Biotin-targeted
Pluronic
1
P123/F127
mixed
micelles
delivering
niclosamide:
A
repositioning
strategy
to
treat
drug-resistant
lung
cancer
cells
Annapina
Russo
a,1,
Diogo
Silva
Pellosi
b,c,1,
Valentina
Pagliara
a,
Maria
Rita
Milone
d,
Biagio
Pucci
d,
Wilker
Caetano
c,
Noboru
Hioka
c,
Alfredo
Budillon
d,
Francesca
Ungaro
b,*
,
Giulia
Russo
a,*
,
Fabiana
Quaglia
ba
LaboratoryofBiochemistry,DepartmentofPharmacy,UniversityofNapoliFedericoII,ViaDomenicoMontesano49,80131Napoli,Italy
b
LaboratoryofDrugDelivery,DepartmentofPharmacy,UniversityofNapoliFedericoII,ViaDomenicoMontesano49,80131Napoli,Italy
cResearchNucleusofPhotodynamicTherapy,ChemistryDepartmentStateUniversityofMaringá,Av.Colombo5.790,87020-900,Maringá,Paraná,Brazil d
ExperimentalPharmacologyUnit,DepartmentofResearch,IstitutoNazionaleTumori-IRCCSG.PascaleViaM.Semmola,80131Napoli,Italy
ARTICLE INFO Articlehistory: Received6June2016
Receivedinrevisedform24June2016 Accepted26June2016
Availableonline29June2016 Keywords:
Micelle Pluronic1
Niclosamide Lungcancercells Multidrugresistance
ABSTRACT
Withtheaimtodevelopalternativetherapeutictoolsforthetreatmentofresistantcancers,herewe propose targeted Pluronic1 P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMMfortargetingand imaging,Pluronic1F127 wasconjugatedwithbiotin,while Pluronic1P123wasfluorescentlytaggedwithrhodamineB,inbothcasesatoneofthetwohydroxylend groups.ThisdesignintendedtoavoidanyinterferenceofrhodamineBonbiotinexpositiononPMM surface,whichisakeyfundamentalforcelltraffickingstudies.Biotin-decoratedPMMwereinternalized moreefficientlythannon-targetedPMMinA549lungcancercells,whileverylowinternalizationwas found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustaineddrug releaseinprotein-rich mediaand improvedcytotoxicityin A549cellsas comparedtofreeNCL(P<0.01).TogoindepthintotheactualtherapeuticpotentialofNCL-loadedPMM, acisplatin-resistantA549lungcancercellline(CPr-A549)wasdevelopedanditsmultidrugresistance testedagainstcommonchemotherapeutics.FreeNCLwasabletoovercomechemoresistanceshowing cytotoxiceffectsinthiscelllineascribabletonucleolarstress,whichwasassociatedtoasignificant increaseoftheribosomalproteinrpL3andconsequentup-regulationofp21.Itisnoteworthythat biotin-decoratedPMMcarryingNCLatlowdosesdemonstratedasignificantlyhighercytotoxicitythanfreeNCL inCPr-A549.TheseresultspointatNCL-basedregimenwithtargetedPMMasapossiblesecond-line chemotherapyforlungcancershowingcisplatinormultidrugresistance.
ã2016ElsevierB.V.Allrightsreserved.
1.Introduction
Cancer is the leading cause of mortality worldwide and its incidence continues to rise (Siegel et al., 2015). Although the discovery of novel and effective anticancer drugs is a major research area, it is well known that the development of new chemotherapeuticsinoncologyisalengthyandextremelycostly process.Furthermore,itoftenresultsinonlyslightreductionof
chemotherapy-related side effects and minimal increase in the overallsurvivalrate.Therefore,alternativeapproachesto antican-cerdrugdiscoveryareneeded.
To reduce pharmaceutical research costs and development timelines,considerableattentionhasbeendevotedtothesocalled drug “repositioning” or “repurposing” approach, which entails findingnoveltherapeuticindicationsforalreadyapproveddrugs (AshburnandThor,2004).Somesuccessfulrepositioningstories, suchasthoseofminoxidil,sildenafilandraloxifene,areverywell known.Nevertheless,despiteseveralinitiatives,onlyafewdrugs have been successfully approved for new indications (Novac, 2013).Inrecentyears,thisapproachhasbeenverypromisingto identify approved non-cancer drugs that possess anticancer
* Corresponding authors at: Department of Pharmacy, University ofNapoli FedericoII,ViaDomenicoMontesano49,80131Napoli,Italy.
E-mailaddresses:ungaro@unina.it(F.Ungaro),giulia.russo@unina.it(G.Russo).
1
Theauthorsequallycontributedtothiswork.
http://dx.doi.org/10.1016/j.ijpharm.2016.06.118
0378-5173/ã2016ElsevierB.V.Allrightsreserved.
ContentslistsavailableatScienceDirect
International
Journal
of
Pharmaceutics
activity,thusabbreviatingthedevelopmentprocess(ShimandLiu, 2014).
Somestudieshavereportedtheinhibitoryeffects of niclosa-mide (5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenza-mide)(NCL),ananthelminticdrugapprovedfor useinhumans fornearly50years,onmultipleintracellularsignallingpathways (Balgietal.,2009;Liuetal.,2015;Renetal.,2010;Sacketal.,2011; Xiangetal.,2015).Thesignalingmoleculesinthesepathwaysare eitherover-expressed,constitutivelyactivatedormutatedinmany cancercells,thus making NCLa veryintriguing tool for cancer treatment(Lietal.,2014).Indeed,NCLinhibitstheATP-binding cassetteeffluxpumpABCG2(breastcancerresistanceproteinor BCRP)(Strouseetal.,2013),whichisaproteinabletoconferMulti DrugResistance(MDR)toabroadspectrumofanticancerdrugs (Staud and Pavek, 2005). Furthermore,NCL exhibits antitumor activity in vitro and in vivo by suppressing the expression of lipoproteinreceptor-relatedprotein (LRP), thus inhibiting Wnt/
b
-cateninsignalinginhumanprostateandbreasttumorcells.LRP has been involved in the resistance of ovarian cancer cells to cisplatinandshowntoplayanimportantroleinthetransportof cisplatin(Wangetal.,2004).ThemajorconcernrelatedtothetherapeuticuseofNCLisits verylowsolubilityandconsequentpoordissolutionpropertiesin biological fluids. Furthermore, the bioaccessibility of systemic anticancerdrugstotumourtissueremainslimitedandlargedrug dosesarecommonlyrequiredtoexertatherapeuticeffect,with consequenthightoxicitytonormalcellsandincreasedincidenceof MDR.
