Nanomedicine as a potent strategy in melanoma tumor microenvironment

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Pharmacological Research

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / y p h r s

Review

Nanomedicine as a potent strategy in melanoma tumor microenvironment

Vincent Pautu

^a,1

, Daniela Leonetti

^b,1

, Elise Lepeltier

^a

, Nicolas Clere

^a

, Catherine Passirani

^a,∗

aMINT,UNIVAngers,INSERM,CNRS,UniversitéBretagneLoire,IBS-CHU,4rueLarrey,F-49933Angers,France

bCRCINA,UMRINSERM1232,CNRSERL-UniversityofNantes,France

a r t i c l e i n f o

Articlehistory:

Received5January2017

Receivedinrevisedform14February2017 Accepted14February2017

Availableonlinexxx

Keywords:

Melanoma Nanoparticles

Tumormicroenvironment Drugresistance Nanotherapies Targeting Formulation Nanotheranostics Drugdelivery Metastasis

a b s t r a c t

Melanomaoriginatedfrommelanocytesisthemostaggressivetypeofskincancer.Despiteconsiderable progressesinclinicaltreatmentwiththediscoveryofBRAForMEKinhibitorsandmonoclonalantibodies, thedurabilityofresponsetotreatmentisoftenlimitedtothedevelopmentofacquiredresistanceandsys- temictoxicity.Thelimitedsuccessofconventionaltreatmenthighlightstheimportanceofunderstanding theroleofmelanomatumormicroenvironmentintumordevelopementanddrugresistance.

Nanoparticlesrepresentapromisingstrategyforthedevelopmentofnewcancertreatmentsableto improvethebioavailabilityofdrugsandincreasetheirpenetrationbytargetingspeciﬁcallytumorscells and/ortumorenvironment.Inthisreview,wewilldiscussthemaininﬂuenceoftumormicroenvironment inmelanomagrowthandtreatmentoutcome.Furthermore,thirdgenerationloadednanotechnologies representanexcitingtoolfordetection,treatment,andescapefrompossiblemechanismofresistance mediatedbytumormicroenvironment,andwillbehighlightedinthisreview.

Contents

1. Introduction...00

2. Nanocarriers:maincharacteristics...00

2.1. Surfacecharacteristics...00

2.1.1. Surfacecharge...00

2.1.2. Coating...00

2.2. Size...00

2.2.1. Escapetorenalandhepaticclearance...00

2.2.2. Cellinternalization...00

2.3. Shape...00

2.4. Encapsulationproperties...00

Abbreviations: ␣-SMA,␣-smoothmuscleactin;MCP1,monocytechemotacticprotein1;FAP,ﬁbroblastactivationprotein;PDGF,platelet-derivedgrowthfactor;HGF, hepatocytegrowthfactor;CAPS,cancerassociateprotease;PLGA,poly(lactic-co-glycolicacid);ESMSVs,enzyme-stimulatedmultistagevectors;NF-␬B,nuclearfactor-␬

B;NHE,Na+/H+exchanger;MCTs,monocarboxylatetransporters;pHe,extracellularpH;EMT,epithelial–mesenchymaltransition;pGP,p-glycoprotein;PEO,poly(ethylene oxide);P2VP,poly(2-vinylpyridine);PICs,polyioncomplexes;HH,histidineconjugate;PH,polyethylenimine-histidineconjugate;HD-DOX,doxorubicinloadedmicelles;

ODN,decoyoligonucleotide;PEI,polyethylenimine;MDSC,myeloid-derivedsuppressorcells;Treg,regulatoryTcells;NKcells,naturalkillercells;iDC,immaturedendritic cells;TAF,activatedﬁbroblaststromalcells;ARF,alternative-readingframegene;PD-L,ProgrammedCellDeathLigand;CpG-ODN,CpG-oligonucleotides;DR5,deathreceptors 5;MC1R,melanocortin-1receptor;NLG,N-laurylglucosamine.

∗Correspondingauthorat:INSERMU1066,CNRS6021IBS−CHU,4RueLarrey,49933AngersCedex9,France.

E-mailaddress:catherine.passirani@univ-angers.fr(C.Passirani).

1 Theseauthorscontributeequally.

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3. Tumormicroenvironmentastargetofnanoparticles...00

3.1. Vasculature...00

3.2. NanoparticlesandEPReffect-passivetargeting...00

3.3. Fibroblastsandextracellularmatrix(ECM)...00

3.4. NanoparticlesandECMproteins...00

3.5. Acidiﬁcationoftumormicroenvironnement...00

3.6. pH-sensitivenanocarriers...00

3.6.1. Protonablegroups...00

3.6.2. Redox-responsivesystem...00

3.6.3. pH-sensitivepeptide...00

4. Immunecells...00

4.1. Nanoparticlesandmonoclonalantibodies...00

5. Nanoparticlesandtumorcell-activetargeting...00

5.1. Monoclonalantibodies...00

5.2. Peptides...00

5.3. Others...00

6. Conclusionsandperspectives...00

Conﬂictofinterest...00

Acknowledgements ... 00

References...00

1. Introduction

Melanomaisaheterogeneousdiseasewhichrepresentsoneof themostdeadlycancersinitsmetastaticform.Overthepastyears, theincidenceofmelanomahasrisenfasterindevelopedcountries thananyothercancertype,particularlyinthepopulationover50 yearsold.On aglobalscale,malignantmelanomawasthe16th and15thmostcommonlydiagnosedcancerinmalesandfemales respectively[1,2].

Despiteconsiderableprogressesinclinicaltreatmentswithin recentyears,overallprognosisforadvanced melanomaremains poor. Consequently, the development of targeted treatments appearstobeessentialtoimprovemelanomasurvival.Melanoma beginsinthelowestlayerof theepidermisandoriginatesfrom melanocytes,acelltypeinvolvedinmelaninapigmentproduction.

Themelanocytelineageisderivedfromtheneuralcrest.Afterdif- ferentiation,progenitorcells, calledmelanoblasts,migratefrom theneuralcrest toward theepidermiswhere theyare stopped upon contact with keratinocytes. After differentiation, human melanocytes are speciﬁcally localized to the basement mem- braneandcansurvivewithintheupperepidermallayersonlyif transformedintomelanomacells[3].Melanomaresultsfromaneo- plastic transformation of pigment-producingmelanocytes, with cutaneousneoplasms of stratiﬁedepithelium.Most melanomas haveacutaneousorigin(>90%),particularlysun-exposedsiteson thelimbs,trunk,andface.However,itcanalsooccurinvarious extracutaneoussiteswherepigmentcells arepresentincluding, mucousmembranesofthesinusesandoropharynx,esophagus,rectum,vagina,ocularmelanomasandrarecasesofmelanomainsome internalorgans[4,5].

Most patients are diagnosed with localized melanoma and can be early treated successfully by a wide local excision fol- lowedbylymphnodemanagement[6];nonetheless,theprognosis ofpatientswithadvancedstagemelanomais poorwithhalfof patientsdyingwithin8–10monthsofdiagnosis[7,8].

The complexity of the treatment of advanced or metastatic melanomaisrelated totheevolution and heterogeneityof this canceraswellasthedevelopmentoftherapy resistancetrough anumberofmolecularand cellularmechanisms,includinghigh mutationrate,alterationofmembranedrugtransporters,increased DNArepairandevasionofapoptosis[9,10].

However,since2011,thetherapeuticlandscapeofmelanoma hasbeenrevolutionized withtheapproval of two newdistinct approaches.

