Easyhigh-throughput-Indirectmeasure

microscopy

SIINFEKLasantigenquantiﬁcation

-LimitedtoantigendeliverytoAPCspresentationassay

-Fixedendtime-point

Cellularfractionation

QuantitativePCR

-AmountofDNA

[82,83]

-Cytosol<>endosomes-Labor-intensivecellular-Easyhigh-throughputfractionation

quantiﬁcation

-Carrier-boundDNA?-Invivomeasurements-Fixedendtime-pointStem-loopPCR

-Amountofbiologically[84]

-Cytosol<>endosomes-Labor-intensivecellularactivesiRNAboundto-Easyhigh-throughputfractionation

RISC

quantiﬁcation

-Immunoprecipitationstepnecessary-Invivomeasurements-Fixedendtime-point-Onlyactivecargo

-LimitedtosiRNA

Radio-activity

-AmountofDNA[50]-Cytosol<>endosomes-Labor-intensivecellularfractionation

-Amountofproteins

[86]-Easyhigh-throughputquantiﬁcation

Intracellular

Wide-ﬁeldepi-ﬂuorescenceor-Visualscoring[53,65,81]-InCytosolvivomeasurements<>endosomes-Limitedtosmallcargoﬂuorescenceproﬁle

confocalmicroscopy

-Quantiﬁcation

[47,87,88]

-Easyread-out-Difﬁcultquantiﬁcation-Speciﬁclabeling

-Fluorescentlabeling

356T.F.Martensetal.Table3(Continued)

Distinguishcytosolic

MeasuringtechniqueUse

ReferencePro’sCon’s

fractionfrom

sequesteredfraction

Fluorescent

Fluorescence-Measurereductive[89]

-Easyread-out-Indirectmeasure

microenvironmentmicroscopy

environment

-Speciﬁclabeling

-Standardcurvenecessarysensors

-Measuredifferencein[26,51,71,90—92]-Livecells-NoquantiﬁcationpH

-Fluorescentlabeling

Real-timevisualizationLive-cell-videoVisualizeburstingof[66,84,93]

-Cytosol<>endosomes-Limitedtosmallcargoofendosomalescape

microscopy

endosomes

-Mechanismof-Lowthroughputendosomalescapequantiﬁcation

--Livecells

-Fluorescentlabeling

Visualizefusionwith[69—71]endosomes-Speciﬁclabeling

ColocalizationConfocalmicroscopy

-Visualscoring

[10,96—98]-Cytosol<>endosomes-Difﬁcultquantiﬁcation-Easyread-out-Fluorescentlabeling

-[51,79,82,100]

Quantiﬁcation

-Speciﬁclabeling-Limitedtemporalresolution-Allcargo

Fixationsometimesnecessary

Visualassessment

Electronmicroscopy

-Visualscoring

[14,49,54,84,-Veryhighresolution-Fixationartifacts

105,108,109]-Labelingnotalwaysnecessary-Lowthroughputquantiﬁcation

Intracellulardeliveryofnanomaterials357358

[46]oreGFP[78].Theextentofreporterproteinexpres-sionisthenameasureforsuccessfuldeliverytothecytosolformRNAandfurthertothenucleusinthecaseofpDNA,whichonlycouldhavehappenedifendosomalescapehasoccurred.ForsiRNA,amutantcelllinecanbeusedthat(stably)expressesareporterprotein.TheextenttowhichthedeliveredsiRNAsilencesthereporterproteinexpressionisthenagainameasureforsuccessfulcytosolicdeliveryofsiRNA[79].Alternatively,knockdownofahousekeepinggenecanbequantiﬁedbyRT-PCR[80].Forproteindelivery,onecanmakeuseofmodelenzymes,suchasthe␤-galactosidaseenzyme,whosebiologicalactivitycanbemeasuredastheintensityofbluecoloraftercleavageofX-Galsubstrate[81].Whendealingwithantigendelivery,theuniquepropertiesofAPCsallowdifferentantigenpresentationassaysasbio-logicalread-out.Whenusingovalbumin(OVA)asamodelantigenicprotein,theextentofOVA-deliverytoAPCscanberelatedtoeithertheproliferationofandIL-2secretionbyOVA-peptidespeciﬁcCD8+T-cells[17],ortotheamountofMHC-IdependentSIINFEKLpresentationbyAPCsasmea-suredbyimmunohistochemicalanalysis[35].