Overwhelming attention is nowadays devoted to novel polymeric carriers with tailored properties to attain efficient transportofchemotherapeuticsinthebody(d’Angeloetal.,2014). Pluronic1polymersare amphiphilictriblock copolymersofpoly (ethyleneoxide) (PEO) and poly(propyleneoxide) (PPO), which self-assemble in aqueous media forming core-shell micelles (Kabanov et al., 2002). Due to their ability to accommodate hydrophobicdrugsinthePPOcore,Pluronic1micelleshavebeen proposed to solve solubility issues of several drugs ( Alvarez-Lorenzo et al., 2011). Furthermore, Pluronic1 micelles possess uniquebeneficialfeaturesforcancertherapy,thatis:(i)smallsize (<100nm)usefultoprolongdrugcirculationtimeandtopromote itspassivetargetingtosolidtumors(BatrakovaandKabanov,2008; Zhu et al., 2016); (ii) inhibition of drug efflux transporters overcomingMDR(AlakhovaandKabanov,2014;Alvarez-Lorenzo etal., 2011); iii)presence ofhydroxylend groupsallowingthe covalentbindingoftargetingligandsandimagingagentsproviding multifunctionality(Dingetal.,2011;Jungetal.,2013;Zhangetal., 2011).
RecentlyourgrouphasdevelopedPluronic1P123/F127mixed micellesthatarestableinbiologically-relevantmediaandableto load and stabilize hydrophobic benzoporphyrin derivatives for photodynamictherapyapplication(Pellosietal.,2016).Inanalogy to all PEO-coated nanocarriers, Pluronic1 micelles suffer poor uptake in cancer cells due to increased surface hydrophilicity, whichcanbeadrawbackwhentryingtoaccumulatedrugcargo intracellularly. To overcome this issue, and hopefully improve therapeuticeffectsinabiologicalenvironment,surfacedecoration withsmalltargetingligandsisacommonstrategytoencourage carrieraccumulation in targeted cells (Stylianopoulos and Jain, 2015).Thevitaminbiotin,anessentialmicronutrientthat could givetumor-targetingproperties,hasbeenrecentlyproposedtothis purpose. In fact, several cancer cell lines overexpress biotin-specificreceptorssuchasavidin,neutravidin,andstreptavidinthat facilitate receptor-mediated endocytosis of biotinylated nano-particlesincancercells(Chenetal.,2010;Morral-Ruizetal.,2015). Totrackmicellesinabiologicalmilieu,afluorescentprobecanbe covalently linked to polymer unimers providing fluorescent
micelles.Infact,imaging-basedtechniquesallowunderstanding ofmicelledynamic,monitoringtheirbodydistributionand real-timecelluptake,thusbringingtremendousadvantagesinrelating traffickingtobiologicaleffects(Wangetal.,2014).
Withthisideainmind,herewehavedevelopeda multifunc-tionalplatformforthedeliveryofNCLtocancercellscomprising Pluronic1 P123/F127 mixed micelles (PMM) surface-decorated withthevitaminbiotinastargetingagentandfurtherengineered withafluorescenttagforcellimagingpurposes.Afterformulation studies aimed at assessing the best conditions to entrap and release NCL, NCL-loaded biotin-decorated PMM have been obtained.TheroleplayedbybiotindecorationonPMMselectivity towardlungcancercells,anditsconsequentcytotoxicityincells either sensitive or resistant to cisplatin, were elucidated. We generatedandcharacterizedacisplatin-resistantnon-smalllung cancercellline,providingavaluabletoolforinvestigatingboththe therapeuticpotentialofNCLtoovercomeMDRincancercellsand themechanismthroughwhichthedrugexertsitscytotoxiceffect. 2.Materialsandmethods
2.1.Materials
Pluronic1P123(EO20-PO65-EO20,MW=5750gmol1)andF127
(EO100-PO65-EO100, MW=12600gmol1), biotin, HABA/avidin
reagent,N,N’-dicyclohexylcarbodiimide(DCC), 4-dimethylamino-pyridine(DMAP),niclosamide(NCL),rhodamineBandtrehalose were obtained from Sigma-Aldrich (Italy). Ethanol, dichloro-methane,diethylether,acetoneandacetonitrilewerepurchased fromCarloErbaReagents(Italy).Dimethylsulfoxide-d6 (DMSO-d6), D2O and all deuterated solvents for nuclear magnetic
resonance(NMR)spectroscopywerepurchasedfromMerck(Italy). Alltheotherchemicalswereofanalyticalreagentgradeandused withoutpreviouspurification.
2.2.Synthesisofbiotin-conjugatedandrhodamine-conjugated Pluronic1
Thesynthetic procedurewas adapted fromtheliterature(Li etal.,2010)andwassimilarforbothP123andF127.For biotin-conjugatedPluronic1, F127 (5.0g,0.40mmol) was dissolved in 75mL of dichloromethane followed by the addition of biotin (0.12g,0.48mmol). For Rhodamine-conjugated Pluronic1, P123 (3.0g, 0.40mmol) was dissolved in 75mL of dichloromethane followedbytheadditionofrhodamineB(0.44g,0.48mmol).After that,DMAP(0.004g,0.03mmol)wasaddedtoeachreactionflask andthesolutioncooledto0C.DCC(0.08g,0.40mmol)wasadded dropwise viaa dropping funnel over 30min, and thereactions werecarriedoutfor48hatroomtemperatureunderstirring.The reaction mixtures were then extracted with a NaHCO3 water
solution(10%v/v).Afterthisstep,theorganicphaseswerefrozen overnightandtheinsolublesubstanceswereremovedbyfiltration. Theorganicsolutionswerethenprecipitatedtwiceincolddiethyl ether. The functionalized polymers were filtered and dried overnightundervacuum.
Biotin-F127reactionyield=82%.1HNMR(300MHz,DMSO-d6,
TMS),
d
(ppm):1.000-1.485(s,–CH3b),1.493(m,H5,2H),2.200(m,H3,2H),2.821(m,H5,2H),3.105(m,H4,2H),3.260–3.619(m,
–O-CH2-CH2-a),4.138(m,H60,1H),4.300(m,H6,1H),6.363(s,HN2,1H) e6.437(s,HN1,1H).