Ontheonehand,thedevelopmentsinwhole-genomesequenc- inghaveallowedtoidentifyanumberofsomaticgeneticalterations inB-Raf,NRASandRAC1genes,germlinemutationsassociatedwith familialmelanomasuchasCDKN2A,CDK4genes,BRCA1-associated protein 1 (BAP1) and microphthalmia-associated transcription factor (MITF) [11], with a critical role in driving tumorigene- sis.Theidentificationofthesesomaticgeneticalterations,which contributetomelanocytetransformationsby modulatingMAPK pathway,hasledtothedevelopmentofmoleculartargetedtreat- mentapproaches.Thesenewtherapies,whicharebasedontheuse ofmultiplepotentkinaseinhibitorstosuppresstheMAPKpath- waydownstreamsuchasvemurafenib(aselectiveandreversible V600BRAFkinaseinhibitor),orcobimetinib(aselectiveMEK1/2 inhibitor),havetranslatedintoasubstantialimprovementinthe outcomesofpatientswithmetastaticmelanoma[12–14].How- ever,targetedmonotherapyapproachaswellasthecombination oftargetedtherapiesasvemurafenibandcobimetinib,haveledto cutaneoustoxicities,andthedurabilityofresponsetotreatmentis oftenlimitedtothedevelopmentofacquiredresistance[15,16].On theotherhand,theadvancementsmadeintargetedtherapyhave alsoinvolveda significantevolutioninthefieldofimmunobiol- ogyandresultantimmunotherapyagainstmelanoma.Asignificant improvementofthemedianoverallsurvival(OS)hasbeenobserved aftertreatmentwithipilimumab,amonoclonalantibodyagainst cytotoxicT-lymphocyteantigen-4(CTLA-4),whichisabletoexploit thenatural ability of the immune systemto eradicateprimary cancercells[17].Despitethepositiveimpactonthelong-termsur- vival,theresponserateforipilimumabinadvancedmelanomais onlyaround15%,andlong-termdurabilityofresponseoccursin aminorityofpatients.Furthermore,thetreatmentofipilimumab isassociatedwithinflammatoryadversereactionsresultingfrom increasedorexcessiveimmuneactivitywhichcanaffectthegas- trointestinal,liver,skin,nervous,andendocrinesystems[18].On thebasisofstudiessuggestingtheabilityofkinaseinhibitorsto modulatetumor-associatedimmuneresponse[19]andgiventhe highresponseratesandthelong-termdurabilityofresponsenoted respectivelyinpatientstreatedwithacombinedtargetedtherapy andipilimumab,combinationstrategies,suchasvemurafenibplus ipilimumab,havealsobeenstudiedwithoutsuccess[20].Further- more,varioustrialsdesignedtoassessthesafetyandefficacyof newcombinationsoftherapiesarecurrentlyinprogress[21].

Thelimitedsuccessusingconventionaltherapeuticshighlights theimportanceofunderstandingtheroleofthetumormicroenvi- ronmentanditsprecisecontributiontocarcinogenesis.Inrecent

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years, severalstudieshave supported theidea thatstroma can beinfluencedand promotetumorgrowthand theformationof tumormetastases[22–24].Tumormicroenvironmentdoesnotonly contributetotumorgrowth,invasion,andmetastasis,butininter- actingwithtumors,caninduceimportantmechanismsunderlying drug resistance[25,26]. The environment-mediated drug resis- tanceisinducedbysignalingeventsthatareinitiatedbysoluble factorspresentinthetumormicroenvironmentsuchascytokines, chemokinesandgrowthfactorssecretedbyfibroblast-liketumor stromaorbytumor-associatedmacrophages(TAMs).Theadhesion oftumor cellintegrins tostromalfibroblasts ortocomponents of the extracellular matrix (ECM), such as fibronectin, laminin andcollagen[27,28]canalsobepartofthesefactors.Moreover, microenvironmentcouldaffecttumorvasculature,whichservesas abarriertooptimaldrugdelivery[29].Thus,microenvironment constituentscanbekeyregulatorsofthesensitivitytocancercell targetedtherapy.

Theuseofnanomedicinesrepresentsapromisingstrategyfor thedevelopmentofnewcancertreatments.Nanotechnologyrefers toobjectssizedundermicrometerorwithonedimensionunder 100nm. Duetotheirnanoscalesize,nanocarriersbring numer- ousadvantages.Theycanprotectsensitivetherapeuticagentsfrom degradation,improvethebioavailability,enhancetheefﬁcacyand increasethetolerabilityofdrugs.Nanocarrierscantargetspeciﬁ- callytumorcellsandtumortissuetoenhancetheiraccumulationin tumorenvironementandincreasetheirpenetrationincells.Finally, nanocarrierscandeliverawiderangeofagents,suchaschemother- apeuticsandimagingcontrastagents[30].

In this review, after the description of nanocarrier characteristics and properties, the nanoparticle-based strategies for melanomatreatmentwillbedeveloped. Theinvolvmentof different constituents of tumor microenvironment in melanoma developpementaswellastheirinﬂuenceintreatmentoutcome willbediscussed.Thirdgenerationnanotechnologies,bydirectly targetingtumorcellsorusingstimulusprovidedbytumormicroen- vironment,isapromisingapproachfordetection,treatment,and escape frommechanismof resistancein melanomaandwillbe highlighted.

2. Nanocarriers:maincharacteristics

In the last decades, advancement in material and nano- technology science has allowed to develop a large variety of nanomedicines.Nowadays,threegenerationsofnanomedicinecan bedescribed.

First generation of nanomedicine, mainly nanoparticles and liposomes,targetsthereticuloendothelialsystem(RES)andisused todeliverdrugslocally intosomeorganssuchastheliver.Sec- ondgenerationofnanomedicineisusuallycoatedwithastealth layer,topreventdegradationbytheRESandthusincreasethecir- culationtimeinbloodstreamleadingtoanincreasedaccumulation intumorsitesthroughEnhancedPermeablityandRetention(EPR) effect.Thirdgenerationconsistsinaddingfunctionalities,likelig- andsortriggersinordertotargetspeciﬁcallytumortissuesand/or tumorenvironment.Ligandgrafted tonanocarriers,canbindto speciﬁcsurfacemarkersexpressedontargetcells,orexpressedin tumorenvironment,leadingtoaprecisetargetingofnanocarri- ers.Allthesecharacteristicshaveanimpactonbiodistributionand canofferinterestingstrategiesfortheuseofnanomedicineinsolid tumors,particularlyinmelanoma(Table1).

SincetheclinicalapproveofDoxil^® in1995,apegylatedlipo- some,drug-loadednanocarriersrepresentapromisingstrategyto overcomeside effectsand toincreasethetherapeuticresponse.

Doxil^® isananosystemencapsulatingdoxorubicin,awidelyused chemotherapeutic agent in many types of cancers as bladder,

Fig.1.Numberofpublicationsofnanomedicinesinthecontextofmelanomaper year.PubMedwww.ncbi.nlm.nih.gov/pubmed.

breast, lung, stomach,and ovarian cancer. However,efﬁcacyof doxorubicinislimitedduetoitshighcardiotoxicity[63].Doxil^® reducedsigniﬁcantlythiscardiotoxicitycomparedtothefreedrug [64].

Since the last 20 years, number of studies covering nanomedicines as a potential therapy in cancer has consider- ably increased, especially in melanoma over the last 5 years (Fig.1).Organicnanomedicines,suchasmicelles,liposomesand lipidnanoparticles,havebeenlargelystudiedascarriersincancer therapy. In addition, inorganic nanomedicines, including metal nanoparticles,quantumdotsandmagneticnanoparticlesoffered promisingresultsincancertreatment(Fig.2).Theseobjectscan arbourvariousphysicalandchemicalpropertiesassurface,size, shapeandencapsulationproperties.Thesecharacteristicsarekey factorsfortheformulationofnanomedicinesduetotheirinﬂuence onbioavailability.

2.1. Surfacecharacteristics

Surfacecharacteristicsofnanocarriersplayanimportantroleon thebiodistributionandtimeresidenceinblood.Themainobsta- cle to a long-time circulation is theclearance of nanoparticles bytheRES. Thissystemcorrespondstothephagocytic cells,as macrophages. Itsmaingoal istoprotect thebodyfrom foreign objectslikesyntheticparticles.

Afterintravenousadministration,adsorptionofplasmaproteins named opsonins, suchas antibodiesand complement proteins, occursatthenanoparticlesurface.Thisassociationwillleadtothe eliminationofnanocarriersthroughphagocytosisbymacrophages.

Throughthismechanism,ithasbeenwidelyshowedthatnanopar- ticles are rapidly cleared few minutes after injection [65], if nanoparticles can not prevent opsonisation. Therefore, surface characteristicsmodiﬁcationofnanocarriersisawidelyusedstrat- egy to prevent opsonisation and extend theresidence time in bloodstream(Table1).

2.1.1. Surfacecharge

Thesurfacechargeofnanocarriersrepresentscriticalparameter actinginthehalflifecirculationtimeofnanocarriersandtheircell internalization.Ithasbeenshownthatplasmaticproteins(egalbu- min)canaggregatetopositivelychargedcomponentsduetotheir negativecharges.Thisphenomenonknownasproteincorona[66], leadstoformlargecomplexes,abletoberecognizedbytheimmune system,resultingin theireliminationandtherefore adecreased therapeuticeffectofthenanomedicines.Itispossibletomodify thechargeofthenano-objectbymodifyingitssurface(Table2).

For instance, gold nanoparticles,withcore diameteraround 2nm, were coated with tetraethylene glycol containing vari-

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Table1

Maincharacteristicsofnanomedicines.

Characteristics Strategies Effect Ref.

