Efficient ferrocifen anticancer drug and Bcl-2 gene therapy using lipid nanocapsules on human melanoma xenograft in mouse

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Pharmacological Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / lo c a t e / y p h r s

Research article

Efﬁcient ferrocifen anticancer drug and Bcl-2 gene therapy using lipid nanocapsules on human melanoma xenograft in mouse

Pauline Resnier

^a

, Natacha Galopin

^b

, Yann Sibiril

^c,d

, Anne Clavreul

^a

, Jérôme Cayon

^a,e

, Alessandro Briganti

^a

, Pierre Legras

^b

, Anne Vessières

^f

, Tristan Montier

^c,d

, Gérard Jaouen

^f

, Jean-Pierre Benoit

^a

, Catherine Passirani

^a^,∗

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

bSCAHU–FacultédeMédecine,PavillonOllivier,rueHautedeReculée,F-49933Angers,France

cINSERMU1078–Equipe‘Transfertdegènesetthérapiegénique’,FacultédeMédecine,22avenueCamilleDesmoulins,CS93837,F-29238Brest,Cedex3, France

dCHRUdeBrest,ServicedeGénétiqueMoléculaireetd’histocompatibilité,5avenueMaréchalFoch,29609Brest,France

ePACeM(Plateformed’AnalyseCellulaireetMoléculaire),SFRICAT4208,Universitéd’Angers,4rueLarrey,F-49933Angers,France

fCNRS,UMR8232,ENSCP,11rueP.etM.Curie,F-75231ParisCedex05,France

a r t i c l e i n f o

Articlehistory:

Received8January2017

Receivedinrevisedform26January2017 Accepted27January2017

Availableonline31January2017

Keywords:

Genetherapy Metal-baseddrug SK-Mel28 Nanoparticles Passivetargeting

a b s t r a c t

Metastaticmelanomahasbeendescribedasahighlyaggressivecancerwithlowsensibilitytochemother- apeuticagents.Newtypesofdrug,suchasmetal-baseddrugs(ferrocifens)haveemergedandcould representanalternativeformelanomatreatmentsincetheyshowinterestinganticancerpotential.Fur- thermore,molecularanalysishasevidencedtheroleofapoptosisinthelowsensibilityofmelanomasand especiallyofthekeyregulator,Bcl-2.Theobjectiveofthisstudywastocombinetwostrategiesinthesame lipidnanocapsules(LNCs):i)genetherapytomodulateanti-apoptoticproteinsbytheuseofBcl-2siRNA, andii)ferrocifensasanewtypeofanticanceragent.TheefficientgenesilencingwithLNCswasverified bythespecificextinctionofBcl-2inmelanomacells.Thecellulartoxicityofferrocifens(ferrociphenol (FcDiOH)orAnsa-FcDiOH)wasdemonstrated,showinghigherefficacythandacarbazine.Interestingly, theassociationofsiBcl-2LNCswithAnsa-FcDiOHdemonstratedasignificanteffectonmelanomacell viability.Moreover,theco-encapsulationofsiRNAandferrocifenswassuccessfullyperformedintoLNCs foranimalexperiments.AreductionoftumorvolumeandmasswasprovedaftersiBcl-2LNCtreatment andAnsa-FcDiOHLNCtreatment,individually(around25%).Finally,theassociationofbothcomponents intothesameLNCsincreasedthereductionoftumorvolumetoabout50%comparedtothecontrolgroup.

Inconclusion,LNCsappearedtoprovideapromisingtoolfortheco-encapsulationofametal-baseddrug andsiRNA.

1. Introduction

Althoughsigniﬁcantprogresshasbeenmadeoverthelastfew years,malignantmelanomaisstilltheleadingcauseofdeathfrom skincancer,witha5-yearsurvivalrateoflessthan10%[1].Indeed,

∗Correspondingauthorat:INSERMU1066,IBS-IRIS,4rueLarrey,49933Angers, Cedex9,France.

E-mailaddresses:[email protected](P.Resnier), [email protected](N.Galopin),[email protected](Y.Sibiril), [email protected](A.Clavreul),[email protected] (J.Cayon),[email protected](A.Briganti),[email protected] (P.Legras),[email protected](A.Vessières),[email protected] (T.Montier),[email protected](G.Jaouen),[email protected] (J.-P.Benoit),[email protected](C.Passirani).

dacarbazine (DTIC), the FDA approved chemotherapeutic treat- mentformetastaticmelanoma,hasbeenknowntoinducealow responserate(16%)[2].Otherwise,immunityhasbeenknownto play animportant role in melanomapromotion and alsoin its eradication,inspiringthedevelopmentofnewtherapeutics[3,4].

Since2011,twotargetedtherapies,vemurafenibandipilumimab, whichcorrespondtoaB-RafinhibitorandaCTLA-4blockinganti- bodyrespectively,havealsobeenevaluatedonmelanomapatients.

However,resistancephenomenawererapidlydescribedforB-Raf inhibitorswitharecurrenceofmetastasis,andCTLA-4showedlow- responseratesasforDTIC[5,6],withasurvivalmedianinferiorto 13months.Thechallengeofmelanomatherapyconsistsinbypass- ingthehigh-resistancephenomenathatlimitorcompletelyavoid theefﬁcacyofchemotherapy.

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Theloweffectofanticancerdrugsisexplainedbytheintrinsic resistancetoapoptosis,oneoftheimportanthallmarksofcancer, andespecially inmelanoma[3,7].Thiscelldeathmechanism is controlledbypro-apoptotic(Bax,Bak) andanti-apoptoticmembers(Bcl-2,Bcl-XL,Mcl-1)[8].Bcl-2(B-celllymphoma2)isakey regulatorinapoptosispathwaysblockingtheeffectiveoligomer- ization of Bax and Bak, and thus protecting against cell death [9]. Molecularanalysishas evidenced theup-regulation of Bcl- 2inmelanomametastasis,andthishighlevelwasassociatedto chemoresistance[10,11].Interestingly,theoverexpressionofBcl-2 hasbeencorrelatedwithpoorprognosesandshortsurvivalrates inmelanomas[10,12,13].Bcl-2inhibitionwasassessedearlyby antisenseoligonucleotide(oblimersen)incancerapplicationsand especiallyinmelanomas[14,15].Althoughoblimersenincreased apoptosisinmelanomaxenograftsandimprovedtheirsensitivity toDTIC[16],itonlyhadmodestimpactonpatientsurvivalinclinical trials[17].Thislimitedeffectcanbeexplainedbythelowbioavail- abilityofoligonucleotideintothebloodstreamanditsincapacity topassthroughthebiologicallipidmembranes.