Rhodamine-P123 reaction yield=68%. 1H NMR (300MHz,
DMSO-d6, TMS),
d
(ppm): 1.000-1.126(s, –CH3 b),1.209(t, N-CH3,12H),3.260-3.549(m,–O-CH2-CH2-a),3.641(d,N-CH2,8H),6.980(m,H2-H7-H4-H5,4H),7.094(d,H8-H1,2H),7.473(dd,H60,
2.3.Preparationofmicelles
Unloaded and NCL-loaded Pluronic1 mixed micelles (PMM) werepreparedbythin-filmhydrationmethod(Zhangetal.,1996). Briefly,differentamountsofaP123/F127mixture(2:1w/w)were dissolvedinethanolinaround-bottomflasktogivesolutionsat different concentrations (10–50mgmL1). For drug-loaded micelles, different amounts of NCL (0.1–1.0mgmL1) were dissolvedinethanoland addedtoPluronic1solution.Then, the solvent was removed by rotary evaporation at 50C for about 20min. After that, the dried film was hydrated with filtered distilledwaterand thesamplewassonicatedfor5min.Micelle dispersion was filtered through Phenex1-RC 0.22
m
m filters (Phenomenex, Italy) to remove the unincorporated drug or possible large cylindrical aggregates formed by P123. When necessary,theresultingsolutionwasfreeze-driedfor24h.ForBiotin-decoratedPMM(Bio-PMM),biotin-conjugatedF127 (20%of totalF127 wt)was employed.For RhodamineB-tagged PMM (Rho-PMM or Bio/Rho-PMM) rhodaminated-P123 (1% of total P123 wt)was used togive rhodamine Bconcentrationof 5.0
m
M.Inthecase ofBio/Rho-PMMmicellesat10mgmL1,for example,2.66mgofF127and0.66mgofbiotin-conjugatedF127, 6.60mgofP123and0.066mgofrhodamineB-conjugatedP123(a stocksolutioninethanolofallthecomponents)wereused.Recoveryyieldoftheproductionprocesswasevaluatedonan aliquotof PMMdispersionby weightingthesolid residue after freeze-drying.Resultsareexpressedastheratioofactualweightto thetheoreticalpolymerweight100.Formulationoptimization wasassessedonthebasisofmicellesize,polydispersityindexand NCLencapsulationefficiency.
2.4.Micellecharacterization
Hydrodynamic diameter (DH), polydispersity index (PI) and
zetapotentialofmicellesweredeterminedusingaZetasizerNano ZS(MalvernInstrumentsLtd.,UK).Thefreeze-driedformulations were dispersed in Milli-Q water and measurements were performedat37.0Con90 angle.Resultsarereportedasmean of three separated measurements on three different micelle batches(n=9)standarddeviation(SD).Bio/Rho-PMM morphol-ogywas evaluatedby Transmission ElectronMicroscopy (TEM, JEOLJEM-1400,JeolLtd.-Japan)afternegativestainingwitha2% phosphotungsticacidsolution.
The critical micelle concentration (CMC) of the Pluronic1 mixtureinaqueousPBSsolutionswasdeterminedbyfluorescence measurements using pyrene as fluorescent probe ( Kalyanasun-daramand Thomas,1977).An aliquot(5
m
L) of a pyrenestock solutioninacetone(3.610–4molL1)wasaddedintoaseriesof testtubes,andtheacetonewasevaporated.Followingthis,micelle solutionsatvariousconcentrations(3.0mL)wereaddedtoeach testtubeandsonicatedfor2h([pyrene]=6.0107molL1).The excitationspectrawererecorded(from200nmto360nm)with theemission wavelength fixed at 372nm. TheCMC was deter-mined by I337/I334 intensity ratios from excitation spectra ofpyrene.
TheamountofbiotinavailableonthesurfaceofBio-PMMwas quantifiedby a competitive binding assay(HABA/avidin) (Bian etal.,2012).RhodamineB-conjugatedP123wasnotincludedinthe micellestoavoidinterferenceinHABA/Avidincomplexabsorption. ThepowderedHABA/avidinreagentwasreconstitutedwith10mL of deionized water. Thereafter, 900
m
L of HABA/avidin solution werepouredintoa1mLcuvette.Theabsorptionspectrumfrom 450nmto700nmwascollectedandtheabsorbanceatl
=500nm wasrecordedandindicatedasA500HABA/Avid.Then,100m
LofBio-PMMwereadded intothis solution.Afterextensivemixing,the
absorbance at
l
= 500nm was recorded and indicated asAHABA=Avidþsample500 .Theabsorbanceoftheblankmicellesolutionat
l
=500nmwasindicatedasAblanksample500 .Theamountofthebiotinactuallyexposedontomicelleswas calculatedbythefollowingequation:
m
mol biotin mL ¼ 34D
10 ð1Þ whereD
A500¼0:9A HABA=Avid 500 A HABA=Avidþsample500 . The resultsare reportedasmeanofthreeseparatemeasurementsSD. 2.5.Niclosamideloadingandrelease
NCL-loadingwasevaluatedbymeasuringtheUV–vis absorp-tion after diluting the freshly prepared NCL-loaded PMM formulationsinethanol(1:100v/v).
InvitroreleaseofNCLfrommicelleswasassessedbyadialysis method. Aknown amountof NCL-loadedmicelles (40mg)was dispersedin1mLofPBSorDulbecco’sModifiedEagle’sMedium (DMEM)supplementedwith10%offetalbovineserum(FBS)and placed in a dialysis bag (MWCO=3500Da, Spectra/Por1). The sampleswereplungedin5mLof10mMphosphatebufferatpH7.4 containing137mMNaCland2.7mMKCl(PBS)containing0.5%v/v ofpolysorbate80inordertoensuresinkconditionsandtoavoid NCLprecipitationandkeptat37C.Atselectedtimeintervals,1mL ofreleasemediumwaswithdrawn,replacedwithanequalvolume offreshmediumandanalysed.ReleaseprofileoffreeNCL(0.7mg dissolvedin1mLofethanolorDMEMwith10%FBSmedium)are reportedforcomparison.Resultsareexpressedasmeanrelease% overtime(n=3)SD.