Passive Size Escapedfromrenal,hepaticandRES^*

clearance

Prolongedcirculation [31–33]

EPR^**effect Increasedtumoraccumulation [34,35]

Coating ReducedRESclearance Prolongedcirculation [36]

Surfacecharge [37,38]

Encapsulation properties

Encapsulatedpoorlysolubledrugs Increasedtherapeutic response

[39–43]

Protecteddrugfromdegradation [44]

Active Redoxcapabilities TumorpHe^***:disassemblyofnanocarriers, drugrelease

Drugreleaseintumorenvironment [45,46]

TumorpHe:removedpolymerprotection Increasedcellularuptake [47]

Protonation capabilities

TumorpHe:surfacechargeandsize modiﬁcation

Increasedcellularuptake [48–51]

TumorpHe:disassemblyofnanocarriers Drugreleaseintumorenvironment [52–54]

Enzymesensitivity TumorECM^****composition:disassembly ofnanocarriers;releasednanoparticles frommicroplatform

Drug/nanocarriersreleasedin tumorenvironment

[55,56]

TumorECMcomposition:removed polymerprotection

Increasedcellularuptake [57]

Activetargeting (ligand)

Targetedspeciﬁcmoleculesorligands overexpressedontumorcells

Increasedcellularuptake [58–62]

* RES:ReticuloEndothelialSystem.

** EPR:EnhancedPermeabilityandRetention.

***pHe:extracellularpH.

****ECM:ExtraCellularMatrix.

Fig.2. Varioustypesandshapesofnanoparticles:Organicnanomedicine,suchasliposome,micelleandlipidnanoparticle.Inorganicnanomedicines,includingmetal nanoparticles,quantumdots,magneticnanoparticles,silicananoparticle,calciumphosphatenanoparticleandcarbonnanotube.

ous chemical functions in order to provide different particle charges (alcohol: −1.1mV, ammonium ion: +24,4mV and car- boxylicacid: −37,9mV).In vivostudies onxenografted-ovarian bearingmiceshowedtheimpactoftheseparticlechargesoncell

uptakeandbiodistribution.Slightlynegativegoldnanoparticles(- 1.1mV)showedahighertumoruptakeandanincreasedcirculation timecomparedtohighlynegativeorpositivesurfaces(-37.9mV, +24,4mV) after intravenous or intraperitoneal injections [37].

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Table2

Sizeandsurfacechargeofvariousnanoparticlesinfunctionoftheircomposition.

Nanoparticles Composition Diameter(nm) Charge(mV) Ref

Lipidnanocapsules(LNC) PTX-loadedLNC 55.4±1.5 −6.5±1.8 [40]

PTX-loadedLNC-SDS 53.1±1.5 −22.9±1.2

PTX-loadedCS-LNC 69.9±3.4 +30.8±5.9

SolidLipidNanoparticles(SLN) ASP-loadedSLN 361±78.6 −10.6±4.21 [67]

ASP-loadedCS-SLN 430±51.1 +16.0±5.78

CUR-loadedSLN 400±58.3 −13.3±3.26

CUR-loadedCS-SLN 440±65.9 +17.0±4.69

Liposomes CL-DOX 116.8±8.2 −25.75±1.6 [68]

CH-DOX 118.9±6.4 −27.12±2.8

PEG-DOX 126.6±5.4 −13.6±4.5

PEtOz-DOX 128.5±4.6 −14.9±1.3

Micelles DSPE-b-PEG2k(90%)

DSPE-b-PEG3.5k-Tatp(1%)

64±1.31 5.1±1.42 [69]

DSPE-b-PEG2k(48%) DSPE-b-PEG3.5k-Tatp(1%) PGA5.6k-b-PLA1.8k(42%)

95±1.72 −12.6±1.95

DSPE-b-PEG2k(42%) DSPE-b-PEG3.5k-Tatp(1%) PGA12k-b-PLA6k(48%)

165±1.55 −25.2±1.74

Gold nanoparticles Au–tiopronin 2.7±0.9(Aucoresize) −16.79±1.94 [70]

Au–PEG–COOH −42.33±8.39

Au–myxoma −22.10±3.55

Au–PEG–myxoma −25.56±4.10

Au–cRGD −16.58±4.57

Au–PEG–cRGD −18.40±4.64

Abbreviations:LNC:lipidnanocapsules,SDS:Sodiumdodecylsulphate,PTXPaclitaxel,CS:chitosan,ASP:aspirin,CUR:curcumin,DOX:Doxorubicin,CL:Conventional Liposomes,CH:cholesterolhemisuccinatemodiﬁedliposomes,PEG:Polyethyleneglycol,PEtOX:Poly(2-ethyl-2-oxazoline),DSPE-b-PEG:1,2-Distearoyl-sn-glycero-3- phosphoethanolamine-b-poly(ethyleneglycol),Tatp:transactivatorTatpeptide,PGA:poly(L-glutamicacid),PLA:poly(D,L-lacticacid).

Neutralorslightlynegativechargednanoparticlescantherefore increasetheirbiodistributionbylimitingtheirRESrecognition.Xia andco-workers[38]observedthatmicellarnanoparticleswithsim- ilarparticlesizesbutvarioussurfacecharges,showedsigniﬁcant differencesinmacrophageuptakeandinorganaccumulation.Pos- itivelycharged(+18.5mV,+29.5mVand+37mV)micellesshowed, respectively,anuptakeinRAW264.7macrophages,by1.9folds, 3.5-folds and 5.9 folds higher, respectively, compared to neu- tralmicelles(+3.6mV).Oncontrary,theyobservedadecreaseof macrophageuptake withnegatively chargedmicelles(-26.9mV,

−17.5mV,−8.5mV). Biodistribution analysis in SKOV-3 human ovariancancerxenograftbearingnudemicealsoshowedinfluence ofthesechargesonbiodistributionprofile,24hafterintravenous injection.Neutralandnegativelychargedmicellesdemonstrated atumoruptakehigherthanpositivelychargedmicelles.Further- more,slightlynegativeandneutralmicellesshowedasignificant liveruptakecomparedtohighlycharged(positiveandnegative) micelles.Thesurfacechargeofnanocarrierscanimpactnotonly tumoraccumulationandbiodistribution,butalsotheirinternaliza- tionintumorcells.Researchersshowedinvitroandinvivothat positivelychargednanoparticlessuchasmicelles[71],quantum dots[72],liposomes[73],hydroxyapatitenanoparticles[74],have highercellularuptakecomparedtoneutralornegativelycharged nanoparticles, possibly due to electrostatic attraction between thepositivechargesofnanoparticlesandnegativechargesofthe cell membrane. A study demonstrated this electrostatic attraction:positivelychargedgoldnanoparticlesshowedanincreased cellularuptakecomparedtonegativelyandneutralgoldnanopar- ticles.Inaddition,depolarizationofcellmembranewithKClledto reducetheuptakeofpositivelychargednanoparticles,confirming theimpactoftheelectrostaticinteractionsontheinternalization mechanism[75].

2.1.2. Coating

In order toreduce the blood clearance, nanocarriers canbe coated with hydrophilic polymer to provide stealth effect to nanoparticles.Polyethyleneoxide,alsocalledpolyethyleneglycol

(PEG)but differingfromthemonomerused,are repeatedunits ofethyleneoxide.Botharenon-ionichydrophilicpolymerswith stealthcapabilities[76],firstlyreportedbyAbuchowskietal.in 1977.Theyshowedanextendedbloodcirculationtime(morethan 50h)forenzyme(catalase)modifiedwithaPEG-5000comparedto unmodifiedenzyme(12h)[77].Whenphysicallyaddedorcova- lentlygrafted onnanocarriers,PEGforms a flexiblehydrophilic layeratthesurface,andthereforeprevents,viasterichindrance, adsorptionofopsonins.Poly(␧-caprolactone)(PCL)nanoparticles modifiedwithPEG,werefoundtoreduceinvitrotheopsonisation anddecreasedthecomplementactivationcleavagecomparetoPCL nanoparticles[36].Furthermore,inthesamepublication,biodis- tributionstudyinmiceofPCLandPEGmodifiedPCLnanoparticles showedarapidaccumulationofPCLnanoparticlesinthemononu- clearphagocytesystemcomparetoPEG-PCLnanoparticles.

ThisPEGprotectionhasbeenshowntobedependentof the PEGconformationat thesurfaceofnanoparticles:PEGcouldbe inbrushormushroomconformations[78],dependingofitscon- centrationatthenanoparticlesurface.Recentworksshowedthe impactofPEGstateonthebindingofhumanserumalbumin(HSA).