Itisclearthatthenanomaterialsinvestigatedwiththeseassaysshouldconsistofcargowithbiologicalactivity(suchaspDNA,siRNAorproteins),regardlessofwhetherthiscargoisdeliveredbyliposomes[17],polymersomes[35]orcationicpolymerssuchasPEI[46]orchitosan[78].Itmustbenotedhoweverthatthebiologicalactivityisusuallytheend-pointofanintracellularcascade,fromwhichendosomalescapeisonlyoneaspect.Nanoparticleuptake,dissociationofthecargofromitscarrier,cytosolictranslocationtothenucleus(forpDNA),transcription,translation,etc.canallinﬂuencetheﬁnaloutcome.Therefore,thebiologicalassaysareonlyanindirectmeasureforendosomalescape,warrantingtheuseofothermorespeciﬁcassays,asdescribedfurtheron.

Cellularfractionation

Todistinguishnanoparticulatecargointhecytosolfromthatsequesteredintheendolysosomalcompartment,afre-quentlyusedassayinvolvesfractionationofthecellsandmeasuringthecargointhecytosolicandendosomalfractionswithinvitroassays.Forexample,theamountofnucleicacids[82,83]incytosolandendosomeswasquantiﬁedwithquantitativePCRafterfractionation.TomeasuretheamountofbiologicallyactivesiRNAinthecytosol,theAgo-proteinoftheRISC-complexwasimmunoprecipitatedfromtissuelysates,followedbystem-loopPCRquantiﬁcationoftheamountoftargetsiRNApresentintheRISC-complex[84].InsteadofPCRquantiﬁcation,itisalsopossibletouseﬂuorescentlylabeledcargoandquantifytheﬂuorescenceindifferentcellularfractions/compartments.Forexam-ple,ﬂuorescentlylabeleddextransandPEG-particleshavebeenquantiﬁedthiswayaftercellularfractionation[85].Radio-activelabelinghasbeenusedforbothpDNA[50]andproteins[86]aswell.Itwasevenshownthatradio-activelabeledproteinscanbequantiﬁedincellularfractionsfromtissuelysatesafterinvivoadministration[86].

Wewouldliketohighlightthatforinvestigatingendo-somalescape,cellularfractionationismerelyameansofdistinguishingcytosolicfromendosomalcargo,andshouldalwaysbecomplementedwithinvitroassaystomeasure

T.F.Martensetal.

toamountofcargoineachfraction.Thoughthenanoma-terialswhichcanbeinvestigatedbythisendosomalescapeassayarethereforeonlylimitedtowhetherthecargocanbemeasuredornot,typicalconcernsaboutcellularfrac-tionationarethelabor-intensivenessoftheassay,aswellastheuncertaintyofcollectingallthefractionswhilstavoid-ingcontaminationfromtheendolysosomalfractioninthecytosolicfraction[83].Also,questionshavebeenraisedaboutthefractionationprocess,whereuptakeofmacro-moleculesornanomaterialsmightalterthevesiclebuoyantdensityandthereforenecessitateanadaptedpuriﬁcationprotocolforeachnanoparticle[85].Thereforeitseemsadvisabletoconﬁrmﬁndingsbythecellularfractionationassaywithotherassays,astheonesdescribedbelow.