TheamountofNCLinthesampleswasmeasuredat331nmona Shimadzu1800spectrophotometer(Shimadzu,Italy)ina1.0cm quartz cuvette as previously reported (Costabile et al., 2015). Briefly,a calibrationcurvewasobtainedbyplotting absorbance versustheconcentrationofNCLstandardsolutionsinethanol.The linearityoftheresponsewasverifiedoveraconcentrationrangeof 0.2–10
m
g/mL(r2=0.998).Thelimitofdetectionandquantificationwere0.24
m
g/Land0.80m
g/L,respectively.Unloadedmicellesand releasemediahadnegligibleabsorptionat331nm.2.6.Cellcultures
Adenocarcinoma humanalveolarbasalepithelial (A549) and mouseembryofibroblast(NIH3T3)celllineswerepurchasedfrom AmericanTypeCultureCollection(Rockville,MD,USA)andwere authenticatedbyLGCStandards(SestoSanGiovanni,Italy).A549 and NIH3T3 cells were cultured in DMEM with glutamax (Invitrogen, Carlsbad, California) supplemented with 10% FBS, 2mML-glutamineandpenicillin-streptomycin50U/ml.
2.7.Micelleuptakebyfluorescencemicroscopy
A549andNIH3T3cellswereplatedoncoverslipsatadensityof 2104cellsperwellin12-wellplates.Then,cellswereincubated
withRho-PMMandBio/Rho-PMM(0.5mg/mL)for1,4and24h. Intracellular localization of micelles was monitored using a fluorescent microscope (Leica Microsystems GmbH, Wetzlar, Germany) to visualize 40,6-diamidino-2-phenylindole (DAPI) (345/661nm) and Rhodamine-labelled micelles (557/571nm). FluorescenceimageswerecollectedandprocessedbyAxiovision 4.8.1software.Imageswereprocessed,mergedandreconstructed usingAdobePhotoshopsoftware.Thescalebarsonalltheimages correspondto10
m
m.2.8.Cisplatin-resistantcellselection
Acisplatin-resistantA549cellline(CPr-A549)wasgeneratedby continuouslyculturingthedrug-sensitiveparentalcelllineA549in amediumcontainingincreasingconcentrationsofcisplatin(from 0.5
m
Mto14m
M)inastepwiseprocedureover10months.After10 months,theselectedcellswere‘pooled',inordertoavoidclonality, andweretestedtoevaluatetherateofdrugresistance,calculated astheresistanceindex,RI=IC50CPr-A549/IC50A549.Bothcelllinesweregrownin DMEMmedium containing10% heatinactivated FBS,50U/mlpenicillin,500
m
g/mlstreptomycin,20mMHepes(pH 7.4)and4mMglutamine.Thecells weregrowninahumidified atmospherecomposedof95%airand5%CO2at37C.2.9.Cellviabilityassays
ForMTTassay,A549,CPr-A549andNIH3T3cellswereseeded onto 96-well plates (2104 cells/well) and incubated with micelles for 24 and 72h. Then, cell viability was evaluated as mitochondrialactivityusingtheMTTassayaspreviouslyreported (Maiolinoetal.,2015).Theabsorbancewasmeasuredat540nm usingamicroplatereader(LabsystemsMultiskan,MS).
ForsulforhodamineB(SRB)colorimetricassay,cellviabilityof A549 and CPr-A549 cells was measured after 96h of drug treatment in 96-well plates, as described previously (Milone etal.,2013).
2.10.Proteinextractionandwesternblotting
Cells were grown and treated as indicated above, collected, lysedandseparatedonSDSpolyacrylamideelectrophoresisgels, andthenproteinsweretransferredtonitrocellulosemembranes (d’Emmanueleetal.,2015).Westernblottingassaywasperformed asdescribedpreviously(DeFilippisetal.,2016)usingantibodies anti-rpL3(Primm,Milan,Italy),anti-p53, anti-p21,anti-LRP and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, California), anti-ABCG2(CellSignaling),anti-MRP1andanti-MDR1(Abcam). 2.11.Clonogenicassay
Forclonogenicassay,CPr-A549cellswereplatedintriplicateat 4103 in6-wellmultidishes. At10days,colonies werestained
with 1% methylene blue in 50% ethanol. Percent survival was normalizedtotheobservednumber ofcoloniesgeneratedfrom untreatedcells.
2.12.Statisticalanalysis
Errorbarsrepresentmeanstandarderrormean(SEM)from n=3biologicalreplicates. Statisticalcomparisonsweremadeby one-way ANOVA followed by Bonferroni’s test for multiple comparisons. * #P<0.05, ** ## P<0.01, *** P<0.005. P>0.05
wasconsiderednotstatisticallysignificant. 3.Results
3.1.Pluronic1functionalizedwithbiotinorrhodamineB:synthesis, assemblingpropertiesandcelluptake
Multifunctional micelles werepreparedfromF127and P123 reactedathydroxylendgroupswithbiotinastargetingagentor rhodamineBasfluorescenttag,respectively.1HNMR spectraof
modified polymers (Fig. 1A and B) showed the main peaks correspondingtothePPO (1.0–1.2ppm) and PEO (3.2–3.6ppm) segments.Most of biotin signals were overlapped to polymers signals(Fig.1A)althoughthemultiplesignalsat4.138ppm(H60),
4.300ppm (H6) and thesinglet signals at 6.363ppm (HN2)and
6.437ppm (HN1) confirmed biotin-F127 conjugation. For the
rhodamine-conjugatedP123(Fig.1B),thearomaticsignalsabove 7ppmconfirmRhodamineconjugationtoP123molecules.
Suggestionthatmodifiedpolymerscouldself-assemblewith unmodifiedonestoformmixedmicelleswasgivenbycomparing
1HNMRinDMSO-d6andD
2Oassolvents.Infact,DMSO-d6isa
goodsolventforallcomponentsandsignals referringtoallthe speciescouldbedetected(Fig.1AandB).Nevertheless,D2Oisa
good solvent for biotin, rhodamine B and PEO blocks while lipophilic PPO blocks are poorly soluble in water, driving the formationofthemicellecore.Infact,NMRsignalsoriginatingfrom hydrophobic PPO blocks weresuppressed inwater due to PPO restrictedmotioninthemicellecore(Fig.1C).