Nowadays,innovativenanomedicinescanimprovethebioavail- abilityofencapsulateddrugssuchasanticancerdrugs,ornucleic acids, in orderto limit systemic side effects and increase drug concentrationintargetedsiteswithaloweradministeredquan- tity of drugs [18]. Lipid nanocapsules (LNCs), nanomedicines basedonphase-inversion emulsions,wereadapted recentlyfor smallinterferingRNA(siRNA)encapsulation,toprovidespeciﬁc geneextinction.Theyshowedefﬁcienttargetingofsubcutaneous melanomacells after intravenous injection[19,20].In addition, thesenanoparticleshaveanoilycoresuitablefortheencapsulation andthedeliveryoflipophilicdrugssuchasanticancercompounds inordertodevelopconcomitantstrategyincancertherapy[21].

Metal-baseddrugsrepresentapromisingfamilyofcompounds for cancerapplicationand especially for ferrocifen-based drugs [22]. These bio-organometallic molecules are deﬁned as active moleculesthatcontainatleastonecarbonatomdirectlyboundtoa metalormetalloid.Inferrocifens,themetalstudiedisiron[7],and themetallocenderivativeis ferrocen[␩5-Fe(C5H5)2]chemically graftedontoapolyphenolicskeleton,resultinginferrocenylphenol derivatives[23].Multipleferrocifenshavebeensuccessfullyencap- sulatedintoLNCsandsomeofthemhavedemonstratedpromising anticancerpropertiesonresistantcancerssuchastriple-negative breastandgliomacancermodels[24,25].

Inordertoimprovetheresponserate,associationofdifferent drugsrepresentsarelevantstrategy.Multiplepre-clinicalandclin- icalstudieshavealreadybeenfocusedontheanalysisofinnovative combinatorytherapeutic[26–28].TheassociationofBcl-2siRNA andnewanticancerdrugssuchasferrocifencompoundsrepresents anewopportunityformelanomatreatment.Forthis,twodifferent promisingferrocifens wereusedcorrespondingtoferrociphenol (FcDiOH)orAnsa-FcDiOHdifferentiatedbytheirspecificchemi- calstructure(Scheme1).Theobjectiveofthisworkconsistedof developingLNCsasnewnanomedicinesforinnovativeandefficient combinedapplication,i.e.genetherapywithsiRNAandanticancer drugdeliverywithferrocifen.TheefficacyofLNCstodeliverBcl-2 siRNAandferrocifenswastestedonhumanSK-Mel28melanoma cells.Finally,asubcutaneousmelanomamodelimplantedinnude micewasusedtostudyitsinvitroandinvivoanticancerproper- tiesandpossiblesynergiceffects.Thecellulartoxicityofferrocifens (FcDiOHorAnsa-FcDiOH)wasdemonstrated,showinghighereffi- cacythanDTIC.Interestingly,theassociationofsiBcl-2LNCswith Ansa-FcDiOHdemonstratedasignificanteffectonmelanomacell viability.Moreover,theco-encapsulationofsiRNAandferrocifens wassuccessfullyperformedintoLNCsforanimalexperiments.A reductionoftumorvolumeandmasswasprovedaftersiBcl-2LNC treatmentandAnsa-FcDiOHLNCtreatment,individually(around 25%).Finally,theassociationofbothcomponentsintothesame

Scheme1.Chemicalstructureofferrociphenolandansa-ferrociphenol.Chemical structureoftwoderivatives(ferrociphenol,FcDiOH;ansa-Ferrociphenol, ansa- FcDiOH)withcharacteristicferrocengroups.

LNCsincreasedthereductionoftumorvolumetoabout50%comparedtothecontrolgroup.

2. Materialsandmethods

2.1. Chemicalmaterials

FcDiOHandAnsa-FcDiOHwerepreparedbyaMcMurrycross- couplingreaction,aspreviouslydescribed[29,30].Theirstructures are presented in Scheme 1. The lipophilic Labrafac^® WL1349 (caprylic-capric acid triglycerides) was purchased from Gatte- fosseS.A.(Saint-Priest,France),Solutol^® HS15(amixtureoffree polyethylene glycol 660 and polyethylene glycol 660 hydrox- ystearate) by BASF (Ludwigshafen, Germany), Lipoïd^® S75-3 (correspondingtosoybeanlecithinat69%ofphosphatidylcholine) byLipoïdGmbh(Ludwigshafen, Germany) andNaCl byProlabo (Fontenay-sous-bois,France).Deionizedwaterwasacquiredfrom aMilli-Qplussystem(Millipore,Paris,France).

1,2-DiStearoyl-sn-glycero-3-PhosphoEthanolamine-N- [methoxy-(polyethyleneglycol)-2000] (DSPE-PEG) (Mean Molecular Weight (MMW)=2805g/mol), cationic lipid as 1,2-dioleoyl-3-trimethylammoniumpropane(DOTAP) and zwit- terioniclipidas 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)werepurchasedfromAvantiPolarLipids(Alabaster,USA).

In this study, the siRNA sequence targeting human Bcl-2 protein (sense sequence: 5-GUGAUGAAGUACAUCCAUUdTdT-3; antisense: 5- AAUGGAUGUACUUCAUCACdTdT-3; reference NM000633) and scramble sequence as control (sense sequence: 5-UCUACGAGGCACGAGACUUdTdT-3; antisense: 5-AACUCUCGUGCCUCGUAGAdTdT-3; reference NM002046/NM001256799) were purchased from Eurogentec (Seraing,Belgium).

2.2. FormulationofLNCs 2.2.1. BlankLNCs

LNCswerepreparedaccordingtoaphase-inversionmethodas previouslydescribed[31].Brieﬂy,thepreparationprocessinvolved 2steps.Step1consistedofmixingalltheexcipients(Kolliphor^® HS15(17.2%w/w),Labrafac^® (20.8%w/w),NaCl(1.8%w/w)and water(60.2%w/w)undermagneticstirringandheatingfromroom temperatureto90^◦C.Threecyclesofprogressivecoolingandheat- ingbetween90 and60^◦Cwerethencarriedout.Step2wasan irreversibleshockinducedbyrapiddilutionwithwater(33%v/v) appliedtothemixtureat70–75^◦C.Slowmagneticstirringwasthen appliedtothesuspensionfor5min.

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2.2.2. siRNALNCs

Association siRNA/lipids – for lipidpreparation, the cationic lipidDOTAP,solubilizedinchloroform,wasweightedattheratio 1/1(Mol/Mol)withtheneutrallipidDOPEtoobtainafinalcon- centrationof30mMofcationiclipidcharge,basedonthenumber oflipidchargespermolecule.Aftertheevaporationofchloroform undervacuum,deionizedwaterwasaddedtorehydratethelipid filmovernightat4^◦C.Thelipidfilmwasthensonicatedfor15min.