PEGgraftedlipidbilayersinmushroomconformationshowedbind- ingofhumanserumalbumin.Onthecontrary,PEGinbrushstate supressedthebindingofHSA[79].Pegylationofnanocarriersby providingprotectionagainstRES,canincreasethetimeresidence ofnanocarriersinplasmaandthereforeincreasetheirtherapeu- ticeffects.SurfacemodiﬁcationwithPEGisnowwidelyused,and numerouspegylatednanocarriersaretestedinclinicaltrialsand havebeenapprouved(Table3).

Surfacemodificationcanbeusedtoenhanceefficacyofdrugs containedinnanomedicine,bybringingforexamplestealthprop- erties compared tonon-modified nanocarriers. PEG hasshown its capability to provide protection to nanocarriers, but other polymershavebeenstudiedtobringotherpropertiessuchasa controlleddrugrelease:chitosan[85],poly(2-methyl-2-oxazoline (PMOX), polyvinylpyrrolidone (PVP), poly[N-(2-hydroxypropyl) methacrylamide] (HPMA), poly(N-acryloyl morpholine (PAcM), poly(N,N-dimethyl acrylamide) [86], (PDMA), poloxamer 188

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Table3

MarketedPegylatednanocarriersandinclinicaltrials.

Name Type Drug Statut Indication Clinicaltrials

identiﬁer/Reference IHL-305 Pegylatedliposome Irinotecan Phase-1 solidtumors,ovarian,breast,lung

cancer,bladder,headandneck squamous...

NCT00364143 [80,81]

Plm60-s Pegylatedliposome Mitoxantrone Phase-1 melanoma,squamouscellscarcinoma,

Non-Hodgkin’slymphoma,rectum cancer,pharynxcancer...

NCT02043756.[82]

Doxil^® Pegylatedliposome Doxorubicine Approved ovariancancer

HIV-associatedKaposi’ssarcoma, multiplemyeloma

Onivyde^® Pegylatedliposome Irinotecan Approved metastaticadenocarcinomaofthe pancreas

Promitil^® pegylatedliposome Mitomycin-C Phase-1 recurrent,ormetastaticmalignant solidtumors

NCT01705002[83]

DCR-MYC Pegylatedlipid

nanoparticule

MYC-targeting siRNA

Phase-2 Phase-1

solidtumors;multiplemyeloma;

non-hodgkinslymphoma;pancreatic neuroendocrinetumors...

NCT02314052 NCT02110563

Genexol-PM^® Polyethyleneglycol andpoly(D,L-lactic acid)micelle

Paclitaxel Phase-2

Phase-4

Bladder;ureter,ovarian,breastcancer, adenocarcinomaofthepancreas,head andnecksquamouscellcarcinoma

NCT01426126[84]

NCT02739633 NCT00912639 NCT00877253 NCT01689194

[87].Fluorescentprobe, antibodies[88],peptide[89],andother molecules[90,91],canalsobegraftedatthesurfaceofnanocarri- erstoincreasetheirtherapeuticefﬁcacy.Thesewillbediscussedin thepart6.

2.2. Size

Before reaching tumor site, nanoparticles need to circulate throughthebloodstreamandcrosstheepithelialbarriers.Sizeof nanoparticlesisthereforeakeyparameterinﬂuencingthebiodis- tributionof nanocarriers.These last canbe designed ina large sizescale by changing theformulation process ortheir chemi- calcomposition. For example, a studyshowed theinﬂuence of LNCcompositionontheirsizedistribution.Byadjusting3compo- nentsofLNC(caprylicacidtriglyceride/water/PEG),particlesizes between20and95nmwereobtainedwithoutchangingtheprocess [92].

2.2.1. Escapetorenalandhepaticclearance

Sizeisanimportantfactorineliminationanddegradationof nanoparticles(Table1).Kidneysarecapabletorapidlyeliminate moleculesinthebloodplasmathroughglomerularcapillaryfenes- tration[93].Thesefenestrationsmeasuring4.5–5nmindiameter, canconducttorapidrenaleliminationofnano-objects[94].Renal clearancestudyofquantumdotsizingfrom4.36to8.35nmlabelled withTechnetium99^m, showedvarious accumulationand blood half-lifeinfunctionoftheirsize.Renalclearanceandbodyretention ofquantumdot(excludingbladderandurethra)wasobservedto besizedependent.Fourhoursafteradministration,50%of5.5nm quantumdot,≈80%for4.36nmquantumdotand≈20%for8,35nm quantumdotwereexcretedinurine.Furthermore,quantumdots witha sizeupper8nmdidnotshowrenalfiltrationbutexhib- itedhighuptakeinliver,lungandspleen[31].Theresultsofthis studysuggestedthatnanoparticleslargerthan10nmcanprevent renalelimination.Designofnanomedicinescanconsiderthisphys- iologicalconstrainttoobtainalongcirculationtimebypreventing kidneysandliverfiltration.However,sizeofnanoparticlesshould notexceed200nm,duetosplenicfiltration:interendothelialslits inspleenare sizedfrom200to500nm[95],resultinginfiltra- tionofparticleslargerthan200nm.Furthermore,nanocarrierssize

playsa critical role in complementactivation [32,33].Different studiesshowedasizedependenteffectonthisresponse[78,96].

Forexample,astudyevaluatedthecomplementactivationby20, 50and100nmLNCinaCH50test.Thistestconsistsofmeasur- ingtheamountof serumabletolyse50%ofa ﬁxednumberof sensitizedsheeperythrocytes.TocomparetheLNC,thecomple- ment consumption wasstudied in functionof the surface area of thenanoparticles.For the same surfacearea (3000cm²/mL), 20nmnanocapsulesshowedacomplementconsumptionof10%

(traducedbyanincreaseof10%ofserumamountneededtolyse 50%oferythrocytes),50nmand100nmLNCshowedaconsump- tion of15% and 20% respectively [97].Another study,revealed thatopsonisationofliposomeswasreducedforsmallerliposomes (200nm)comparedtolargerliposome(800nm)[32].Theseresults suggestthatsmallnanoparticleswereabletoescapecomplement activationandthereforeshowedalongercirculationtime,withan increaseoftheirtherapeuticseffects.

2.2.2. Cellinternalization

Nanocarrierscanpenetrateintocellcytoplasmthroughmultiple pathwaysasclathrin,caveolinmediatedendocytosis,micropinocy- tosis and phagocytosis [98,99] Rejman et al. investigated the size-dependent endocytosis pathway in non-phagocytic mouse melanoma B16F10 cells. They observed that clathrin mediated pathway had an upper size limit internalization of fluores- cent polystyrene microspheres (Fluoresbrite^®). Internalization of nanospheres smaller than 200nm was found to involve a clathrinmediatedpathway.Thecellinternalizationofnanospheres largerthan 200nm(500nm)occurredvia a caveolae mediated mechanism [100]. However, size-dependent internalization of nanoparticlesisinfluencedbycelltypesandchemicalcomposition ofthenano-objects[101].Thedesignofnanocarrierscantherefore berealizedbytakingintoconsiderationthecellulartargettobuild efficientdrugdeliverysystemsandreducetheirpotentialtoxicity.

2.3. Shape

Recognitionofnanocarriersbycomplementsystemisdepen- dentonthesizeofnanocarriers.Smallerobjectsshowingahigh radiusofcurvature,leadtopreventrecognitionbycomplement