Bio-PMMwerepreparedat2:1(w/w)P123andF127ratioon thebasisofourpreviousstudy(Pellosietal.,2016).TheCMCof P123/F127mixtureinthepresenceofBiotin-conjugatedF127was 0.0031%w/V,asevaluatedbypyrenemethod(Fig.2A). Analogous-ly, addition of Rho-conjugated P123 did not alter Bio-PMM properties(datanotshown).Theavailabilityofbiotinonmicelle surfacewasalsoquantifiedbymeasuringtheabsorbanceofthe HABA/avidincomplexat500nm(Fig.2B)(Bianetal.,2012).The theoreticalamountofavailablebiotinonBio-PMMwas1.25
m
mol mL1,correspondingto10%ofbiotinatamicelleconcentrationof 10mgmL1.Theactualavailableamountofbiotinwas1.04m
mol mL1,whichmeansthatapproximately8.3%ofthemicellesurface iscoveredwithbiotin.Bio-PMMwerealmostsphericalasshownin theTEMimagereportedinFig.2C.Theuptakeandthesubcellulardistributionofunloaded Rho-PMMandBio/Rho-PMMwereassessedinbiotinreceptor-positive A549cellsandinanon-cancerouscelllineNIH3T3ascontrol.After atime-dependentincubationwithmicelles(1,4and24h),thecells werestainedwithDAPIandobservedbyfluorescencemicroscopy. InA549cells,thelargemajorityofthefluorescenceassociatedto Bio/Rho-PMM was distributed in the cytoplasm, confirming micelleinternalization(Fig.3).Thequantificationoffluorescence revealed a time-dependent uptake of the micelles. In normal NIH3T3 fibroblasts, a very limited internalization of both Rho-PMMandBio/Rho-PMMwasfound(Fig.4).
3.2.NCL-loadedPMM:propertiesandcytotoxicityinA549cells Table 1 summarizesthe physicochemicalcharacteristicsand drug-loadingparametersofNCL-loadedPMM.Ineachcase,PMM withincreasedPIandahydrodynamicdiameterwithintherange 25–35nmwereachievedascomparedtounloadedPMM(Table1). Zetapotentialvalueswereslightlynegativeindependentlyofthe drugamountloaded(Table1)asgenerallyfoundforuncharged PEO-coatednanocarriersduethehighelectronicdensityofoxygen (Conteetal.,2014).Ofnote,bothmicellesizeandchargewerenot affectedbythepresenceofthesmallandneutralbiotinmolecule on micelle surface at present conditions. The encapsulation efficiencystronglydependeduponPluronic1/NCLratiobyweight. Highratiosresultedingoodencapsulationefficiencieswhilethe oppositeeffectwasobservedatlowerPluronic1/NCLratios,likely due to drug precipitation during preparation. The optimal formulationconditionforNCLencapsulationwas at40mgmL1 forPluronic1mixtureand0.7mgmL1forNCL.Blankmicellesat samepolymerconcentration(controlexperiment)presentedasize of29.5nm(PI=0.142)andzetapotentialof3.81mV.
ReleaseprofileofNCLfromNCL-loadedBio-PMMinPBSatpH 7.4 was evaluated bydialysis method(Fig.5).NCL displayeda biphasicreleaseprofilewithaninitialburst(first6h)followedbya sustainedreleasephaselastingupto48h.Thedrugcouldfreely diffusethroughthedialysismembraneasdemonstratedincontrol experiments.ReleaseprofileintheDMEMsupplementedwith10%
ofFBSfollowedabiphasicpatternagain,whichoccurredatarate slowerthaninPBS(Fig.5).
Inthelightofthepromisingtechnologicalfeatures,wetested thecytotoxicity of theoptimized NCL-loadedmicelles in A549
cancer cells and normal NIH3T3 fibroblasts. To this aim, cytotoxicityofNCL-loadedmicellesincubatedinawiderangeof concentrations(1.3–112
m
gmL1correspondingto0.07–6m
MofFig.1.1
Fig.3. FluorescencemicroscopyimagesofA549cellsafterincubationwithRho-taggedmicelles.A549cellswereincubatedwithRho-PMMandBio/Rho-PMM(0.5mg/mL)for 1,4and24h.CellnucleiwerestainedwithDAPI.FluorescenceimageswerecollectedandprocessedbyAxiovision4.8.1software.Allmeasurementswerenormalizedtothe fluorescenceofBio/Rho-PMMincellmediumsetas100%.BarsrepresentmeanvaluesSEMofexperimentsdoneintriplicate.*P<0.05,***P<0.005.
Fig.2.PropertiesofBio-PMM.(A)Fluorescenceexcitationspectraofpyrene(6.0107molL1)asafunctionoftheconcentrationofBio-PMMinPBSatpH7.4and37C.The
insetshowstheplotoftheintensityratio(I337/I334)fromthefluorescenceexcitationspectraofpyreneat372nmasafunctionoflogarithmconcentrationofPMM.(B)UV–vis
NCL)wasevaluatedafter72hexposurebyusingMTTassayand comparedtothatoffreeNCL(Fig.6).
Results indicated that NCL-loaded Bio-PMM micelles were significantly morecytotoxic than NCL and NCL-loaded PMM in A549 cells (Fig. 6A). No significant difference in cytotoxicity betweenfreeNCLandNCL-loadedmicelleswasfoundinNIH3T3. UnloadedPMMandBio-PMMdidnotshowanycytotoxicitytoward bothcelllinesatanyconcentrationtested(Fig.S1,Supplementary material).DatafromMTTat24hhighlightthatcytotoxicitywas
again significantly higher for NCL-loaded Bio-PMM (Fig. S2, Supplementarymaterial).
3.3.InvitroactivityofNCL-loadedPMMagainstresistantlungcancer cells
Acisplatin-resistantsublinederivedfromtheparentalsensitive cell line A549 was successfully established. A549 cells were continuouslyselectedwithcisplatinoveraperiodof10monthsin
Fig.4.FluorescencemicroscopyimagesofNIH3T3cellsafterincubationwithRho-taggedmicelles.NIH3T3cellswereincubatedwithRho-PMMandBio/Rho-PMM(0.5mg/ mL)for1,4and24h.CellnucleiwerestainedwithDAPI.FluorescenceimageswerecollectedandprocessedbyAxiovision4.8.1software.Allmeasurementswerenormalized tothefluorescenceofBio/Rho-PMMincellmediumsetas100%.BarsrepresentmeanvaluesSEMofexperimentsdoneintriplicate.