BeforeincorporationintothesiRNALNCformulation,lipoplexes were formulated as a simple equivolume mix of siRNA and DOTAP/DOPE lipids. The complexes were characterized by the chargeratio[32],i.e.,theratiobetweenthepositivechargesofthe lipidsandnegativechargesofthenucleicacids(+/−ratio)ﬁxed at5,basedonourpreviousresults[33].Pre-formulatedlipoplexes inaqueoussolutionwereintroducedduringstep2,asdeveloped inourpreviousworks [19,32].ThissiRNA LNCformulationwas recentlypatented(ref:1458991,September24th2014).

2.2.3. FerrocifenLNCs

Ferrocifen LNCs were prepared according to a phase inver- sionmethodaspreviouslydescribed[31].Ferrocifencompounds (FcDiOHandAnsa-Fc-DiOH),asasolidpowder,wereaddedatstep 1withotherexcipientsinordertoobtainaﬁnalconcentrationof 6mg/mL(2%w/w)[24].Thedrugwassolubilizedduringtheheating stepandultimatelyencapsulatedoverthelastcoolingstep.

2.2.4. FerrocifensiRNALNCs

Ferrocifen-siRNALNCswereformulatedbycombiningthepro- cessof ferrocifen and siRNA encapsulation. For this, ferrocifen powderwasaddedatstep1withalltheexcipients,andthreecycles ofprogressivecoolingandheatingbetween90and60^◦Cwereper- formed.ThesiRNAs,complexedwithlipidsinaqueoussolution wereintroducedduringstep2,inducingtheirreversibleshockin ordertoformulatetheFerrocifen-siRNALNCs.

2.3. Puriﬁcationandpost-insertionwithDSPE-PEG

Purificationwasperformedinwater(obtainedfromaMilli-Q system,Millipore,Paris, France)witha PD10 Sephadex column (AmershamBiosciencesEurope,Orsay,France)toremovethenon- encapsulatedsiRNAandlipoplexes[19].TheNaClconcentration wasadjustedafterthispurificationtophysiologicalconcentration (150mM).The post-insertion of 1,2- distearoyl – sn – glycerol –3 –phosphoethanolamine–N- [methoxy(polyethyleneglycol)- 2000](DSPE-PEG)(MeanMolecularWeight(MMw=2805g/mol) wasperformedbyaincubationofLNCsat37^◦Cfor4hwithafinal PEGpolymerconcentrationat5mM.

2.4. CharacterizationofsiRNALNCs 2.4.1. Sizeandzetapotential

ThesizeandZetapotentialofLNCsweremeasuredbyusingthe DynamicLightScattering(DLS)methodusingaMalvernZetasizer^® apparatus(NanoSeriesZS,MalvernInstrumentsS.A.,Worcester- shire,UK)at25^◦C,intriplicate,afterdilutionataratioof1:200 withdeionizedwater[34].

2.4.2. Encapsulationefﬁciency

2.4.2.1. siRNAquantification.Aspectrophotometricmethodbased onaworkrecentlydescribedbyDavidetal.wasusedtoevaluatethe encapsulationefficiency[33].Briefly,siRNALNCsweremixedwith chloroformandwatertoseparatehydrophilicandlipophiliccom- ponents,respectively.Sodiumhydroxidewasaddedtodestabilize lipoplexes,andfinallyabsoluteethanolwasaddedtodestroythe LNCs.Aftertwocentrifugations,twofractionswereobtained:free

siRNAandencapsulatedsiRNAinLNCs.Todeterminetheconcen- trationofsiRNA,theopticaldensityofeachsamplewasdetermined at260nm(UV-2600,Shimadzu,Noisiel,France)intriplicatecon- ditions.Theencapsulationefﬁciency[16]wasdeterminedbythe ratio(%)betweentheencapsulatedquantityofsiRNAandthetotal amountofsiRNAintroducedintheformulation.

2.4.2.2. Ferrocifenquantification.Thedeterminationof theferro- cifen loaded into LNCs was achieved by spectrophotometry at 450nmafterdissolving10␮LofLNCsinto5mLofmethanol(Fisher chemical)beforeandafterLNCfiltrationwitha0.2␮mfilter(GHP Pall,Acrodisc,VWRinternational,Fontenay-sous-Bois,France).The calibrationcurve,rangedfrom10␮Mto100␮M,wasalsopre- paredinmethanol.Theencapsulationefficiencywasdetermined bytheratio(%)betweentheencapsulatedquantityofferrocifen (afterfiltration)andthetotalamountofferrocifenintroducedin theformulation(beforefiltration).

2.5. SK-Mel28melanomacellculture

TheSK-Mel28humanmelanomacelllinewasgrowninRoswell Park MemorialInstitute (RPMI) 1640medium (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum (Lonza, Verviers,Belgium),1%antibiotics(10unitsofpenicillin,10mgof streptomycin,25␮gofamphotericinB/mL;Sigma-Aldrich,Saint Louis,USA)and1%non-essential aminoacids(Lonza).Celllines wereculturedaccordingtoATCCprotocolduringpassagenumbers 10until20,andmaintainedat37^◦Cinahumidiﬁedatmosphere with5%CO₂.

InvitroTransfection

SK-Mel28cellswereseededonto6-wellplatesatthedensity of2×10⁵ cells/wellor inﬂask(25cm²)with5×10⁶ cells/ﬂask andpreculturedovernight.Beforetransfection,themediumwas changedtoafreshonecontainingnoserum.Differenttreatments (control,scrambledsiRNALNCs,andBcl-2siRNALNCs)wereincu- batedwithcellsandremoved8hpost-transfectionreplacedwith freshcomplementedmedium.Apositivecontrolwascarriedout with oligofectamine^® according tothe manufacturer’s protocol withscrambled and Bcl-2 siRNA.Transfections (with LNCs and oligofectamine^®)werecarriedoutforthreeconsecutivedaysunder thesameconditionseachday.

2.6. Westernblot

Afterinvitrotransfections orinvivoexperiments, totalpro- teinswereextractedfrommelanoma:cellsbyscrapingwithacell lysisbuffer(10mmol/LTris-Base,1mmol/LNa₃O₄,and1%SDS,pH 7.4)andstoredat−20^◦C;andthemelanomatumorsweregrinded withUltra-turrax(Ikaimlab,Lille,France)inthesamebufferlysis.