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systembypreventingopsonisation[102].Shapeofnanocarrierscan thereforeplayaroleinthedistributionandcirculationofnanocar- riers[103,104].Duetothewidediversityofmaterialsandtechnics, nanocarrierscanexhibitalargevarietyofshapes(sphere,ellipse, disk,cone,filament,cube,rod).Adhesionand internalizationby macrophagesofvariousparticleswithdifferentshapeshavebeen studied.Polystyreneparticlesasspheres,oblateellipsoids,prolate ellipsoids weredesigned through stretchingmethods described byChampionandMitragotri[105].Resultsshowedthatparticles withlongestdimensions exhibitedhigher attachmenttendency tomacrophagescompared toparticleswithshorter dimensions [106,107].Internalizationofparticlesbymacrophagesshowedto bedependentofthetangentangleatthecontactpointbetween cellmembraneandparticle.Macrophagesattachedattheellipse extremity(wherethetangentangleisthemostelevated)inter- nalizedtheparticlesinfewminutes,whilewhenmacrophagesare attachedatflatregionofellipse(wherethetangentangleisthe smallest),thenanoparticleswerenotinternalizedaftermorethan 12h.Sphericalparticles,duetotheirsymmetry,wereinternalized bymacrophagesfromanypointsofattachment[105].Gengand co-workersevaluatedtheinvitrouptakebymacrophagesoffilomi- celleshavingthesamediameter,comprisedbetween22–60nm, butwithdifferentlengths(2␮m,4␮m,8␮mand18␮m).Under flow conditions, no filomicelle with a length superior to 3␮m showedamacrophageuptake,whileshortermicellesappearedto betakenupbymacrophage.Internalizationbymacrophagesand time circulation ofthenanocarriers aretherefore dependentof theirshape.Inthesamestudies,researchersevaluatedtheimpact ofthelengthoffilomicellesonthebiodistributioninC57mice.The resultsshowedthattheshorterfilomicelles<4␮mwereslowly fragmentedcompared tolongerfilomicelles.Inaddition,filomi- cellesshowedanimprovedcirculationtimecomparedtospherical micelles.Inthesestudies,authorsshowedtheimpactofnanocarri- ersshapeontheirclearanceandtimecirculationinthebloodstream [108].Inaninvivostudy,Blacketal.evaluatedthebiodistribu- tionoffourdifferenttypesofAu¹⁹⁸nanostructures(nanospheres, nanodisks, nanorods and cubic nanocages) in a murine EMT6 breastcancermodel.Distributionofthesenanocarrierswasper- formedbymeasuringthe␥radiation.Nanospheresshowedalonger timecirculationinthebloodstreamthannanodisks,nanorodsand cubicnanocages andexhibited a highertumoruptake thanthe othernanostructures.Inaddition,differencesoftumordistribution dependingofthenanostructureshape wereobserved.Nanorods and cubic nanocages were localized at the core of the tumor whereasnanospheresandnanodiskswereobservedattheperiph- eryofthetumor[109].Furthermore,Kolharetal.comparedthe targetingabilitiesofantibodies-coatednanospheresandnanorods againstneovascularmarkersandintercellularadhesionmolecules expressedby endothelial cells. The in vitroand invivo studies revealed higher specific endothelialaccumulation for nanorods comparedtonanospheres.Thisdifferencewasexplained bythe surface interactions of thenanocarriers [88]. Nanorods, due to theirshape,exhibitedanhighersurfacecontactthannanospheres [110],andthereforeincreasedtheinteractionbetweenantibodies andmarkersexpressedonendothelialcells.Surfaceinteractions betweennanoparticlesandcellscouldthereforebedependentof theshape.Qiuetal.showedthatlongernanorodswerelessinter- nalizedinHeLacellscomparedwithshorternanorodshavingthe samesurfacecharge[111].Haoetal.havestudiedtheimpactof mesoporoussilicananoparticleshapesonthecellularuptakemech- anism.Theyreportedthatmesoporoussilicananorodspreferredto beinternalizedthroughcaveolae-mediatedpathway,comparedto sphericalmesoporoussilicananoparticleswhichwerepreferen- tiallyinternalizedviatheclathrin-mediatedpathway[112].Such variationbetweenthesedifferentstudiesindicatedthat1)thesize

and2)thesurfaceofnano-objectsshouldbetakenintoconsidera- tionsforthedevelopmentofnanocarriers.

2.4. Encapsulationproperties

Due to their variety of physicochemical properties, nanomedicinecanbeusedtodeliveralargerangeofmoleculesin tumors,whileincreasingtheirefﬁcacyandtolerability(Table1).

Therapeuticagents canbeencapsulated,adsorbedorcovalently attachedonnanocarriersinordertoovercomethewaterinsolubil- ityofdrugssuchaspaclitaxel,docetaxel,etoposide,curcumin... Forexample,LNC,duetotheirtriglyceridecore,canencapsulate awidevarietyoflipophilicdrugsandenhancetheireffects.Ithas beenreportedthatpaclitaxelandetoposidewhereencapsulated inLNCwithanencapsulationratevaryingfrom82%to93.4%,for etoposideandpaclitaxel,dependingonthedrugusedandtheLNC size(25nm,50nm,100nm)[39].InvitrostudiesonC6ratglioma showed inhibition of cell growth 4 folds higher for etoposide loadedLNCand40foldshigherforpaclitaxelloadedLNCcompared tofreedrugs.Nowadays,numerousstudiesshowedthepossibility touselipid nanocarriers forlipophilic drugdelivery: paclitaxel [40–43],docetaxel[113–115],curcumin[67,116,117].Lipidcar- rierslikesolidlipidnanoparticles(SLN)orLNCofferinteresting perspectiveinthedeliveryofpoorlywatersolubledrugs,through awidediversityofnanocarriers.Polymericmicellescomposedof poly (ethylene glycol) methyl ether-b-(poly lactic acid-co-poly (b-aminoesters))(MPEG-b-(PLA-co-PAE))loadedwithpaclitaxel, showedahighercytotoxiceffectonK562leukaemiacells,com- paredtofreepaclitaxel.Inaddition,pharmacokineticanalysisin ratshowedanincreasedhalftime(8folds)forpaclitaxelloaded micellescomparedtofreepaclitaxel[118].NK105,anotherpacli- taxelloadedmicellarnanoparticles,usedinphase Istudyon10 breast cancerpatients, showeda partialresponse on6patients andastabilisationofthediseaseon4patients[119].Numerous othernanocarriersencapsulatingpoorly solubledrugs areactu- allystudied,asforexample liposomes[120,121]and polymeric nanoparticles[122–124].Besidesencapsulatinglipophilicdrugs, nanocarriers canbe used todeliverhydrophilic agents suchas nucleicacid.Sincemanyyears,awidevarietyofnucleicacidloaded nano-systemshasbeendevelopedandoptimisedinthescopeof anti-cancertherapies[125].Lipidsnanocapsules,forexample,are able todeliver small interfering RNA(siRNA) in thecytoplasm of SK-Mel28 melanoma cells [44]. The polytherapy strategy in melanoma offers a new perspective for the development of nanomedicines.Drugcombinationcanbringbetterresponserates thanmonotherapy[126].Duetotheircapabilitytoencapsulate hydrophilicandhydrophobicdrugs,theuseofnanocarriersseems tobeapromisingstrategyinordertoobtainasynergisticeffect.In theirwork,Maandco-workersusedcore-matchednanoemulsions todeliverhydrophilic(ﬂuoroucacil)andhydrophobic(paclitaxel) drugs in humanepidermal carcinoma implantedin nude mice.

They showed a significant reduction (57,7%) of tumor growth (KB-8-5cellline)inmicetreatedwithpaclitaxel-co-fluorouracil nanoemulsions compared to other groups (treated with paclitaxel alone,paclitaxelnanoemulsion,fluorouracilnanoemulsion and untreated)[127]. Nanocarrierscanalsocombinedrugsand diagnostic agents. This strategy called nanotheranostic [128]is abletoenhancedeliveryoftherapeuticsinmonitoringthedrug distribution.Rizzitellietal.showedtheimpactofatumorultra- soundapplicationonthedrugreleasecontainedinliposome.To followthisrelease,theydevelopeddoxorubicinloadedliposome (as Doxil^®) co-encapsulating gadoteridol (an MRI agent). After injection of doxorubicin gadoteridol loaded liposomes (Doxo- Gado-Lipo)intomammary adenocarcinomasbearing mice,they showedthatultrasounds(1MHzand/or3MHz),whenappliedat thetumorsurface,canincreasethedrugamountintothetumor

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environment. This drug release from liposomes was followed byacontrastincrease onMR images.In addition,after11days, mice that received Doxo-Gado-Lipo and ultrasounds showed a tumor volume reduction of 55 folds compared to untreated mice.Incomparison,micetreatedwithDoxo-Gado-Lipowithout ultrasoundtreatmentshowedatumorvolumereductionofonly 2.5folds[129].

3. Tumormicroenvironmentastargetofnanoparticles

Tumormicroenvironmentincludescancercells,variousstromal cells suchasvascular endothelialcells, lymphaticcells, cancer- associated ﬁbroblasts (CAFs), and different types of immune cells,includingTAMs(Fig.3).Thearchitecturalscaffoldoftumor microenvironmentisprovidedbyECMcomponentswhichaffect cellmorphologyandimpactonmanyimportantcellfunctionssuch asproliferationanddifferentiation[25,130].

Anadaptive,continuousdialogueexistsbetweencancercells, stromalcells and ECM, mediatedthrough direct cellcontact or through secreted signaling factors (cytokines, chemokines and growthfactors)[131,132].Besides,recentstudieshaveidentiﬁed other key players such as exosomes, miRNAs and metabolites involvedinthiscomplexinteractionandcontibutingtotumorpro- gressionandmetastasis[133–135].

Furthermore, the molecularinteraction between cancer cell metabolismandthetumormicroenvironmentleadstotheacidiﬁ- cationoftumor-microenvironmentwhichpromotestheformation ofan acid-resistant tumorcell populationwithincreasedinva- siveand metastaticpotentialinsolid tumorssuchasmalignant melanoma[136].