Table1
CompositionandpropertiesofNCL-loadedBio-PMM.Resultsreportedasaverageofthreeseparatedmeasurementsonthreedifferentbatches(n=9)SD. Pluronic1 (mgmL1)a NCL (mgmL1) ratio Plur/NCL
Yield(%) NCLactualloadingSDb
(EESD)c
MeanDH
(nmSD)
P.I. Zetapotential (mVSD) 10 0.1 100 95 0.9130.043 (91.51.2) 27.41.8 0.132 3.910.67 10 0.2 50 89 1.330.10 (67.81.8) 35.92.4 0.266 4.591.15 10 0.3 33 89 1.720.26 (59.03.1) 28.24.7 0.345 3.331.89 20 0.1 200 92 0.495 0.014 (99.2 1.2) 25.21.2 0.138 4.010.88 20 0.3 67 85 1.240.052 (88.43.4) 24.22.3 0.178 3.891.01 20 0.5 40 91 1.620.073 (66.44.1) 20.23.7 0.351 5.492.48 30 0.5 60 90 1.540.38 (93.92.0) 28.42.5 0.152 4.621.61 40 0.7 57 94 1.580.55 (91.91.9) 31.81.7 0.131 3.371.08 50 0.7 71 88 1.290.27 (93.44.1) 26.52.5 0.129 5.492.07 50 1.0 50 93 1.810.49 (92.34.1) 33.43.9 0.246 2.210.88 a
P123/F127mixture2:1w/wwherebiotin-conjugatedF127wasusedat20%ofF127mass.
b Actualloadingisexpressedastheamount(mg)ofdrugencapsulatedper100mgofBio-PMMSD.
order to generate corresponding age and passage-matched cisplatin-resistantcellline,designatedasCPr-A549.Maintenance of the resistant subline was continued at 14
m
M. The SRBproliferation assay demonstrated an increased resistance to cisplatin-induced cell death (Fig. 7A). Specifically, the drug-adapted CPr-A549 cell line showed a significantly higher IC50
compared with parental A459 cells (13.34 versus 3.80.4, respectively;P<0.05),resultinginmorethan3-foldresistanceto cisplatin (Table 2). In addition, we demonstrated a significant increase in the IC50 of CPr-A549 cells compared to A549 cells
(Table2)tootherconventionalchemotherapeuticdrugssuchas docetaxel(Fig.7B), 50-deoxy-5-fluorouridine(50-DFUR)(Fig.7C) and5-fluorouracil(5-FU)(Fig.7D).Theseresultsindicatethat CPr-A549 cells show resistance to multiple drugs representing an interestingtooltostudyMDRinlungcancer.
TheeffectoffreeNCLonCPr-A549celllineviabilitywasfirst investigated.Tothispurpose,cellswereexposedtoawiderangeof NCL concentrations (ranging from 0.07 to 6
m
M) and in vitro cytotoxicityevaluatedbyusingMTTassay.Fig.8Ashowsthe dose-responseeffectafter72hoftreatmentwithfreeNCLgivinganIC50valueof1.8
m
M.ThisresultdemonstratedthatNCLcanovercome resistancetodifferentchemotherapeuticsinCPr-A549cells.TogoindepthintomolecularmechanismunderlyingMDRin Crp-A549cells,theexpressionprofileofATPbindingcassette(ABC) effluxpumpsABCG2,lipoproteinreceptor-relatedprotein(LRP) wasevaluated.Theexpressionlevelofmulti-drugresistance 1/P-glycoprotein(MDR1)andMDR-associatedprotein1(MRP1)was alsoanalyzed.Westernblottinganalysisshowednodifferencein theexpressionofsuchproteinsinCPr-A549cellsascomparedwith theparentalA549cells(Fig.8B).
ToinvestigatewhetherNCLcytotoxiceffectonCPr-A549cells wasassociatedtonucleolarstress,proteinextractsfromCPr-A549 cells treated or not with free NCL were analyzed by western blottingfortheexpressionprofileofrpL3anditstargetp21protein. WefoundthattheresistanceofA549cellstocisplatincorrelatedto the loss of rpL3 expression(Fig. 8C). NCL treatment caused a significant increaseofrpL3levelthat wasassociatedtothe up-regulationofp21.
Theantiproliferativeactivity ofNCL-loaded micellestowards multidrug-resistantCPr-A549cancercellswasfinallyinvestigated (Fig.9).After3daysof treatment, invitrocytotoxicity was first evaluated by MTT assay. Extrapolationfrom the dose-response curvedemonstratedthatcytotoxicityofNCL-loadedBio-PMMwas higherthanthatofNCL-loadedPMM(andfreeNCL)dependingon thedose(Fig.9A).Toexcludedefinitivelythattreatedcellsretained anycapacitytodivideandtoproliferate,theinvitroeffectivenessof NCL-loadedBio-PMM against CPr-A549 cells was also assessed through a clonogenic assay. The assay was performed after treatment of CPr-A549 cells with 0.3, 0.9 and 1.8
m
M of NCL-loadedBio-PMMfor72h.AscanbeseeninFig.9B,after10daysthe colony number was strongly reduced upon exposure to drug-loaded Bio-PMM confirming the ability of NCL to inhibit clonogenicity.4.Discussion
Anincreasingresistanceofcancercellstothetreatmentwith commonlyusedchemotherapeuticdrugshasraisedtheurgencyin the search for newanti-cancer agents. Several drugs originally approvedforindicationsotherthancancertherapyhaverecently beenfoundtoexertacytotoxiceffectoncancercells(ShimandLiu, 2014; Stenvang et al., 2013). Cisplatin represents the first line chemotherapyforthetreatmentofavarietyoftumors,including non-smallcellslungcancer(NSCLC),andplatinumresistanceisa major limitation in the treatment of NSCLC. Recent studies focusingontheanthelminticdrugNCLdemonstrateitsabilityto overcomedrugresistanceindifferentcancers(Lietal.,2013;Liu etal.,2015,2014). 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 NCL release (%) Time (hours) NCL Bio-PMM in PBS NCL in PBS NCL Bio-PMM in DMEM NCL in DMEM
Fig.5.ReleaseprofilesofNCLandNCL-loadedBio-PMMdispersedinPBSandin DMEMsupplementedwithFBS10%.FreeNCLisreportedascontrol.Theexternal mediumusedfordialysiswasPBSwithpolysorbate80(0.5%v/v)atpH7.4andat 37C.NCLconcentrationinthebagwas0.7mgmL1.Dataarereportedasmeanof
threeindependentexperiments(n=3)SD.