Twentymicrogramsofproteinswereresolvedon10%(v/v)SDS- PAGEgelandtransferredtoanitrocellulosemembrane(0.45␮m poresize)(AmershamGEHealthcare).Mouse anti-humanBcl-2 (ab694,Abcam)andmouseanti-humanactin(CloneC4;Millipore), wereusedasloadingcontrols,andwerethendiluted,respectively, at ratios of 1:500 and 1:5000 according to themanufacturer’s instructions,andincubatedovernightat4^◦C.Asecondmouseanti- body(goatanti-mouseIgG,secondaryantibody,HRPconjugate,Life Technologies)wasusedatadilutionof1:2000.Detection(using LAS4000,GEHealthCare)wasperformedusingenhancedchemo- luminescence(ECL;FisherScientiﬁc,Pierce).Ineachcase,protein levelswerenormalizedtothehousekeepingproteinactinandthe Bcl-2proteinexpressionfolddifference,betweentestedconditions andthecontrolcells,asplottedbyImageJsoftware.

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2.7. RT-q-PCR

ThetotalRNAofcellswasextractedandpurifiedusingRNeasy Microkit(Qiagen,Courtaboeuf, France), andtreated withDNase (10UDNaseI/␮gtotalRNA).RNAconcentrationsweredetermined usingaND-2000NanoDrop(ThermoFisherScientific,Wilmington, DelawareUSA)andusedfornormalizationoftheinputRNAinthe Reversetranscription.FirststrandcDNAsynthesiswasperformed withaSuperScript^TMIIReverseTranscriptase(Invitrogen),incom- binationwithrandomhexamers,accordingtothemanufacturer’s instructions. Following first-strand cDNA synthesis, the cDNAs werepurified(QiaquickPCRpurificationkit,Qiagen,Courtaboeuf, France)andelutedin40␮LRNAsefreewater(Gibco).Aquantityof 3ngofcDNAwasmixedwithMaxima^TMSYBRGreenqPCRMaster Mix(Fermentas)andprimermix(0.3␮M)inafinalvolumeof10␮L.

PrimersequencesforGAPDHandBcl-2arelisted:GAPDHforward CAAAAGGGTCATCATCTCTGC, GAPDH reverse AGTTGTCATGGAT- GACCTTGG, Bcl-2 forward GTGGAGGAGCTCTTCAGG and Bcl-2 reverseCAGGAGAAATCAAACAGAGG.Ampliconsizeare155bpfor GAPDHand242bpforBcl-2,Amplificationwascarriedoutona LightCycler480(Roche)withafirstdenaturationstepat95^◦Cfor 10minand40cyclesof95^◦Cfor15s,and60^◦Cfor30s.Afterampli- fication,ameltingcurveoftheproductsdeterminedthespecificity oftheprimersforthetargetedgenes.Ameancyclethresholdvalue wasobtainedfrom2measurementsforeachcDNA.Specificgene expressionwascalculatedusingthe2⁻^CTmethodusingGAPDH ascalibrator.

2.8. Determinationofcellviability

SK-Mel28cells wereseededonto 24-wellplates at theden- sityof 5×10⁴ cells/well and precultured overnight. Beforethe treatmentwithdifferentdrugs,theculturemediumwasremoved and the cells were treated with DTIC, free FcDiOH, free Ansa- FcDiOH, FcDiOH LNCs or Ansa-FcDiOH LNCs at concentrations of 0.0001–100␮g/mL. Unloaded (blank) LNCs were also tested withthesameexcipientconcentrationasforFcDiOHLNCs.After 72h, the medium was removed from the wells and replaced with fresh medium. Cytotoxicity assays were performed using MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2Htetrazolium)(Promega, Madison, USA). For this,100␮LofMTS/wellweredisposedandplateswereincubated for2.5hà37^◦Cinahumidiﬁedatmospherewith5%CO₂.TheOD wasevaluatedbyMutliskanAscent(Labsystems,FisherScientiﬁc, Wilmington,USA)at492nm.Cellswithoutanytreatmentwere usedasacontrolandcorrespondedtoacellviabilityof100%.

2.9. Invivoexperiments

Six-toeight-week-oldfemale,nudeNMRImice(ElevageJanvier, France)werehousedandmaintainedattheSCAHU;theywerepro- cessedinaccordancewiththeLaboratoryAnimalCareGuidelines (NIHPublication85-23,revised1985)andwiththeagreementof thenationalethiccommittee(authorizationN^◦01315.01,12/2013, France).Tumor-bearingmicewerepreparedbyinjectingsubcuta- neouslyasuspensionof3×10⁶SK-Mel28melanomacellsin100␮L ofRPMIintotherightﬂankofmice(20–24g).Afterthreeweeks of tumoral growth, different treatments (blank PEG LNCs, PEG ferrociphenol-LNCs,PEGsiRNALNCsorpegylatedco-encapsulated forms)wereappliedintravenouslywithdailyinjectionsfor5days (siRNALNCs)or3weeks(dailyinjectionsfor5daysfollowedby aninterruptionof2days,repeatedfor3weeks).Tenanimalswere usedasacontrolanddidnotreceiveanytreatment,10animals receivedblank PEG LNCs, 8 animals receivedscrambled siRNA PEGLNCs,8 animalsreceivedBcl-2 siRNAPEGLNCs, 8animals receivedFcDioHPEGLNCs,8animalsreceivedAnsa-FcDiOHPEG

Table1

Physico-chemicalcharacterizationandencapsulationyieldofsiRNALNCs,twofer- rocifenLNCsandtheirco-encapsulatedforms.Alllipidnanocapsules(LNCs)ledto monodispersednanomedicineswithsizesrangingbetween60and80nm.SiRNA encapsulationledtoapositivezetapotential,contrarytoblankandferrocifen encapsulationintoLNCs.Interestingly,theco-encapsulationofsiRNAandferro- cifeninducedasigniﬁcantlyhighersiRNAencapsulationefﬁciency(EE,%).Results areexpressedasthemeanmeasure±standarddeviation(SD)(n≥3).Statistical analysiswasperformedusingat-test,*p≤0.05.FcDiOH:Ferrociphenol,Ansa:Ansa- ferrociphenol,LNCs:lipidnanocapsules.

Formulation Size(nm) PDI Zeta(mV) siRNAEE(%) FcEE(%) BlankLNCs 77±2 0.08±0.01 −7±2 / / siRNALNCs 74±4 0.05±0.01 +13±2 35±5 / FcDiOHLNCs 65±2 0.07±0.01 −4±1 / 92±2 AnsaLNCs 64±4 0.05±0.01 −4±2 / 96±1 Si+FcDiOHLNCs 65±1 0.04±0.01 +15±15 63±5* 82±5* Si+AnsaLNCs 69±5 0.11±0.03 +11±13 49±12* 85±7*

LNCs, 10 animalsreceived DTIC, and 8 animals received Ansa- FcDiOHBcl-2siRNAPEGLNCs.Todeterminethetumorvolume, each mouse was monitoredat theindicated time by measure- mentoftumorsize,andthetumorvolumewasestimatedusingthe formula:V=6/␲*length*width².Threeweeksaftertheﬁrstintra- venousinjection,theanimalsweresacriﬁcedandthetumorswere dissectedtoevaluatetheirmass.