Therefore,thetumorand itssurroundingmicroenvironment, togetherplayanimportantroleinthetreatmentoutcome.Conse- quently,understandingandmanagingstroma-mediatedresistance couldprofoundlyimprovetherapeuticstrategies thattargetthis compartment.

3.1. Vasculature

Theconceptthattumorgrowthwasdependentonangiogenesis (tumorangiogenesis)hasbeenproposedfortheﬁrsttimebyFolk- mansuggestingthattumorbloodvesselsrepresentanimportant targetforcancertherapy(anti-angiogenictherapy)[137].Conse- quently,theeffectofvariousantiangiogenic therapieshasbeen andisbeinginvestigatedinpreclinicalandclinicaltrials.Themost studiesarefocusoninhibitionofvascularendothelialgrowthfactor (VEGF)signaling,howeverotherstudiesareaimedatdetermining theeffectofmultikinaseinhibitorsortheinhibitionofangiogenic integrinactivity[138].

Melanomacellshavebeenshowntoexpressand/orsecretea widevarietyofangiogenicfactorsand/orreceptors,includingVEGF, placentalgrowthfactor,basicﬁbroblastgrowthfactor(bFGF)/FGF- 2,acidFGF/FGF-1,transforminggrowthfactor(TGF)-b,angiopoietin (Ang)-1andAng-2,differentinterleukinssuchas(IL)-1,IL-6,IL-8 andIL-35,andMatrixMetalloProtease(MMPs)(MMP-1,MMP-2, MMP-9,MMP-13)andmembranetype1MMP[139].

Furthermore, the expression of several factors involved in melanoma-associatedangiogenesissuchasHIF-1␣,HIF-2␣,VEGF andMMP-2havebeencorrelatedwithpoorerprognosisinhuman cutaneousmelanomapatients[140–142].

Asyet,nostudyhasreportedasigniﬁcantlyprolongoverallsur- vivalinmelanomapatientstreatedwithangiogenesisinhibitors.

Indeed,itiswellrecognizedthatmanypatientsdonotrespond toVEGF-targeted therapy andlimitedefﬁcacyand resistanceto anti-angiogenic therapy remain unsolved problems [139]. One potentialexplanation ofthelimitedefﬁciencyof antiangiogenic

monotherapyistheprocessknownas“vascularnormalization”due toheterogeneityofbloodvesselnetworks.Tumorscontainregions of hypervascularization as well as hypovascularization. During antiangiogenictherapy, vascular normalizationcouldcontribute to improve circulation within a tumor improving the efﬁcacy of chemotherapy/radiationtherapy. However,during prolonged antiangiogenictherapy,thenormalizationeffectdisappearsanda vascularregressioncanbeobserved[143,144].

Vascularnetworksarederivedthroughmodiﬁcationofexist- ing vessels within tissue, or recruitment and differentiation of endothelialprecursorsfrombonemarrow(vasculogenesis),con- tributingtovascular heterogeneityoftumormicroenvironment.

Intumors,anunregulatedgrowthofneoplasticallytransformed cellsoverexpressingpro-angiogenicfactorsleadstothedevelop- mentofdisorganizedbloodvesselnetworksthatarefundamentally differentfromnormalvasculature[25].

Tumorendothelialcellsdisplayphenotypicdifferencesatthe molecularandfunctionallevelswhencomparedtonormalcellsin termsofgeneexpressionproﬁle,proliferationandmigrationability andresponsestogrowthfactorsandseveraldrugs[145–147].

Comparedtonormalendothelialcells,whichformanuniform andcontinuousmonolayer,tumorendothelialcellshaveanirreg- ularshapeandsizewithlongcytoplasmicprojectionsextending acrossthelumen,structuralabnormalitiesinthebasementmem- braneandabnormalpericytes[148].Furthermore,inmelanoma, theheterogeneouscharacteristicsofendothelialcellsdependalso ondifferentmalignancy statusoftumor.Indeed,a recentstudy hasreportedthathigh-metastatictumor-derivedendothelialcells (HM-TECs)showedhigherproliferativeactivityandinvasiveactiv- itythanlow-metastatictumor-derivedendothelialcells(LM-TECs).

Inaddition,themRNAexpressionlevelsofpro-angiogenicgenes suchasVEGFreceptors1and2(VEGFR-1;−2),VEGFandHIF-1␣ werehigherinHM-TECsthaninLM-TECs,suggestingthatHM-TECs haveamorepro-angiogenicphenotype[149].

Tumorbloodvesselwallofsolidtumorisoftencharacterized bythepresenceofendothelialgapsandfenestrae.Intheonehand, thisleakyvasculaturecouldbeinvolvedinamechanismoftumor cellintravasationandinitialstepofmetastasis.Intheotherhand, thisenhancedvascularpermeabilityofferstheadvantageofdrug deliverybypromotingnanomedecineaccumulationinthetumor tissue.Thisphenomenonisknownas“enhancedpermeabilityand retentioneffect(EPR)”andithasprovidedthebasisfordevelopinga passivetargetingofdrugsintotumors[150,151].Passivetargeting throughEPReffecthasbeenwidelydescribedsinceitsdiscovery.

Thiseffectenablesnanocarriersandmacromoleculestoaccumulate intotumorenvironmentthroughleakinessoftumorvasculature anddefectoflymphaticdrainage(Fig.4)[152].

Thevasculatureinfluencesthesensitivityofthetumortodrugs becauseanticancerdrugs,inordertoreachallviablecellsinthe tumor,mustbedeliveredefficientlythroughthetumorvasculature, mustcrossthevesselwall,andtraversethetumortissue.Theinad- equatefunctionofpoorlyorganizedtumorvasculaturesresultsin metabolicchangessuchashypoxiaandlimitedsupplyofnutrients whichleadtoadecreseoftumorcellproliferationandconsequently influencedrugsensitivity[153].

Furthermore,insolidtumors,suchasmelanoma,thehetero- geneityofvascularmaturityiscombinedwithalackorreduced functionallymph vesselswhich leadtoanincreasedinterstitial ﬂuidpressure. Consequently,blood vessels arecompressedand bloodisdivertedawayfromthecenterofthetumortowardthe periphery,contributingtoformabarriertotranscapillarytransport [154].

Finally,inmelanoma,thedrugresistancemediatedbyendothe- lialcells,couldalsodependontheactivationofmolecularpathways intothetumor microenvironment.Arecent studyhasreported that,inamodel ofmelanoma,theacquiredresistanceoftumor

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Fig.3.Tumormicroenvironmentinmelanoma:melanomagrowthandprogression,resultfromtheinteractionoftumorcellswithdifferentconstituentsoftumormicroen- vironment.Theinﬁltrationofimmunosuppressivecells,canexertsuppressiveeffectsoneffectorCD8+andCD4+Tcellseitherthroughdirectcell-to-cellcontactorindirectly bygeneratingcytokinesorgrowthfactorsinsitu,whichhelpstumorcellstoevadetheimmunesystem.Duringtheadvancedstageoftumorprogression,CAFsbecome activatedandcontributetobothtumorgrowthandprogressioninavariouswaysincludingECMremodeling,paracrineanddirectinteractionswithcancercells,bysecreting solublefactors(cytokines,hormonesandgrowthfactors),andpathogenicangiogenesisbyliberatingproangiogenicfactors.Extracellularacidityisapathologicalfeatureof melanoma.ThealterationsofcellularmetabolismsuchasupregulationofglycolysisandoverproductionofCO2andglutaminearethemajorcauseofextracellularacidosis.

AcidicpHeisinvolvedinlocaltumorinvasionandtissueremodeling.Thetumorvasculatureischaracterizedbyadisorganizednetworksandthepresenceofendothelial gapsandfenestrae.Intheonehand,thisleakyvasculaturecouldbeinvolvedinamechanismfortumorcellintravasationandinitialstepofmetastasis;intheotherhand,this enhancedvascularpermeabilityofferstheadvantageofdrugdeliverybypromotingnanodrugaccumulationinthetumortissue(EPReffect).Theheterogeneityofvascular maturityiscombinedwithalackorareducedfunctionallymphvesselswhichladstoanincreasedinterstitialﬂuidpressure.

Fig.4.EnhancedPermeabilityandRetentioneffect(EPR).

endothelialcellstopaclitaxelwasmediatedbyanup-regulation ofmultidrugresistance(MDR)1viatheactivation,inthetumor microenvironment,ofVEGF-VEGFRpathway[147].

Takentogether,theseobservationshighlightthecomplexityof vascularnetworksand regulationofangiogenesisinmelanoma.