Fluorescencemicroscopy

Sincemostnanoparticlesforintracellulardeliverycanbeﬂuorescentlylabeled,orinsomecaseshaveintrinsicﬂuo-rescentproperties,themostfrequentlyusedtechniquefordiscriminatingcytosolicfromendosomalcargoisviaﬂuores-cencemicroscopy.Giventhatthecargoissmallenoughsoitcandiffusethroughoutthecytosol,adiffuseorpunctateIFPcanberelatedtotheefﬁciencyofendosomalescape(cfr.section‘Assaysforstudyingporeformation’).Thishasbeenusedtoassessendosomalescapeofpeptides[53,65],siRNA[87],Qdots[8]andsmallerproteins[47,88].Inmoststud-ies,thediffuseorpunctateﬂuorescencepatternisscoredvisually,sothattheseexperimentsremainratherqualitative[53,65,81].Yet,insomestudiesitwasattemptedtoquantifytheIFP.Forexample,themedianﬂuorescencevalueofcom-pleteconfocalimageswasproposedasasimpleparametertodistinguishapunctatepatternfromadiffuseintracellu-lardistribution[88].Alternatively,theaverageﬂuorescenceintensityinregionsofinterestbelongingtothediffuseandpunctatestainingcanbequantiﬁedandcompared[47,87].Fluorescentprobeshavealsobeenusedinadifferentapproachtosenseachangeinthenanoparticlemicroen-vironment.Forexample,byattachingaquencherviaadisulﬁdebondtoﬂuorescentlytaggedproteins,transfertothecytosoloftheseproteinstriggeredanincreaseinﬂuores-cenceduetoreductionofthedisulﬁdebondsbycytosolicglutathioneandreleaseofthisquencher[89].Otherwise,thechangeinmicroenvironmentpHbetweenacidicendo-somesandcytosolhasbeensensedbyusingﬂuorophoreswithapH-dependentﬂuorescenceemission[71,90].Theseﬂuorophorescanbeeitherattachedtothenanoparticle,orbeusedasananosensoritself.ToincreasesensitivitytoabroaderpHrange,thesesensorsaremadeofapH-independentreferenceﬂuorophoreandone[26,51,91]ortwo[92]dyeswhoseemissionsareaffecteddifferentlybythepH.Theseratiometricﬂuorescentsensorshavebeenemployedonseveraloccasions,andmorespeciﬁcallytoinvestigatetheendosomalescapeofprotonsponge-basedPEI-deliveryvectors[51,91,92].Nevertheless,whenusingsuchnanosensors,itshouldbenotedthatastandardcurveisalwaysrequiredtorelateﬂuorescenceemissionratiotopH(Fig.6A).ThepHobtainedbythesenanosensorscanthenbevisualizedintheacquiredimagesbymeansofcolor-coding,asdemonstratedinFig.6B.

Intracellulardeliveryofnanomaterials

Figure6(A)Exampleofastandardcurveobtainedinvitro,whereﬂuorescenceemissionintensityratioisrelatedtopHofbuffersandﬁttedtoatheoreticalmodel.(B)Colorcodedmicro-scopicimageoftheintracellularpHfollowingincubationwithathree-ﬂuorophorenanosensor.Thetoprowshowstheimagesasacquiredbyﬂuorescencemicroscopy,whilethebottomrowarecolorcodedimagesaccordingtothepHstandardcurveinA.Scalebar10␮m.N=nucleus.ReprintedbypermissionfromMacmillanPublishersLtd:MolecularTherapy[92],©2012.

Endosomalescapecanalsobeinvestigatedbylive-cellvideomicroscopy.Forinstance,thetransferofsmallnanoparticulatecargosuchasODNs[66],ribozymes[93]andsiRNA[84]towardthecytosolbyPEI-mediatedendosomalrupturewasvisualizedbyvideomicroscopyasaburstofﬂu-orescencefromtheendosomestothecytosol.ThereleaseofpDNA,however,cannotbedirectlyvisualizedsinceithasverylimitedmobilityinthecytoplasm[34,66].Thesamealsoappliestoaggregatesofcargodeliveredtothecytosol,ashasbeenobservedforQdotsdeliveredbylipofectamine,polymers,CPPsandphysicaltechniquessuchaselectropora-tion[9,94].ThisproblemwascircumventedbyRehmanandcolleagues,whopreparedPEI-pDNAcomplexescontaininginadditionself-quenchedﬂuorescentlylabeledODNsastracercompounds[66].Thisallowedtodetectburstingofendo-somesbydequenchingofthesmallODNs,inasimilarwayascalceincouldbeused.Interestingly,inthiswaytheycouldactuallycountthenumberofendosomalescapeeventspercell.Furthermore,byvisualizingtherateofODNaccumu-lationinthenucleus(Fig.4D),theycouldconﬁrmthatPEIinducessuddenendosomalburstingandimmediaterelease

359

ofcargointhecytosol,whileamoregradualleakageofODNswasobservedwhenlipidcarrierswereused.Likewise,Remautetal.madeuseofFRET-ODNsthatcouldestimateboththecytosolicdeliveryoftheODNsbasedonthenuclearaccumulation,andtheintegrityofthedeliveredODNsbasedontheFRETsignal[95].Itwasfoundthatthecompositionofthepoly-beta-aminoestercarriercangreatlyinﬂuencetheamountandintegrityofODNsthataredeliveredtothecytosol.