2.10. ALAT—ASATdetermination

Bloodsampleswerecollectedfromthelateralsaphenousvein fromanimalsreceivingnoinjection(n=4),siRNALNCs(n=8),and PEGsiRNALNCs(n=8).BloodwascollectedbeforeandonDays2,4 and9afterLNCinjectionsondifferentanimals(twopercondition perday)tobecompatiblewithanimalwelfare.Theywerecollected in Microvettecollection tubes (Sarstedt, Numbrecht,Germany).

Afterwards,thesampleswerecentrifugedfor2minat10,000gat 4^◦Candtheplasmawasremovedforfurtheranalysis.TheALATand ASATvaluesweredeterminedusingaSelectra-E(Elitech,Signes, France).

2.11. Statisticalanalysis

Comparisonsbetweenallgroups,supposedtohavenormaldis- tribution, wereperformedusing a classicalanalysisof variance (one-wayANOVA)followedbyaTukey’spost-hocanalysis.Statis- ticalsigniﬁcancewasascribedtoathresholdof0.05.

3. Results

3.1. DescriptionofLNCsandco-encapsulation

ThesizeoftheLNCswasnotsignificantlymodifiedbytheencap- sulationofalltesteddrugs(Table1).Ontheotherhand,thezeta potentialwasinfluencedinfunctionofthedrugencapsulated.Con- trarytonegativeblankandferrocifenLNCs,thecationiclipidused forsiRNAbindinginducedapositivechargeofLNCs.Concerningthe encapsulation,siRNAandthetwoferrocifendrugsalonewereeffi- cientlyencapsulatedindividually,asshowninourpreviousworks [19,24].TheformulationledtoanencapsulationefficiencyofsiRNA evaluatedat35%andmorethan90%forferrocifens(FcDiOH,Ansa- FcDiOH)(Table1).Interestingly,theco-encapsulationofsiRNAand ferrocifendrugs inducedsignificantly higherencapsulation effi- ciencyofsiRNA(49%or63%vs35%),andferrocifenencapsulation wasgreaterthan80%(Table1).

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Fig.1. ProteinandgeneextinctionofBcl-2bysiRNAlipidnanocapsules.Bcl-2expressionwastestedbywesternblot(A)withdensitometryanalysis(B)andRT-q-PCRanalysis (B).Cells,Oligofectamine^®withsiCTRL,Oligofectamine^®withsiBcl-2,siCTRLLNCsandsiBcl-2LNCsweretested.mRNAlevelswereexpressedasthemeanmeasure±standard errorofthemean(SEM)(n≥3).StatisticalanalysiswasperformedbyANOVA1W,post-hocTukey,**p≤0.01,*p≤0.05.

3.2. ExtinctionofBcl-2

The extinction of the Bcl-2 protein was tested using siRNA tools. The transfection efficacy of LNCs was compared to a commercialagent(Oligofectamine^®)(Fig.1).ProteinandmRNA analysesevidencedthesignificantinhibitionofBcl-2proteinwith Oligofectamine^®andsiRNALNCswithareductionof60%ofprotein synthesisand50%ofmRNAproduction(Fig.1B/C).Thecontrolcon- ditionsinducednon-significantmodificationsoftheBcl-2protein ormRNAlevel(Fig.1).

3.3. Ferrocifenactivityandchemo-sensitizationonmelanoma cells

TheactionofFcDiOHandAnsa-FcDiOHwastestedandcom- paredtoreferencechemotherapyusedformetastaticmelanomas, dacarbazine(DTIC,rangefrom0.001to100␮g/mL).HumanSK- Mel28melanoma cells showedlow sensitivitytoDTIC, even at highconcentrations(superiorto100␮g/mL)(Fig.2).FcDiOHand Ansa-FcDiOHcompoundsshowedsigniﬁcantlyhighcytotoxicity compared toDTIConSK-Mel28 melanomacells withIC₅₀ esti- matedat1.3␮g/mLand0.5␮g/mLrespectively(correspondingto 3␮Mand 1.2␮M)(Fig.2A).Encapsulated forms ofFcDiOH and Ansa-FcDiOH into LNCs presented the same toxicity proﬁle as freedrugs,evidencingthecapacityofLNCs todeliverferrocifen drugsintomelanomacells withoutadditionaltoxicity (Fig.2B).

ThecombinationofBcl-2siRNAandferrocifenswasassessedon humanmelanomacellsinordertostudyapossiblesynergiceffect (Fig.2C).TheassociationofBcl-2siRNALNCswasassessedwith DTIC,FcDiOHandAnsa-FcDiOH.ThetransfectionofBcl-2siRNALNC inducedlittletoxicityasscrambleandblankLNCs(79%versus89%

and94%).ThecombinationofBcl-2siRNALNCwithDTICorFcDiOH didnotshowanysynergiceffect.Finally,theconcomitanttreat- mentwithBcl-2siRNALNCandAnsa-FcDiOHledtolowersurvival

ratecomparedtoindividualtreatment(46%versus79%forBcl-2and 90%forAnsa-FcDiOH),demonstratingasigniﬁcantsynergiceffect comparedtocontrolconditions.

3.4. Bcl-2siRNALNCtreatmentontumorgrowth

Threeweeksaftercellimplantation,repeatedintravenousinjec- tions(IV)ofsiRNAPEGLNCswereperformedinthetailveinof nudemicefor5days(1IV/day)correspondingto0.75mg/kg/day ofsiRNA.Nodeathorclinicalsideeffectswereobservedonmice during all experimental protocols. The control group (without treatment,n=10)wascomparedtoblankPEGLNCs(n=10),siCTRL PEGLNCs(n=8)andsiBcl-2PEGLNCs(n=8)(Fig.3A).Anevalua- tionoftumorvolumeevidencedtheabsenceofeffectusingblank andsiCTRLPEGLNCs.siBcl-2PEGLNCsinducedatleast25%volume reductioncomparedtoothergroups(Fig.3A).Furthermore,thedis- sectionoftumorsonD21conﬁrmedthelowermassoftumorfor thegroupthatreceivedthesiBcl-2PEGLNCs(Fig.3B).

TheextractionofproteinsintumorsonD6andD21wasper- formedtostudytheinvivotransfectionefﬁcacyofsiRNAPEGLNCs.

