Development of noncytotoxic PLGA nanoparticles to improve the effect of a new inhibitor of p53-MDM2 interaction

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ContentslistsavailableatSciVerseScienceDirect

International

Journal

of

Pharmaceutics

j o u r n al hom ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / i j p h a r m

Pharmaceutical

Nanotechnology

Development

of

noncytotoxic

PLGA

nanoparticles

to

improve

the

effect

of

a

new

inhibitor

of

p53–MDM2

interaction

Ana

M. Paiva

a,b

,

Rita

A. Pinto

c

_,

_Maribel

_Teixeira

a,d,∗

_,

_Carlos

_M.

_Barbosa

a,e

_,

Raquel

T. Lima

a,c

,

M. Helena

Vasconcelos

a,c,f

,

Emília

Sousa

a,b,∗∗

,

Madalena

Pinto

a,b

a_Centro_de_Química_Medicinal_–_Universidade_do_Porto_{(CEQUIMED-UP),}_Departamento_de_Ciências_Químicas,_Faculdade_de_Farmácia,_Universidade_do

Porto,RuaJorgeViterboFerreiran◦_228,_4050-313_Porto,_Portugal

b_Laboratório_de_Química_Orgânica_e_{Farmacêutica,}_Departamento_de_Ciências_Químicas,_Faculdade_de_Farmácia,_Universidade_do_Porto,_Rua_Jorge_Viterbo

Ferreiran◦_228,_4050-313_Porto,_Portugal

c_Cancer_Drug_Resistance_Group,_IPATIMUP_–_Institute_of_Molecular_Pathology_and_Immunology_of_the_University_of_Porto,_Rua_Dr._Roberto_Frias,_S/N,

4200-465Porto,Portugal

d_Centro_de_Investigac¸_ão_em_Ciências_da_Saúde_(CICS),_Instituto_Superior_de_Ciências_da_Saúde_–_Norte,_CESPU,_Rua_Central_de_Gandra_1317,_4585-116

GandraPRD,Portugal

e_Laboratório_de_Tecnologia_{Farmacêutica,}_Departamento_de_Ciências_do_Medicamento,_Faculdade_de_Farmácia,_Universidade_do_Porto,_Portugal f_Laboratório_de_{Microbiologia,}_Departmento_de_Ciências_Biológicas,_Faculdade_de_Farmácia,_Universidade_do_Porto,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received23May2013

Receivedinrevisedform5July2013 Accepted8July2013

Available online 12 July 2013 Keywords: Antitumor Poly(d,l-lactide-co-glycolide) Polymericnanoparticles Xanthones

a

b

s

t

r

a

c

t

Onepossibleapproachtoovercomesolubilitycomplicationsandenhancethebiologicalactivityofdrugs istheirincorporationintodrugdeliverysystems.Withinthisscope,severalnanosphereand nanocap-suleformulationsofanewinhibitorofp53–MDM2interaction(xanthone1)weredevelopedandtheir physicochemicalpropertiesanalyzed.Throughtheinvestigationoftheeffectofseveralempty nanopar-ticlesonthegrowthofMCF-7cells,itwaspossibletoobservethatfouroutofﬁveformulationswere cytotoxicandthatsomecorrelationsbetweenthetoxicpotentialofthesepolymericnanoparticlesand theirproperties/compositioncouldbeextrapolated.Oneemptyformulationofnanocapsulesdeveloped byemulsiﬁcation/solventevaporationandcontainingPLGA,PVAandMygliol®₈₁₂_was_found_to_be

non-cytotoxictothiscellline.Thecorrespondingcompound1-loadednanocapsulesshowedanincorporation efficiencyof77%andrevealedtobemorepotentthanthefreedrugagainstcellgrowthinhibition,which mayberelatedtotheenhancementinitsintracellulardelivery.Inanintegrativestudy,theintracellular uptakeofnanocapsuleswasconfirmedusingfluorescent6-coumarinandwellascompound1release fromnanocapsules.Overall,itwaspossibletoenhancetheeffectofthehitinhibitorofp53–MDM2 interactionthroughthedevelopmentofsuitablenoncytotoxicpolymericnanoparticles.