Dual-colorﬂuorescencemicroscopyandcolocalizationanalysisiswidelyusedtodistinguishcytosolicnanoparticlesfromthosethatarestillsequesteredintheendolysosomalcompartments,especiallythosetoolargetodiffusethrough-outthecytosol,suchaspDNApolyplexesandlipoplexes.Often,(thelackof)colocalizationbetweennanoparticlesandendosomesisregardedasameasureforendosomalescape.Althoughcolocalizationisfrequentlyvisuallyscored[10,96—98],itcanbeverywellquantiﬁedbydifferentapproaches[99].Themostrudimentarywayistoquantifycolocalizationonaper-pixelbasis,wherethepercentageofoverlappingpixelsinbothchannelsistakenasameasureforcolocalization[79,82].OtherfrequentlyusedparameterstoquantifycolocalizationbetweenpixelsarethePearson’s[100]andManders’correlationcoefﬁcient[51].Inourgroup,wehaverecentlyoptimizedadual-colordynamiccolocal-izationtechnique,whichallowsquantifyingtheamountofcolocalizationbetweentwoﬂuorescentlabelsbasedontheirmovementduringacertaintimeframe,inthiscasethecolocalizationbetweenlabeledendosomesandlabeledpDNA-polyplexes[101,102].Intheory,thismethodshouldalsobecapableofdetectingcorrelatedtrajectorieswhichdivergeovertime,ascouldbethecaseforendosomesandtheirreleasedmacromolecularcargo.

Giventhewidespreaduseofﬂuorescencemicroscopyandcolocalizationanalysisforassayingendosomalescape,itisinstructivetohighlightsomeofthewell-acknowledgedtechnicalchallengesandlimitationsofthetechnique[103].Firstly,duetothelimitedopticalresolutionofthemicro-scope,careshouldbetakenwhenevaluatingdiffusevs.punctatestaining,asout-of-focuslightmightfalselygivetheimpressionofadiffusestainingorapparentcolocalizedpixels.Furthermore,sub-resolutionobjectsthatarelocatedclosertogetherthanthemicroscope’sresolutionwillalwaysappeartobecolocalized,whichcanbeespeciallyprob-lematicintheperinuclearregionthattypicallycontainsahighdensityofendosomes.Confocalmicroscopycertainlyispreferredoverwideﬁeldepi-ﬂuorescencemicroscopyasiteliminatesout-of-focusﬂuorescencetoalargeextent.How-ever,duetoarelativelylowsensitivity,confocalmicroscopycaneasilymissdimfeatureslikesmallendosomesandnanoparticles.Thiscanbemitigatedtosomeextentbyusingslowscanningspeedstocollectmorephotons,iftheﬂuores-centmoleculesdonotbleachtooquickly.Anotherfrequentlyencounteredproblemiscrosstalkofdifferentﬂuorophoresbetweendetectionchannelswhichagainmayleadtofalsecolocalization.Thiscanbelimitedbyrecordingeachchan-nelseparatelywithsequentialexcitationofeachofthedifferentﬂuorophores[100],thoughthegeneralguidelineistouseﬂuorophoresthathaveaminimalspectralover-lap.Nevertheless,confocalmicroscopyisusuallyassociatedwithpoortemporalresolutionandhencetheneedforﬁxa-tion,whichimportantlyhasbeenshowntoleadtoartifacts

360

concerningtheendosomalsequestration[54,104].Nonethe-less,fastconfocalimageacquisitioninlivecellsisnowmadepossibleusingspinningdiskconfocalmicroscopesequippedwiththenewestgenerationofsensitiveCCDcameras,liketheelectron-multiplyingCCDsorscientiﬁcCMOScameras[66,84].