WhileanyinhibitionwasobservedatD21,atearlytime,afterdaily repeatedintravenousinjectionsofsiBcl-2PEGLNCs,speciﬁcinhi- bitionwasevidenced onhalfoftheanimals,demonstratingthe potentialinvivotransfectionefﬁcacyofsiRNAPEGLNCsafterintra- venousinjections(Fig.3C).

3.5. Ferrocifentreatmentontumorgrowth

Ferrocifen drugs were tested ona subcutaneous melanoma modelengrafted in nudemice.Repeatedintravenous injections wereperformedforthreeweeks(1IV/Day,5daysconsecutively, 3weeks). FcDiOHLNCs (n=8)and Ansa-FcDiOHLNCs (n=8)at 45mg/kgwerecomparedtoacontrolgroup(n=10)andaposi- tivecontrol(n=10)withasinglei.p.injectionofDTICat100mg/kg

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Fig.2.Cytotoxicityoffree,LNC-encapsulatedferrocifensandsynergiceffectwithBcl-2LNConanSK-Mel28humanmelanomacellline.(A)FreeFcDiOH(greydottedline)and Ansa-FcDiOH(greyline)solubilizedinorganicsolventshowedsignificanttoxicityonamelanomacelllinecomparedtoreferencechemotherapy(DTIC,solubilizedinwater) (blackline).Ansa-FcDiOHhadthelowestIC50valuesestimatedat1␮M(comparedto3␮MforFcDiOH).(B)EncapsulatedFcDiOH(greydottedline)andAnsa-FcDiOH(grey line)intoLNCsinducedasignificantspecifictoxicitycomparedtoblankLNCs(blackline).Thepercentageofcellviabilitywasexpressedasthemeanpercentage±standard errorofthemean(SEM)(n≥3).(C)Control,blank;controlsiRNAandBcl-2siRNALNCswereassociatedtochemotherapeuticdrugssuchasDTIC(white),FcDiOH(grey, striped),andAnsa-FcDiOH(blackstriped)toevidenceapossibleadditiveorsynergiceffectofconcomitanttreatment.Resultsshowedthepotentialofsynergicassociation ofBcl-2withAnsa-FcDiOH.Cellswithoutanytreatmentwereconsideredas100%cellviability.StatisticalanalysiswasperformedbyANOVA1Wpost-hocTukey,**p≤0.01,

*p≤0.05.DTIC:dacarbazine,FcDiOH:ferrociphenol,Ansa-FcDiOH:Ansa-ferrociphenol.

(Fig.4A).Tumorvolumeanalysisdemonstratedthebetterefﬁcacy ofAnsa-FcDiOHLNCstoreducetumorprogressionascompared withFcDiOHLNCsandthecontrolgroup.Interestingly,theeffect ontumoralvolumewascorrelatedwithtumormassanalysiswith areductionof30%forAnsa-FcDiOHLNCs(Fig.4B).

3.6. Concomitanttreatmentonmelanomagrowth

Ansa-FcDiOHandsiRNAco-encapsulatedintoPEGLNCswere testedonsubcutaneousmelanomamodelengraftedinnudemice.

Repeatedintravenousinjectionswereperformedforoneweek(1 IV/day,5daysconsecutively)andfollowedbytwoweeksofAnsa- FcDiOHPEGLNCtreatments(Fig.5).Asigniﬁcanteffectontumor volumewasobservedforconcomitanttreatmentcomparedtothe controlgroupfromDay10untiltheendoftheprotocol(*p=0.05,

ANOVA1W,post-hocTukey).Thevolumereductionwasestimatedat 53%comparedtonon-concomitanttreatment(−30%forsiBcl-2PEG LNCsand−37%forAnsa-FcDiOHPEGLNCs)(Fig.5A).Thesigniﬁcant reductioneffectwasalsoobservedontumormasswithcomparable reduction(50%)comparedtothecontrolgroup(*p=0.05,ANOVA 1W,post-hocTukey)(Fig.5B).

3.7. HepatotoxicityofrepeatedIVofLNCs

TodeterminethehepatotoxicityofsiRNALNCsandthepegy- lated form compared to blank LNCs after repeated injections (D1-D5),bloodsampleswerecollectedregularlyduringtheobser- vationperiod(D0,D2,D4,D9)andtheenzymeactivityofalanine aminotransferase(ALAT)andaspartateaminotransferase(ASAT) werequantiﬁed(Fig.6).Forthecontrolgroup(withoutinjections),

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Fig.3.TumorprogressionafterrepeatedintravenousinjectionsofsiRNALNCsinnudemice.(A)Theevaluationofsubcutaneousmelanomaprogressionwasperformedwith anestimationoftumoralvolumeovertimeaftertreatment.LipidnanocapsuleswithBcl-2siRNA(greydashedline)showedareductionofatleast30%oftumorvolume comparedtocontrolconditions(blackline),blankLNCs(blackdashedline)andsiCTRLLNCs(greyline).(B)TumorsweredissectedandweighedonD21aftertreatment,and tumormassanalysisconﬁrmedthereductionof25%foranimalsreceivingthesiBcl-2LNCscomparedtoothercontrolgroups.(C)Proteinanalysiswasperformedontumors onD6,illustratingtheheterogeneityoftheBcl-2responseinmelanomatumors(n=4).Bcl-2inhibitionwasshowninone-halfoftheanimalsreceivingthesiBcl-2LNCs.

Tumorvolumesandmasswereexpressedasmeanvalues±standarderrorofthemean(SEM)(n≥8).NosigniﬁcantdifferenceswereevidencedusingANOVA1W.

ALATandASATshowedabasalconstantlevelaround55U/L.For eachgroupreceivingLNCs orPEGLNCs, ASATand ALATvalues showedaslightincreasecorrespondingtoamaximumof90U/L betweenD2andD4.AtD9,thebasallevelwasrecoveredforall conditionsindicatingaminorandfullyreversiblehepatolysis.

4. Discussion

Non-specific distribution and resistance mechanisms pose manylimitationsthatmayresultinreducedeffectivenessofthe chemotherapeuticagentsincancertreatmentsuchasmelanoma [35,36].Combinationtherapyhasbeenrecommendeddue toits advantageofincreasedefficacybyadditiveorsynergicpossible effects[37].Numerouspre-clinicalandclinicalstudieshavealready focused on the analysis of innovative combinatory therapeutic [26–28].Thedown-regulationof genesby theuseofsiRNAhas emergedasoneofthemostpromisingstrategies foranticancer therapy [36]. However, theirbiological instabilityin the blood streaminvolvedtheirencapsulationintonanomedicines[18].The encapsulationofsiRNA,inordertohaveanefficientcellularand tumordeliveryofnucleicacids,representsanimportantchallenge

incancerresearch[38].PreviousworkonsiRNALNCsdemonstrated theefﬁcientencapsulationandtheinterfaciallocalizationofsiRNA intoLNCs byinteraction withDOTAP/DOPElipidsand wasevi- dencedbyimaginganefﬁcientpassivetargetingofmelanoma[19].