Fig.6.InvitrocytotoxicityoffreeNCLandNCL-loadedmicellestowardA549(A) andNIH3T3(B)cells.ThecellswereexposedtoincreasingconcentrationsofPMM (1.3–112mgmL1correspondingto0.07–6mMofNCL)orNCLinDMSO(0.07–6mM) for72h.Afterincubation,cellviabilitywasevaluatedusingtheMTTassay.Thecell viabilityfromuntreatedcellswassetto100%.Resultsarepresentedaspercentage (meanSEM)(n=3)ofthecontrolcells.*,
**NCL-loadedBio-PMMvsNCL-loaded PMM.#,##
NCL-loadedBio-PMMvsNCL.*#
P<0.05,**##
Despitepromising,thetranslationofNCL-basedchemotherapy invivorunsintospecialdifficulties,mainlyrelatedtotheverypoor solubility profile of NCL in biological fluids. To overcome NCL solubilityconstraints,Pluronic1P123/F127mixedmicelles(PMM) fortargeteddeliveryofNCLtolungcancercellswerebuiltup.
Inapreviouswork,wedemonstratedthatPMMmadeof2:1(w/ w)mixturesofP123andF127displayhighstabilitytodilutionand canloadhighlyhydrophobicbenzoporphyrinderivatives(Pellosi etal.,2016).Here,PMMweresurface-engineeredtoencouragecell internalizationbyexposingbiotinontheirsurface.PMMwerealso madefluorescentbylinking RhodamineBtomicellesurface to tracktheirtraffickinginsidecells.Biotinand rhodamineBwere linkedtoPluronic1unimersattheirhydroxyl-terminationusing carbodiimidechemistry(Lietal.,2010).Wedecidedtoconjugate
biotintoF127,bearinglongerPEOblocks,whilerhodamineBwas linkedtoP123,withshorterPEOchains.Throughthisdesign,itwas expected that targeting ligandexposition on micellesurface is attained andany interferencebetweenbiotinand rhodamineis minimized, which is a key prerequisite for micelle-receptor recognition. NMR analysesprovidedinsight intotheoccurrence oftheself-assemblyofthetwoPluronicderivatives,indicatingthe formation of a well-defined core-shell structure in aqueous solutionwherea“water-protected”PPOcoreissurroundedbya PEOhydrophiliccoatingexposingbiotinastargetingelementand rhodamineBasfluorescenttag.Indeed,complexformationof Bio-PMMwithavidinclearlyindicatesthatbiotinwassurface-exposed andpotentiallyabletointeractwithreceptorsoncellmembranes (Bian et al., 2012). Meanwhile, the reasonably low CMC value indicated a good stability of targeted PMM to dilution being promisingfortheirpotentialapplicationsasdrugcarrier.
Aiming at demonstrating thesoundness of this approach to promote NCLaccumulation in cancercells, the uptake and the subcellulardistributionofunloadedRho-PMMandBio/Rho-PMM wereassessedin biotinreceptor-positivelung A549cancercells andinanon-cancerousNIH3T3cellline.Resultsdemonstrateda higher amount of biotin-decorated PMM in cancer cells as compared to normal cells. Thus, the overexpression of biotin receptorsonthesurfaceofA549cancercellsrelativetonormal NIH3T3 cells, induced a higher micelle uptake by
receptor-Fig.7.Developmentofcisplatin-resistantNSCLCcellline.SensitiveA549cellsandCPr-A549cellsweretreatedwithincreasingconcentrationsof(A)Cisplatin,(B)Docetaxel, (C)5-FUor(D)50-DFURfor96handcellgrowthassessedbySRBcolorimetricassay.Cellgrowthisexpressedaspercentageofcontrolforeachtimepoint.Valuesarethe
meanSDfromatleastthreeindependentexperimentsperformedinquadruplicates.*P<0.05,**P<0.01.
Table2
AntiproliferativeeffectofanticancerdrugsonsensitiveA549andcisplatin-resistant A549cells(CPr-A549).Resistanceindex(RI)isreportedasIC50CPr-A549/IC50A549.
IC50(mM) A549 CPr-A549 Cisplatin 3.80.4 13.34(RI=3.5) Docetaxel 0.541030.05103 >64103(RI>164) 5-FU 2.550.07 8.90.28(RI=3.45) 50-DFUR 4.650.35 17.983.7(RI=3.97)
mediatedendocytosis(Heoetal., 2012;Jungetal., 2014;Maiti etal.,2013).
Thefeasibilityoftheproposedapproachforselectivedeliveryof NCL in lung cancer cells was then assessed. To this purpose, preliminarystudiesweredevotedtothedevelopmentofBio-PMM, affordinghighencapsulation efficiency ofNCLat theminimum Pluronic1/NCLratio(i.e.higherdrugloadingformicelle).Thekey
factor for formulation optimizationwas thedependence of the encapsulation efficiency on Pluronic/NCL weight ratio. Values between50and60wereconsideredsuitablefora therapeutically-relevantsystem.Besidesaslightsizeincreaseafterentrapmentof thedrug,likelyascribabletodrugentanglinginthePPOcore,the micelleswerestillsmallenoughfortumor-specificaccumulation viatheEPReffect(BatrakovaandKabanov,2008).Furthermore,the
Fig.8. EffectofNCLinCPr-A549cells.(A)InvitrocytotoxicityofNCL(0.07–6mM)towardCPr-A549cellsafter72h.CellviabilitywasevaluatedbyMTTassay.Thecellviability fromuntreatedcellswassetto100%.Resultsarepresentedaspercentage(meanSEM)(n=3)ofthecontrolcells.*P<0.05,**P<0.01.(B)ExpressionprofileofMRD1,ABCG2, MRP1andLRP,inCpr-A549cells.ProteinextractsfromA549cells(control)andCPr-A549cellswereanalysedbywesternblottingbyusinganti-MRD1,anti-ABCG2, anti-MRP1,anti-LRPandanti-GAPDHtonormalize.(C)AnalysisofrpL3andp21expressionuponNCLtreatmentinCpr-A549cells.ProteinextractsfromA549cells(control)and Cpr-A549cells,untreatedortreatedwithfreeNCL,wereanalysedbywesternblottingbyusinganti-rpL3,anti-p21andanti-b-actintonormalizeresults.Quantificationof signalsisshown.BarsrepresentmeanvaluesSEMofexperimentsdoneintriplicate.***P<0.005.
developeddeliveryplatformallowedforaslowreleaseofNCL,also inacomplexprotein-richmilieu,suchasthecellculturemedium employedforinvitroexperiments.