Ofequalimportanceforproperﬂuorescencemicroscopyisthechoiceofﬂuorescentendosomalmarkers.Dex-trans[97]ortransferrins[54]thatareaddedtothecellmediumareconsideredtobeanon-speciﬁcendo-somelabelingmethod.Alternatively,onecanpulse-chaselipophiliccarbocyaninedyesintheplasmamembrane[10]oruseplasma-membranespeciﬁcdyessuchasPKH67[68],whichwillendupinmostendocyticvesicles.Ontheotherhand,speciﬁclabelingoflysosomescanbeattainedbyusinglysosome-speciﬁcmarkerssuchaslysosomal-associatedmembraneprotein1(LAMP1),forexamplebyimmunoﬂuorescencestainingagainstLAMP1[87]ortrans-geneexpressionofaneGFP-LAMP1-construct[101].AlsoacidotropicdyescanbeusedsuchasLysotrackerBlue[9,93],LysotrackerGreen[79,80],LysotrackerRed[17,96]andLysoSensorGreen[100],whichhavebeenshowntolabelabout70%oftheintracellularvesicles[82].Onacau-tionarynote,itisimportanttokeepinmindthatmostacidotropicdyesareconsideredweakbasesandmightinﬂu-enceendosomalacidiﬁcationafterlongincubationtimes.Therefore,itisrecommendedtofollowthemanufacturer’sinstructions.Furthermore,combiningacidotropicdyeswith‘‘protonsponge’’-nanoparticlesforparticle-lysosomecolo-calizationhasbeenshowntoinﬂuencetheoutcome[105].Indeed,thoughPEI-particlesshowedalackofcolocalizationwithanacidotropicdye,colocalizationwasinfactnoticedwithﬂuorescentlylabeledLAMP1.ThiswasattributedtothefactthatPEIhasabufferingeffectontheendolysosomes,therebyinhibitingstainingbyacidotropicdyes,aconclusionthatisalsoconﬁrmedbyMoandcoworkers[98].

Regardinglabelingofthenanoparticleofinterest,onehasthechoiceofeitherlabelingthecarriermoleculesorthecargo.Ontheonehanditcanbearguedthatitisbesttolabelthecargosinceitwilltypicallybeincorporatedintothecarrierandhavetheleastinﬂuenceonuptakeandintra-cellularprocessingofthenanoparticle.Also,labelingthecarriermightgiverisetofalseconclusions,seeingascarrierdissociationisshowntoalreadyoccurintheendosomesforPEI[66].Ontheotherhand,ﬂuorescentlabelsaretypicallyhydrophobicandmightinteractwiththeendosomalmem-braneandinﬂuencethedisplacement[66,106].Aconsensusonthishasnotbeenreached,however,warrantingfurtherstudiesonthistopic.

Electronmicroscopy

Eventhoughsamplesneedtobeﬁxedandrequireexten-sivepreprocessing,transmissionelectronmicroscopyisstillfrequentlyusedforassayingendosomalescapeduetoitsunparalleledresolution.Itallowstovisuallydis-tinguishifthenanoparticlesarelocatedfreelyinthecytosol,orsequesteredinmembranousvesicles.Forinor-ganicnanoparticleslikeQdots[54],goldnanoparticles[107]andsuperparamagneticironoxideNP’s[14,108],labelingstepsareusuallynotnecessary(Fig.7AandC).However,organicnanoparticlescannotalwaysbeeasilydistinguished

T.F.Martensetal.