Inparallel,consideringthepoorwater-solublepropertyofferro- cifendrugs,LNCsrepresentedasuitabledrugdeliverysystemfor thiskindofdrugthanktoitsoilycore[25].AsshowninFig.2,LNCs appearedasapromisingnanomedicinefortheco-encapsulationof poorly-solubleferrocifenandhydrophilicsiRNA,withasigniﬁcant higherdegreeofsiRNAencapsulation.Thisbetterencapsulation efﬁciencycouldbeexplainedbypossibleinteractionsbetweenfer- rocifenandDOTAP/DOPElipidsboostingsiRNAentrapment.

Moreover,siBcl-2LNCsinmelanomacellsinducedsigniﬁcant geneextinctioncomparedtoacommercialagentatlowsiRNAcon- centrationscomparedtoblockco-polymersor cationicmicelles describedintheliterature(16nMvs25nMor50nMrespectively) [38,39].However,nodifferenceonsurvival ratewasevidenced comparedtoblankLNCs.Furthermore,similarstudiesonsiRNABcl- 2transfection,thankstoPEGcoatedlipoplexes,havedemonstrated theincreaserateofapoptoticcellsafterBcl-2extinctionincolorec- talcancercells[40].Inourwork,duetoobserveddecreaseofinvitro

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Fig.4.Tumorprogressionafterrepeatedintravenousinjectionsofferrocifen-baseddrugsencapsulatedintoLNCsinnudemice.(A)Theevaluationofsubcutaneousmelanoma progressionwasperformedwithanestimationoftumoralvolumeovertimeaftertreatment.DTIC(light-greydottedline)andLNCswithAnsa-FcDiOH(blackdashedline) showedareductionof30%oftumorvolumecomparedtocontrolconditions(blackline),blankLNCs(dark-greyline)andFcDiOHLNCs(dark-greydashedline).(B)Thetumors weredissectedandweighedonD21aftertreatmentandtumormassanalysisconﬁrmedthereductionof30%foranimalsreceivingtheBcl-2siRNALNCsand20%foranimals receivingDTIC(i.p.injection)comparedtoothercontrolgroups.Tumorvolumesandmasswereexpressedasmeanvalues±standarderrorofthemean(SEM)(n≥8).No signiﬁcantdifferenceswereevidencedusingANOVA1W.

Bcl-2 leveland invivotumoral volume,theeffectonapoptosis wasnotchecked.However,thepro-apoptoticpotentialofferro- cifenderivativeswasdemonstratedinmelanomacellsbyTUNEL methodandp53/p21activationinpreviousstudy[41].Neverthe- less,apoptosispathwayswithcaspaseanalysescouldbestudiedin furtherexperimentsinordertoclarifytheinvivoobservations.

Interestingly,DTICshowednoinvitrocytotoxiceffects com- paredtoferrocifen.Moreover,theassociationofBcl-2siRNALNC andDTIConmelanomacellsdidnotinduceanyinterestingaddi- tiveeffects. Moreover, experimentson Nudemiceconclude on theabsenceofsynergyeffectbyDTICassociatedwithsiRNABcl- 2LNCsonasubcutaneousmelanomamodel(supplementarydata).

In the same way, the addition of Oblimersen^® (Bcl-2 antisens oligonucleotide)withDTICdidnotimprovetheoverall survival orprogression-freesurvivalandactuallyincreasedsideeffectsin patientswithadvancedmelanoma[17].

Inthisway,asshowninFig.2,theinvitroassaywithnewpromis- ingferrocifenbaseddrugsevidencedinterestinganti-proliferative effects on the melanoma cell line. Ferrocifen derivatives were tested on numerous cancerous cell lines (lung, breast, ovary), includingmelanomaonessuchasB16f10,WM35,showingeffectin survivalrate[41,42].Inourstudy,theAnsa-FcDiOHconcentration, whichinhibitscellgrowthat50%(IC50),wasevaluatedrespectively

at 1␮M and 3␮M for Ansa-FcDiOH and FcDiOH, demonstrat- ingthebetterefficiencyofAnsa-ferrociphenol(Ansa-FcDiOH)on melanomas.Thislastcompoundwasalsodemonstratedasthemost efficientderivativeinabreastcancercelllineandevidencedcom- parableIC₅₀values[24,25].Moreover,theencapsulationintoLNCs appearedtoprovideapromisingtoolwithasimilarefficacythan free ferrocifens.For thefirsttime,theassociationofferrocifen- baseddrugs wastestedwithsiRNAtherapy. Invitrointeresting additive effect on proliferation activity wasshown with Ansa- FcDiOHcontrarytoFcDiOHandDTIC.ThebenefitofAnsa-FcDiOH andBcl-2siRNA associationcouldbeexplainedbytheactionof thesetwoagentsonapoptosispathways.Bcl-2inhibitionresulted intheoligomerizationofpro-apoptoticproteins(Bax/Bad)inorder toinducethereleaseofcytochromeCtomitochondriaresultingin anincreaseofapoptosis.ArecentstudyshowedthatAnsa-FcDiOH actionpromotedthesenescenceandalsotheapoptosispathways contrarytoFcDiOH, whichmainlyfavorthesenescencemecha- nism [43]. Wecan suggestan explanationfor thedifferencein reactivitybetweenFcDiOHandansa-FcDiOH.Thefirstcompound generatesROS(reactive oxygenspecies)in cancercellsand the prodrugleadstotheplanarelectrophilicquinonemethideasthe mainactivemetabolitewhichexhibitssomedegreeofreactivity withredoxtargertenzymessuchasthioredoxinereductaseover-

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Fig.5. ImpactofconcomitanttreatmentassociatingBcl-2siRNAandAnsa-FCDIOHferrocifendrugsintoLNCsintumorprogressioninmice.(A)Theevaluationofsubcutaneous melanomaprogressionwasperformedwithanestimationoftumoralvolumeovertimeaftertreatment.LNCswithAnsa-FcDiOH(greyline),siBcl-2(blackdashedline) confirmedareductionof30%oftumorvolumecomparedtocontrolconditions(blackline).TheCo-encapsulationofBcl-2siRNAandAnsa-FcDiOHintoLNCs(dark-greyline) showedsignificantreductionsoftumorvolumecomparetocontrolconditions.(B)ThetumorsweredissectedandweighedonD21aftertreatmentandtumormassanalysis confirmedthesignificantreductionoftumormassforanimalsreceivingthesiBcl-2/AnsaFcDioHLNCscomparedtocontrolgroups.Tumorvolumesandmasswereexpressed asmeanvalues±standarderrorofthemean(SEM)(n≥8).StatisticalanalysiswasperformedwithANOVA1W,post-hocTukey,*p≤0.05,**p≤0.01.

expressedincancercells[44,45].Ansa-FcDiOHisalsooxidizedin asimilarmannerincancercellsbutthequinonemethidehasnot beencharacterized.Weassumesthatduetotheconstraintspro- ducedinthecycletheplanarstructurecan’tbereachedandthe activespeciesistheradicalshownintheschemewhichisthepre- cursorofthequinonemethide.Thisstructureisstabilizedbythed orbitalsoftheironatom.Thisalphastabilisationiswellknownin theferroceneseries.