Prompted by these encouraging findings, the in vitro anti-proliferative effect of the selected NCL-loaded Bio-PMM was investigatedinlungcancercells,aswellasinnormalcells. NCL-loadedBio-PMMdisplayedanenhancedcytotoxicactivityinA549 cellsascomparedtohealthycells,whichcorrelatedwellwiththeir higherintracytoplasmicaccumulation.
Forabetterunderstandingofthetherapeuticpotentialof NCL-loadedBio-PMMand toinvestigate the molecularbasis of NCL cytotoxicactivity,wesuccessfullyestablishedacisplatin-resistant lungcancersublinederivedfromtheparentalsensitivecellline A549 (CPr-A549 cells), which demonstrated resistance also to otherconventional chemotherapeutics. Drug resistance may be
due todecreasedinfluxorincreasedeffluxofdrugs (Rabik and Dolan,2007).SeveralABCtransportereffluxinhibitorshavebeen described (Schinkel andJonker,2003)and itis wellestablished thatthetransmembraneATPbindingcassette(ABC)effluxpumps ABCB1(P-glycoprotein,P-gp),ABCC1(MDR-associatedprotein1, MRP1),andABCG2(breastcancerresistanceprotein,BCRP)playa crucial roleinthedevelopment ofdrugresistance(Eckford and Sharom,2009).Ourresultsclearlyindicatethatthedevelopment ofMDRinCPr-A549cellswasnottheresultoftheoverexpression of neitherABCG2and LRP,norMDR1 andMRP1suggestingthe involvementofotherpathways.
Recentlyourstudiesfocusedontheextraribosomalfunctionsof ribosomal proteins (Russo et al., 2011, 2010), with particular attention to their role in chemotherapyand chemoresistance (Pagliara et al., 2016). Specifically, we demonstrated that
Fig.9.InvitrocytotoxicityofNCL-loadedmicellesinCPr-A549cells.(A)CPr-A549cellswereexposedtoincreasingconcentrationsofmicelles(1.3–112mg mL1 correspondingto0.07–6mMofNCL)for72h.Afterincubation,cellviabilitywasevaluatedbyMTTassay.CytotoxicityofNCLfromFig.8isreportedtofacilitatecomparison. Thecellviabilityfromuntreatedcellswassetto100%.Resultsarepresentedaspercentage(meanSEM)(n=3)ofthecontrolcells.*P<0.05,***P<0.005.(B)Clonogenic assayonCPr-A549cellstreatedwith0.3,0.9and1.8mMofNCLfor72horuntreated.After10days,colonieswerestainedwithmethyleneblue,countedandphotographed.Bar chartindicatingclonogenicgrowthisshown.*P<0.05,**P<0.01,***P<0.005.
ribosome-freerpL3playsacrucialroleincellresponsetonucleolar stressinducedbyanticancerdrugssuchas5-Fluorouracil(5-FU), ActinomycinDandoxaliplatin(Espositoetal.,2014;Russoetal., 2016).Infact,upondrug-inducednucleolarstress,freerpL3isable toinducecellcyclearrest,apoptosisandtoinhibitcellproliferation and migration by controlling p21 expression both at post-transcriptional and post-translational levels. Furthermore, we demonstratedthatrpL3statuswasassociatedtochemoresistance sincethelossofrpL3makesthetestedchemotherapeuticdrugs ineffective(Espositoetal.,2014;Russoetal.,2016).Inagreement withourpreviousresults, CPr-A549cells resistance tocisplatin correlatedtothelossofrpL3expression.ItisnoteworthythatNCL causedasignificantincreaseofrpL3levelthatwasassociatedto theup-regulationofp21.Thesedatarevealthatanovelmolecular mechanism implicated in the cytotoxic activityof NCL can be hypothesized, involvingnucleolarstress and rpL3/p21response pathway.
Ofnote,resultsonCPr-A549cellsdemonstratednotonlythat NCLisstillactiveinresistantlungcancercellsbutalsothat biotin-targeted PMM maintain their cytotoxicity profile being more cytotoxicthan non-targeted micelles,especially at low concen-trations. These results point at biotin-decorated PMM as a reasonablestrategytoimproveNCLactivityalsoinresistantlung cancercells,which,aftertreatment,becomedefinitivelyunableto divideandtoproliferate.
5.Conclusions
With thisstudywe demonstratethat NCLdeliveredthrough tailoredPluronic1mixedmicellescanbeavaluabletherapeutic tool in the treatment of multi-drug resistant NSCLC. Biotin-targeted Pluronic mixed micelles designed for intravenous injectionentrappedNCLwithgoodyield,showedsustainedNCL release and werestable inprotein-rich media.As compared to normal NIH3T3fibroblasts, biotin-targetedmicelles were inter-nalized much more efficiently and exerted a more potent cytotoxicitythanthatofbothfreeNCLandnon-targetedmicelles in A549 cells. After selecting a resistant A549 cell line, we identified rpL3 and p21 as new molecular targets of NCL and demonstrated that the activation of rpL3/p21 pathwayby NCL overcomes MDR. Superior cytotoxicity of NCL-loaded biotin-targetedmicelleswasclearlyevidencedinA549cellresistantto cisplatin,5-FUanddocetaxelwhiletheclonogenicassay demon-stratedthat,aftertreatment,cellsbecomesunabletodivideandto proliferate.Takenalltogether, theseresults demonstratethat a NCL-basedregimenwithbiotin-decoratedPluronicmicellesmight beapromisingsecond-linechemotherapyforlungcancershowing cisplatinormulti-drugresistance.
Conflictsofinterest
Allauthorsdeclarenoconflictsofinterest. Acknowledgments
F.Q.andG.R.wishtothanktheItalianMinistryofUniversityand Research(PRIN2010H834LS,PRIN20104AE23N_006).F.Q.wishes also to thank Italian Association for Cancer Research (IG2014, #15764) for financial support. F.U. gratefully acknowledges CompagniadiSan PaoloandIstituto Bancodi Napoli—Fondazione (STARProgram,JuniorPrincipalInvestigatorGrant2013, Napoli_-call2013_35)forfinancialsupport.
D.S.P andN.H. wishestothanktheBrazilian agency Coorde-naçãodeAperfeiçoamentodePessoaldeNívelSuperior(CAPES) processnumberBEX14036/13-14.
AppendixA.Supplementarydata
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2016.06.118.
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