fromthecellularstructures,thoughforpolymericnanopar-ticles,opinionsvary.BieberandcoworkersdidnotlabelPEI-baseddeliveryvectors,arguingthattheelectrondensePEIwouldgivesufﬁcientcontrastintheTEMimages(Fig.7B)[105].Ontheotherhand,osmiumtetroxideisfrequentlyusedtolabelpolymericdeliveryvectorssuchaspoly(lactic-co-glycolicacid)(PLGA)andPEItoenhancetheircontrast[49,109].Goldcanalsobeemployedascontrastagent,asusedforinstancebyGilleronandcolleaguestovisualizesiRNAinthecytosolorinmembranousvesicles[84](Fig.7D).Draw-backsofTEMincludethenecessityforﬁxationandthatbothsamplepreparationandanalysisisverylabor-intensive.Usually,endosomalescapeisscoredvisu-allybyassessingifthemajorityofthenanoparticulatecargoresidesinmembranousvesicles,orfreelyinthecytosol[14,49,54,105,109].Imelliandcoworkerspresentedquan-titativedataontheamountofviralnanoparticlesattheplasmamembrane,inthecytosolandintheendosomalvesicles,basedondatafrom8—10cellsand23—45par-ticlespercell[110].ThemostadvanceduseofTEMinthiscontextwasarguablypresentedbyGilleronandcol-leagueswhoproposedasemi-automaticquantiﬁcationofTEMimages,bywhichsiRNAcoupledto6nmcolloidalgoldnanoparticles(siRNA-GNP’s)areautomaticallydetectedinTEMimagesaftersettingacertainthreshold(Fig.7D,left).Manualassignmentoftheparticlestoeitherthecytosolicorvesicularlocationwasstillrequired(Fig.7D,middle).Interestingly,toassessifendosomalescapeofsiRNA-GNP’soccurredthroughaburst-likemechanisminaspeciﬁccom-partmentorviagradualrelease,Gilleronandco-authorsplottedtheamountofparticlesinthecytosolovertime[84].Mathematicalmodelingshowedthatagradualreleaseofparticlesshouldleadtoalineartrend,whereasaburst-likemechanismshouldgiverisetoasigmoidalcurve.Experi-mentaldatashowedasigmoidalcurveinbothcelllines,indicatingaburst-likemechanism(Fig.7D,right).

Assessingtheendosomaldepotofnon-deliveredcargo

Additionalveriﬁcationoftheefﬁciencyofendosomalescapeisassayedbycheckingtheamountofcargostillsequesteredintheendolysosomalcompartmentsusingpreviouslydis-cussedmethods.Bydeliberatelydisruptingtheremaininglysosomesandcomparingtheamountofcargointhecytosolwithnormalcircumstances,onecangetanideaofthecargowhichisinefﬁcientlydelivered.Thetechniquesusedforthisendosomaldisruption,e.g.protonsponge[50],Leu-Leu-OMe[38],PCI[12,78],osmoticshock[28],etc.havepreviouslybeenmentionedandthereaderisreferredtothissectionformoredetail(seesection‘Enhancingendosomalescape’).

Computationalmodelingofendosomalescape

Experimentalresultsfromdifferentassaysarefrequentlycombinedinamathematicalmodelinordertoestimatethoseparametersthataredifﬁculttomeasuredirectly.Forexample,computationalmodelingallowedVargaetal.[111]toestimatecellbinding,uptake,endosomalescape,vectorunpackagingandnuclearimportbycombiningquan-titativeexperimentalresultsonvectoruptake,amount

Intracellulardeliveryofnanomaterials

361

Figure7(A.)Visualizationofgoldnanoparticles(GNPs)bytransmissionelectronmicroscopy.RedarrowsindicatecytosolicGNPs,bluearrowsindicatesequesteredparticles.Scalebar500nm.Adaptedwithpermissionfrom[107],©2010AmericanChemicalSociety.(B)VisualizationofPEI-particlesbyTEMaselectron-densespots(whitearrows).PEI-particlesareseenassociatedwiththeendosomalmembrane(blackarrow),leadingtomembranedamage(arrowhead).Reprintedfrom[105],©2002,withpermissionfromElsevier.(C)TEM-visualizationofsuperparamagneticironoxidenanoparticles.Adaptedwithpermissionfrom[108],©2010JohnWiley&Sons,Inc.(D)QuantiﬁcationofendosomalescapebyGilleronandcolleagues(left)AutomaticdetectionofGNPscoupledtosiRNA.(middle)Avisualdistinctionwasmadebetweenearlyendosomes,lateendosomesandlysosomes,basedonvesicularmorphology.(right)Gradualreleaseandcompartment-speciﬁcreleasewereplottedovertime(respectivelyalinearcurveandasigmoidalcurve),andexperimentaldatawasfoundtocorrelatetoacompartment-speciﬁcrelease.AdaptedbypermissionfromMacmillanPublishersLtd:NatureBiotechnology[84],©2013.