Oneof themain aspectsthat inﬂuencetherapy outcome isthe heterogeneityofvasculartumormicroenvironment.Consequently, a noveldrug delivery system to actively target heterogeneous

tumorvasculaturewouldbeapromisinganti-angiogenictherapy toimproveoverallanti-tumorefﬁcacy.

3.2. NanoparticlesandEPReffect-passivetargeting

Multiplechemotherapiesasdacarbazine(DTX), doxorubicine (DOX),paclitaxel(PTX)andcombinationofchemotherapieshave beenevaluatedfor thetreatmentofadvanced melanoma[155].

Thesefreedrugs,circulatingthroughoutthebodyviatheblood-

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stream,penetrateintotumorsthroughpassivediffusion.However, theirphysicochemicalpropertiesandtheircytotoxicityonhealthy cells often limit the dose used leading to different physiolog- ical side effects, as for example neuropathy, musculoskeletal pain, intolerance reaction and myelosuppression. Development ofchemotherapyloadednanocarriers isa strategy toovercome problemsoftoxic side effectstoward healthytissues andweak drugdeliveryefficiencyofchemotherapeuticagents.Drugsencap- sulated in nanocarriers can provide longer circulation time in bloodstream,higherstability,controlleddrugrelease,areduction ofdose,andanincreaseofdrugconcentrationintothetumorenvi- ronmentby passivelytargetingthe tumorsthrought EPReffect [34](Table1).Nanomedicine,by theirencapsulationproperties ofmanydrugagents,canprovideanincreasedaccumulationof drugsintumorenvironmentandreducethesideeffectsofcyto- toxicagents.Forexample,cytochalasinD,duetoisactivityonthe actinfilament[156],couldbeapotentialcytotoxicagent.However, thischemicalcompoundisinsolubleinwater,makingitdifficult touseintravenously. Furthermore,cytochalasin Dtargets actin microfilamentswhich arelocalizedin everycells and therefore cancausesignificantsideeffects.Huangetal.,inordertoover- cometheseproblems,developedcytochalasinDloadedliposomes.

Thisliposomalformulationshowedsignificantinvivoantitumor effects.B16melanoma,CT26colorectalcarcinomaandH22hep- atomabearingmicetreatedwithcytochalasinDloadedliposomes showedsignificantinhibitionoftumorvolumesandhighersurvival ratecomparedtomicetreatedwithcytochalasinDalone.Further- more,nosignificantside effectswere observedin micetreated withcytochalasinDloadedliposomes[157].Otherstudyshowed anincreaseantitumorefficacyofdocetaxelloadedinpolymeric micellescomparedtodoxetacelsolution(Duopafei^®).Thetumor volumesofB16melanomabearingmicewerereducedby91.6%on micetreatedwithdocetaxelmicellesversus76.3%formicetreated withDuopafei^® [158].EPReffectcanbealsousedforimagingof melanoma.Micellarformulationofpoly(ethyleneglycol)-block- poly(2-methyl-2-carboxyl-propylenecarbonate)covalentlylinked withindocyaninegreen,wasclinicallyutilizedformedicalimaging oftumors.Nearinfraredfluorescenceimagingshowedaselective tumoraccumulationofmicellarformulationcomparedtosolution ofindocyaninegreen.Thisselectiveaccumulationwascharacta- rizedbyanincreaseoffluorescenceoftumorinvivoandexvivo afterresection[35].TheseresultsshowedthatEPReffectenables theaccumulationofnanoparticule(NP)intotumorenvironment, butthiseffectdoesnotmeananincreaseofthecellularuptakeof nanocarriers.

3.3. Fibroblastsandextracellularmatrix(ECM)

Fibroblastsareabundantmesenchyme-derivedcelltypeswhich representthemaincellularcomponentofconnectivetissue.Fibrob- lastsmaintainthestructuralframeworkintissuesbypreserving ECMhomeostasis,regulateepithelialdifferentiationandinﬂamma- tion.Furthermore,theyrespondtodamage,andbecomeactivated tosupportrepairbystimulatinggrowthofnearbyepithelialcells andfacilitatingangiogenesis[159,160].

Incancertissue,ﬁbroblastsaremajorcomponentsofstromal cellsthatsurroundcancercellsandprovide,notonlyamechanical support,butalsoinhibitcancercellapoptosis,controlcellprolif- erationandsurvival,stimulatetumorangiogenesisandmetastasis, immunogenicity,andareinvolvedintherapyresistances[161,162].

Stromalfibroblastswithintumors showmorphological,phe- notypic and functional variabilities. This heterogeneity reflects the various sources of cellularorigin, including bone marrow- derivedprogenitorcells,localinfiltratingfibroblasts,endothelial cells, smooth muscle cells, pericytes or adventitial fibroblasts of the vascular system, or cancer cells themselves undergo-

ingﬁbroblastic transformation [163].Furthermore, a signiﬁcant cell plasticity exists within this cell population, as demon- stratedbymesenchymal-to-epithelialtransitions,andepithelial- to-mesenchymal transitions, furtherenhancing stromal heterogeneity.

Quiescentﬁbroblastsundergoactivationduringtissueremodel- ingandtheactivationfactorofCAFsmightvary.Severalactivation factorshavebeenfrequentlyreportedinthestromalﬁbroblasts of solid tumors, such as transforming growth factor-␤ (TGF␤),

␣-smooth muscle actin (␣-SMA), chemokines including mono- cyte chemotactic protein 1 (MCP1), ECM-degrading proteases, fibroblast-specificprotein,PDGFreceptors-␤andfibroblastacti- vationprotein(FAP)[160,163].

Theactivatedphenotypeof ﬁbroblasts isassociated withan increasedproliferativeactivityandenhancedsecretionofECMpro- teinssuchastypeIcollagenand tenascinC,ﬁbronectin,SPARC (secretedprotein acidic and rich in cysteine)and stromal cell- derivedfactor1(SDF1),andavariousgrowthfactorsuchasVEGF, platelet-derivedgrowthfactor(PDGF)andhepatocytegrowthfac- tor(HGF)[26,161].

Withregardtotumorprogression,CAFsplayamulti-faceted rolebecausetheycaneitherinhibitorpromotemalignantgrowth.

Intheearlystagesoftumorprogression,CAFsoperateasrepressors bysecretionofTGF-␤andbyinducingacontactinhibitiononcan- cercellsthroughtheformationofgapjunctionsbetweenactivated ﬁbroblasts[164,165].Duringtheadvancedstage,CAFsbecomeacti- vatedandcontributetobothtumorgrowthandprogressionina variouswaysincludingECMremodeling,paracrineanddirectinteractionswithcancercells,bysecretingsolublefactors(cytokines, hormonesandgrowthfactors),pathogenicangiogenesisbyliber- atingproangiogenicfactorsand regulationofimmune response [130,162,166,167].

Themoleculardeterminantthatgovernsthetumorregulatory roleofCAFremainsunknown.However,arecentstudyreported thatinmelanoma,Notch1pathwayactivitycoulddeterminethe tumor-promotingortumor-suppressingphenotypeinCAF.Indeed, ithasbeenreportedthatCAF carryingelevatedNotch1activity signiﬁcantlyinhibitedmelanomagrowthandinvasion,whileCAFs withnullNotch1promotedmelanomainvasion[168].

Duringmelanomaprogression,theECMisremodelledphysi- ologicallyandpathologicallyandCAFsretainamajorroleinthis processsincetheyaremainlyresponsiblefortheproductionofECM proteins(collagens,ﬁbronectin),hyaluronanaswellasMMPsand otherenzymesinvolvedinthepost-transcriptionalmodiﬁcationof ECMproteinsthemselves[169,170].

Changes in the expression of ECM proteins contribute to melanomaprogressionandcanpotentiallybeusedasaparam- eter of cancer aggressiveness. The melanocyte homeostasis is strictlycontrolledbythemicroenvironmentandthemelanocyte phenotype depends to keratinocyte–melanocyte crosstalk. The melanomacellsdifferentiationresultsfromescapeofmelanocytes ofphysiologicalcontrolthroughtheE-cadherintoN-cadherinclass switchwhichallowsmelanomacellstoeludefromkeratinocyte control. Furthermore, qualitative and quantitative changes in matrixcellularproteinexpression,suchasCCN3protein,osteo- pontin,tenascinCandSPARC,bymodulatingcancercellmigration, innate immuneresponse, cancergrowthand metastasisforma- tion,contributetomelanomaprogression[23].Otherdatasupport theideathatthedevelopmentofmelanomaisassociatedtoECM remodeling.Helal-Netoandcoworkershavereportedthat,compared tothe ECM derived from a humanmelanocyte cell line, ECMproducedbya melanomacelllineisconsiderablydifferent inultrastructuralorganizationandcomposition.Theinteractionof thematrix,derivedfromhumanmetastaticmelanomacellswith endothelialcellstriggersintegrin-dependentsignaling,leadingto

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SrcpathwayactivationthatmaypotentiateVEGFR2activationand consequentlyup-regulateangiogenesis[171].