Forinvitroassays,LNCs werestudiedwithoutlongchainsof PEGonthesurfacedue toa completeinhibition oftransfection efﬁcacyusingPEGLNCs[46].Onthecontrary,forinvivoexperi- ments,alltestedformulationswerepost-insertedwithlongchains ofPEGtoensure melanomapassive-targetingafter intravenous targeting.According toourresults onmelanoma targeting,the optimalPEGconcentrationof5mMwasdeterminedinfunctionof tumoraccumulation[20].Thissurfacemodiﬁcationconfersstealth propertiesto siRNA LNCs allowinga passive-targeting strategy.

RepeatedinjectionsofBcl-2siRNALNCsinducedapromisingreduc- tionofmelanomatumorgrowthcomparedtocontrolconditions.

Moreover,thevariationofBcl-2extinction,evidencedbyprotein analysisoftumoronD5,couldexplainthelimitedeffectobserved, andwasnon-correlatedtothetumorsize.Thus,thisinvivotrans-

fectionresult canbeimproved: i) byincreasing theamountof deliveredsiRNAwithoptimizationoftheencapsulationprocess [19],andii)byabettertargetingefﬁciencywithactiveorsmart- targetingstrategiesforspeciﬁcmelanomacelluptake[20].

Furthermore,consideringthebiodistributionprofile,theliver appearedtobethemajororganforLNCelimination.Hepatotoxicity isoneofthemajorissuesencounteredwithmanynanomedicines duetocapitationbytheliver[47].ALATandASATquantification revealednosignificantincreasedlevelevenwithrepeatedintra- venousinjections ofsiRNA LNCs, demonstrating theabsenceof hepatotoxicity,asobservedforDNALNCs[48].

Otherwise, Bcl-2 targeting is nowadays controversial as explainedinrecentclinicaltrials[17].Infact,apoptosispathways representacomplexnetworkregulatedbynumerousantiandpro- apoptoticmembers,leadingtodeﬁciencyapoptosisinmelanomas [49,50].Recentpublicationshaveevidencedthefactthatinhibiting proteins(i.e.Bcl-2)couldpromote theexpressionofotheranti- apoptoticmembers(Mcl-1,A1,Bcl-XL)toreplaceBcl-2andensure thesurvivalofmelanomacells[51].Theperspectiveofthiswork consistsinassociatingmultipleapoptosistargetingsuchasBcl-2, Bcl-XLorMcl-1toimprovethesensitivitytochemotherapy.Inorder todothis,thesolutioncouldbethecombinationofdifferentsiRNA

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Fig.6. EvaluationofhepatotoxicitywithALAT/ASATenzymaticquantiﬁcationafterrepeatedintravenousinjectionsofLNCsinmice.Bloodsamplesofanimalsreceivingno injections(control,blackline),blankLNCs(blackdashedline),siRNALNCs(greydashedline),PEGblankLNCs(blackdottedline)orPEGsiRNALNCs(greydottedline)were collectedonD0,D2,D4andD9duringtheobservationperiodtoanalyzethehepatotoxicityofthesesiRNALNCs,representedbyALAT(A)andASATvalues(B)(n=2for untreatedanimalsandn=4fortreatedanimals).ALAT:alanineaminotransferase;ASAT:aspartateaminotransferase;PEG:polyethyleneglycol.

ormicroRNA(miRNA).Infact,miRNArepresentsnaturalsiRNAand can,contrarytosiRNA,considerablyinhibitproteinsynthesis[52].

Intherecentyears,it hasbecomeevidentthatmiRNAsplayan importantroleincancer,includingmelanomas[53].

SK-Mel28modelwasdevelopedbasedonimmortalizedhuman melanomacells[54].Thisspeciﬁccellularmodelwasusedinour worktostudy thesiRNA strategy withrelevant humanmolec- ular pathways and could be adapted on in vivo nude mouse melanomagraftmodel.Ferrocifenshad notyetbeenstudiedin humanmelanomamodel.However,theuseofonlyonecellular modelandalsoofanonimmune-competentmousemodellim- itedtheinterpretationofthesuccessofthistherapeuticstrategy inhumans.Futureexperimentationsshouldbeextendedtoseveral cellularmodelsandalsoasyngenicmousemodelsuchasB16F10 melanomacells.

5. Conclusion

Theassociationofchemotherapyandgenetherapyhasappeared to be a promising opportunity to improve the efficacy of chemotherapyonmelanomas.Theobjectiveofthisworkwasto developefficientnanomedicinesfortheco-deliveryofBcl-2siRNA andferrocifeninmelanomatreatment.Thisco-encapsulationwas efficientlyperformedthankstoLNCswithefficientgenetherapy

andanti-proliferativeeffectsonahumanSK-Mel28melanomacell line. Anin vitrochemosensitive effect ofBcl-2 siRNAwas con- firmed inassociation withAnsa-FcDiOH.Subcutaneous,human, melanoma-bearingnudemicereceivedBcl-2siRNALNCsorAnsa- FcDiOHLNCs, inducinga significantreduction oftumorvolume by 25% and 30% respectively. Finally, the combination of Bcl-2 siRNA and innovative Ansa-FcDiOH anticancerdrug encapsula- tiontogetherintoPEGLNCsinducedsignificanttumorreduction (55%) representinga promisingfuture alternativetreatmentfor melanoma.Thisinterestingresultshouldbevalidatedonotherani- malmodelsassyngenicone(B16F10)totestthistherapyonmice withcompetentimmunesystem.

Conﬂictofinterest

Theauthorsreportnoconﬂictsofinterestinthiswork.

Financialsupport

CP:Frenchfoundation“ARC”(ref.No:SFI20121205972),association“Laliguecontrelecancer”(ref.No:R12164NN).