ofpDNAinthenucleusandtransfectionefﬁciency.Simi-larly,Mooreandcolleagues[26]wereabletodeterminetheendosomalescaperateconstant(kescape)byﬁttingamathematicalmodeltoexperimentaldataobtainedbyratiometricnanosensorsmeasuringtheintracellularpHwithﬂuorescencemicroscopy.AstudybyDinhandcoworkers[112]furtheremphasizestheimportanceandusefulnessofmathematicalmodeling,butalsohighlightthelimitationthatmostmathematicalmodelsapproachcellulartransportmechanismsbyﬁrst-orderkineticsbetweenwell-deﬁnedcompartmentsonly.Accordingtotheauthors,theirstudyimprovesonpreviousmathematicalmodelsbyincorporat-ingthespatialstructureofthecell,aswellastakingintoaccountthecontinuousmovementofnanoparticles

basedonsingle-particletrackingexperiments.Alterna-tively,computationalmodelshavebeenusedasso-called‘‘computationalmicroscopy’’,demonstratedbyTianandMa[113].Bycoarse-grainmoleculardynamicsmodelingintheMARTINIforceﬁeld,theauthorsinvestigatethemecha-nismofendosomalescapeofpH-responsivedendrimers.Bycomputationalmodeling,theauthorscouldobtainahigh-resolutionsimulationofhowtheendosomalmembraneisdestabilizedbytheprotonateddendrimers.

Conclusionsandfutureperspectives

Nanomaterialsforintracellulardrugdeliveryorcellimag-ingapplicationsrequireanefﬁcientcytosolicdelivery

362

mechanism.Upuntilnow,theescapefromendosomalsequestrationandsubsequentdegradationremainsamajorbottleneck.Inordertodesignimprovednanomaterials,reli-ableassaysfordetectingandquantifyingendosomalescapearenecessary.Inthisreviewwehavegivenanoverviewofassaysthatarecurrentlyavailable.Giventhestrengthsandweaknessesofeachapproach,acombinationofcomplemen-tarymethodsispreferred,dependingonwhichaspectofendosomalescapeisinvestigated.

Notonlythemechanismortheefﬁciencyofendosomalescapeisofimportance.Itwouldbeofinterestinfuturestudiestotryandrelateendosomalescapetothetimeandlocationatwhichithappensinthecell.Suchinforma-tioncouldberelevantforavoidingprematuredegradationbytheacidicenvironmentofthelateendosomesortheharshhydrolyticconditionsofthelysosomes.Itisthereforeexpectedthatlivecellimagingwillonlygaininimportance.Acurrentlimitation,however,isthatthisismostlybasedonﬂuorescentlylabeledcargoorcarriermaterials.Asitmaybethatthisinﬂuencestheintracellularprocessingmechanismsorkinetics,itisofspecialinteresttolookintothecapa-bilitiesoflabel-freemicroscopytechniques,suchasRamanimaging,tofollowuponendosomalescape.

Acknowledgements

FinancialsupportbytheGhentUniversitySpecialResearchFund(01B04912andDEF08/FOP/010)andtheFundforScientiﬁcResearchFlanders(FWO,Belgium;G019711N)isacknowledgedwithgratitude.KRisapost-doctoralfellowoftheFWO.Theauthorshavenootherrelevantafﬁliationsorﬁnancialinvolvementwithanyorganizationorentitywithaﬁnancialinterestinorﬁnancialconﬂictwiththesubjectmatterormaterialsdiscussedinthemanuscriptapartfromthosedisclosed.Nowritingassistancewasutilizedintheproductionofthismanuscript.

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ThomasF.MartensobtainedhisMaster’sDegreeinBioscienceEngineeringfromGhentUniversityin2009.Lateron,hemovedtothefacultyofPharmaceuticalScienceswherehestartedhisPhDresearchunderthesupervisionofProf.KevinBraeckmansintheBio-PhotonicImagingGroup.HisPhDprojectinvestigatestheoptimaldeliveryofnanomedicinestotheretinaviaintraocularadministration.Hisresearchinterestsincludeadvancedlightmicroscopy,oculargenetherapyandintracel-lularprocessingofnanomaterials.