ThemolecularnatureofECMinatumormicroenvironmentas wellasCAFscanalsoinﬂuencethesuccessoftherapeuticapproach foralargenumberofsolidtumors[26,172–174].

With regard to melanoma, CAFs can exertdirect effects on moleculartargetedtherapy bysecreting severalsolublefactors, suchashepatocytegrowthfactor(HGF),orbyinteractingdirectly withcancercellsinducingtheactivationoftumor-survivalsignal- ing[26,175].Furthermore,CAFscouldplayanimportantrolein primaryresistancetocancertherapy alsoindirectlyby produc- ingECMproteinsandbyformingacompactandwell-organized matrixstructuretopreventtherapeuticdrugsfromreachingcan- cercellsinthecoreofmelanomatissues[176].Thereisevidence thatcancercellsactivelyrecruitandactivateprimaryhumanskin fibroblastsandstimulatethemtodepositECMproteins.Theadhe- sionof cancercells to ECM proteinscontributes totherapeutic escape.Inuvealmelanoma,theadhesionofcelllinestolaminin, collagenIVandfibronectinisinvolvedinthereductionofcellapo- ptosisaftercisplatintreatment[177].Furthermore,incutaneous melanoma,adhesiontofibronectinprotectsmelanomacellsfrom anoikisandoverexpressionoftenascin-Ccontributestoresistance todoxorubicintreatment[178,179].

Theinvolvementofstromalﬁbroblastsinmelanomacellpro- liferationaswellasindrugresistancesuggeststhattheycould be promising therapeutic targets to increase the sensitivity of melanomacellstoanti-tumoragentsforcombinedmelanomather- apy.

3.4. NanoparticlesandECMproteins

Themelanomadevelopmentisassociatedwithareorganiza- tion and an altered composition of ECM. Among the different ECM proteins, cancerassociate protease (CAPS) such asMMPs, areoverexpressedintumorenvironmentandtheiroverexpression isoftenrelated tometastasisand cancerprogression[180,181].

MMP9and MMP2are gelatinaseandcollagenaseenzymes.Due totheirdegradationactivityoncollagenand gelatineand their higherconcentrationinthetumorenvironmentthaninthehealthy tissues, they could be used as a trigger to increase the bio- logical activity of nanocarriers (Table 1). Nanoparticles of poly (lactic-co-glycolicacid)(PLGA)-polyethyleneglycol(PEG)loaded with coumarin 6 as a hydrophobic model drug, were conju- gatedwithaMMP2peptidesubstrate(AGFSGPLGMWSAGSFG).The enzyme-stimulated multistagevectors (ESMSVs)were obtained by conjugating thepeptide-PLGA-PEG nanoparticles (45nm)to poroussiliconmicrodisks(2.6␮m×0.7␮m,50–60nmpores).24h after intravenous injectionof ESMSVsin A375 melanoma lung metastasismice,anincreaseofcoumarinamountinmicetreated withESMSVscomparedtomicetreatedwithnon-peptidemodi- ﬁedmultistagevectorwasobserved(64.6%and46.8%respectively werepositivetocoumarin)[55].Thisstudyshowedthepossibility touseMMPsasatriggertoreleasenanoparticlesfromamicroplat- form[56].ToreducethesizeofnanocarriersinremovingthePEG protectionisanotherstrategyusingMMPsinordertoformulate environmentresponsivenanocarriers.Yuandco-workersdevel- opedenzymeresponsivemicellesaccordingtothisstrategy.Long PEGchains,usedtoprotect themicellefromRESsystem,were abletobecleavedfromthePCLcoreintothetumorenvironment, duetotheMMP-2/-9cleavableproteinlinkerbetweenthepoly- merchains.Inthisstudy,theresultshowed1)a sizereduction ofthenanocarriers,2)ahighertumorconcentrationofMMP-2/-9 cleavablemicellesand3)anantitumoreffectincrease(causedby camptothecinloadedinthemicelles)comparedtocontrolgroups.

Unfortunately,inthisstudy,thePEGsuppressionbyMMP2and9in tumorenvironmentcausedaswellaggregationofthenanocarriers,

butledtoincreasetheconcentrationofcamptothecininthetumor [57].

3.5. Acidiﬁcationoftumormicroenvironnement

Extracellular acidityis a pathological featureof solid tumor tissue including melanoma. Among the main causes of extra- cellularacidosis, ithasbeenreportedthat alterationof cellular metabolismcanbeanimportantcause.OttoWarburgwasthefirst toobserveabnormalcancercellmetabolism.DescribedastheWar- burgeffect,melanomacells utilize aerobicglycolysisinsteadof oxidativephosphorylationevenundernormoxicconditionsleading toanenhancedlactic acidproduction [182].Highlactate secre- tionfromtumorcellscontributestotheirimmuneescape,affects inflammation,enhancesthemotilityof tumorcellsand inhibits monocytemigrationandcytokinerelease[183–186].Furthermore, lactateproductioncontributestoangiogenesisthroughinduction ofVEGF,viaanactivationofhypoxia-inducibletranscriptionfactor (HIF)-1,andanupregulationofIL-8transcriptionthroughnuclear factor-␬B(NF-␬B)[187,188].Inadditiontothisupregulationof glycolysis,metabolicalterationsleadingtoanextensiveproduc- tionofacidicmetabolites,includethepentosephosphatepathway, themajorproductivepathwayofcarbondioxide(CO₂),andglu- taminolysiswhich,byproducingglutaminemetabolite,contribute tomelanoma cell growthand proliferation [136,189]. Accumu- latingevidenceshowsthatseveraltransportersandpumps,such asNa⁺/H⁺exchanger(NHE),H⁺-lactateco-transporter,monocarboxylatetransporters(MCTs),orH⁺-ATPasepumps,contributeto secretionofintracellularlyproducedprotons(H⁺)causingfurther acidificationoftheextracellularspace[190].Theincreaseofacid production following metabolic reprogramming of cancer cells resultsinacidificationofextracellularpH(pHe)andalkalinization ofintracellular/cytosolicpH(pHi).AcidicpHeisinvolvedinlocal tumorinvasionand tissueremodeling.Protonsdiffusefromthe proximaltumormicroenvironmentintoadjacentnormaltissues causingtissueremodelingthatpermitslocalinvasion[191].The resultingacidicenvironmentpromotestheexpressionofdegrad- ingenzymessuchasMMPsandlysosomalenzymeswhich,through thedegradationofECM,providethepotentialtodevelopmetas- tases[192,193].AcidicpHeistoxictomanycells,includingtumors.

However,tumorcellsdevelopresistancetoacid-inducedtoxicity duringcarcinogenesis, allowingthem tosurviveand proliferate inlowpHmicroenvironments[194].In melanoma,extracellular acidosis leadstothedevelopmentofa moreaggressivepheno- typeofcells.AcidicpHepromotesmetastasisinhumanmelanoma cellsbyup-regulationoftheproteolyticenzymesMMP-2,MMP- 9,cathepsinB,andcathepsinL,contributingtothedegradationof theECM,and up-regulationofthepro-angiogenicfactorsVEGF- AandIL-8promotingtumorinvasionbystimulatingangiogenesis [187].Insolid tumors,acidityis closelyrelated tohypoxia.The fastproliferationofcancercellsandthechaoticandincomplete vasculatureoftumorarefrequentlyresponsibleforatransientor persistentoxygendeﬁciency.Thehypoxia-induciblefactor-1(HIF- 1)expressionandactivityareincreasedinmelanomamalignant cellsand contributetotheformationofglycolyticphenotypein cancercellsleadingtoacidiﬁcationvialacticacidproduction[195].

HIF promotesthetranscription ofgenesinvolved in cancerini- tiation,progression andmetastases includinggenesinvolved in adaptationtoanunfavourabletumormicroenvironment.Notably, the two subunits HIF-1␣ and HIF-2␣drive melanomainvasion andinvadopodiaformationthroughPDGFR␣andfocal adhesion kinase-mediated (FAK-mediated)activation of SRCand byECM remodeling[196].TheincreaseofextracellularH⁺ionsmodulates several molecules of theECM, including channels, transporters and the plasma membrane. Nishisho and co-workers reported that vacuolar-type H+-ATPase (V-ATPase) isoform a3 promotes