KatrienRemautwasbornonthe10thofNovember1978inGhent.In2001,shegraduatedasPharmacistwiththegreatestdistinctionatGhentUniversity.ShethenstartedresearchworkintheLabofGen-eralBiochemistryandPhysicalPharmacyunderguidanceofProf.DeSmedtandProf.Demeester.In2007,shereceivedthetitleofdoctorinpharmaceuticalscienceswithherdissertation‘Exploringtherelationbetweentheintracellularfateandbiologicalactivity

ofnucleicacidnanoparticles’.Katrienreceivedseveralscientiﬁcprices(e.g.beststudentprice,2posterprices,priceoftheRoyalAcademyofMedicineforScientiﬁcResearchinPharmacy,period2008—2011)andwaselectedin2013asmemberoftheYoungAcademyinFlanders.Asapostdoc,KatrienperformedresearchattheEuropeanMolecularBiologyLaboratory(EMBL,Germany)andiscurrentlybackatGhentUniversity,attheLabofGeneralBiochem-istryandPhysicalPharmacy.

JoDemeester(1951)earnedaPh.D.inPhar-maceuticalScienceswithgreatestdistinctionatGhentUniversity(1980),wherehebecameProfessor(1989)andheadoftheLaboratoryofGeneralBiochemistryandPhysicalPharmacy(1997).HeislaureateoftheBelgianRoyalAcademyofSciencesandFirstLaureateoftheTravelgrantoftheMinistryofEducation.HewasVice-PresidentoftheBelgianBiophysicalSocietyandPresidentoftheBelgianSociety

T.F.Martensetal.

ofPharmaceuticalSciences.Since1994heisDirectoroftheInternationalCentreforStandardsoftheInternationalPharma-ceuticalFederation(F.I.P).Since2003heisPresidentoftheF.I.P.EnzymeCommission.HiscurrentresearchinterestsaredescribedinthedifferentprojectsattheLaboratoryofGeneralBiochem-istryandPhysicalPharmacy.HispublicationsincludecontributionstoChemicalReviews,differentNaturejournals,etc.Hisresearchonencodingmicrocarriersleadtotwopatentapplicationsandtheuniversityspin-offMemobeadTechnologies.

StefaanC.DeSmedt(1967)studiedphar-macyatGhentUniversity(Belgium)andgraduatedin1995.Hejoinedthepharmaceu-ticaldevelopmentgroupofJanssenResearchFoundation.In1999hebecameProfessoratGhentUniversitywhereheischairingtheGhentResearchGrouponNanomedicines.Since2004heservesasEuropeanAssociateEditoroftheJournalofControlledRelease.Hisresearchisattheinterfacebetweendrugdelivery,biophysics,materialsciencesand

advancedopticalimaging.Hereceivedthe2006CRSYoungInves-tigatorAwardandthe2010APVResearchAwardforOutstandingAchievementsinPharmaceuticalSciences.Heisscientiﬁcfounderofspin-offMemobeadTechnologies.

KevinBraeckmanshavingobtainedaMas-terdegreeinPhysicsatGhentUniversity(Belgium)in1999,hejoinedtheLaboratoryofGeneralBiochemistryandPhysicalPhar-macy(GhentUniversity)toperformresearchonadvancedopticalmicroscopymethodsforpharmaceuticalapplications.DuringhisPh.D.hewasinvolvedinthedevelopmentofanewtypeofencodedmicrocarriersfordiagnosticapplications,forwhichhereceivedtheﬁrstpriceforYoungBiotechnologyResearchfrom

theFundsofBiotechnology(FBBF,Belgium)in2005.HavingobtainedhisPh.D.in2004,hereceivedafellowshipoftheFundforScientiﬁcResearch—Flandersasapost-doctoralresearcher.In2005hejoinedthegroupofprof.Braüchle(LehrstuhlfürPhysikalischeChemieI)attheLudwig-MaximiliansUniversitätMünchenwherehewasinvolvedinthedevelopmentofalgorithmsandsoftwareforsingleparticletrackinganalysis.In2008hewasappointedasprofessoratthefac-ultyofPharmaceuticalSciencesofGhentUniversitywhereheiscurrentlyleadingtheBio-PhotonicImagingGroup,hostedbytheLaboratoryofGeneralBiochemistryandPhysicalPharmacy,inclosecollaborationwiththeGhentResearchGrouponNanomedicines.Hiscurrentresearchtopicsinvolvethedevelopmentandapplicationofbio-photonicmethodsforstudyingtheinteractionofnanomaterialsfordrugdeliveryandbio-imagingwithbiologicalbarriers.

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