1. Introduction

Thepharmacologicalrelevanceofxanthonederivativeshasled thescientiﬁccommunitytoisolateorsynthesizexanthonic com-poundsin thesearchfornoveldrugcandidates(Azevedoetal., 2012;Pintoetal.,2005).Inthepastfewyears,alargenumberof naturally-occurringandsyntheticprenylatedxanthoneshasbeen

∗ Correspondingauthorat:CICS,InstitutoSuperiordeCiênciasdaSaúde–Norte, CESPU,RuaCentraldeGandra1317,4585-116GandraPRD,Portugal.

Tel.:+351220428689;fax:+351226093390.

∗∗ Correspondingauthorat:CEQUIMED-UP,LaboratóriodeQuímicaOrgânicae Farmacêutica,DepartamentodeCiênciasQuímicas,FaculdadedeFarmácia, Univer-sidadedoPorto,RuaJorgeViterboFerreiran◦228,4050-313Porto,Portugal. Tel.:+351220428689;fax:+351226093390.

E-mailaddresses:maribel.teixeira@iscsn.cespu.pt(M.Teixeira),esousa@ff.up.pt

(E.Sousa).

reported,particularlysomewithantitumoractivity(Azevedoetal., 2012;PintoandCastanheiro,2009).Pre-clinicalstudiesofnatural prenylatedxanthoneshavealreadysuggestedtheextremelylow oralbioavailabilityforthemostinvestigatedprenylxanthone, ␣-mangostin(Fig.1)(Chitchumroonchokchaietal.,2013;Lietal., 2011).Recently,adihydropyranoxanthone,synthetizedbysome of us, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one(1,Fig.1),presentedsigniﬁcantantiproliferative andapoptoticinducingeffects(Paivaetal.,2012;Palmeiraetal., 2010)inhumantumorcelllines.Both␣-mangostin(Leãoetal., 2013a)andcompound 1(Leãoet al.,2013b)wereshown tobe promisinginhibitorsofp53–MDM2 interaction,withcompound

1showingthehighestinhibitoryactivityinayeasttarget-based assay,mimickingtheactivityofknownp53activators.Inaddition, compound1wasshowntoinhibitP-glycoproteininleukemiacells andpresentedanapparentlyhighpermeabilitycoefﬁcientacross thehumancoloncancercellline(Caco-2)(Sousaetal.,2012).

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Fig.1. Representativeprenylatedxanthonesinhibitorsofp53–MDM2interaction:␣-mangostinandthetargetmoleculeofthisstudy, 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one(1).

Asforthemajorityofpromisingnewcompounds,thesuccess ofcompound1andotherxanthonederivativesmaybe compro-misedbytheirpoorsolubility.Ingeneral,apartfromthedifﬁculty associatedwiththeadministrationofwater-insolubledrug sub-stances,thispropertyisoftenlinkedwithpoorbioavailability.One possibleapproach toovercomepoor physiochemicalproperties andenhancethebioavailabilityofdrugsistoassociatethedrug withapharmaceuticalcarrier–a drugdeliverysystem(DDS)– whichmayenhancedrugpharmacokineticsandcellular penetra-tion(Chenetal.,2011).

Aliphaticpoly(esters)likepoly(lactide),poly(glycolide)and spe-cially poly(d,l-lactide-co-glycolide) (PLGA) have been the most extensivelyinvestigatedpolymersfordrugdelivery,duetotheir excellentbiocompatibilityandbiodegradability.Drugsentrapped inthis type of polyester polymer matrix arereleased ata sus-tainedrate,throughdiffusionofthedruginthepolymermatrix andbydegradationofthepolymermatrix(JongandBorm,2008). Nanoparticlesaresubmicronsizedcolloidalpolymericsystemsand accordingwiththemethodsusedfortheirpreparationnanospheres ornanocapsulescanbeobtained.Nanospheresarematrix-type sys-temsinwhichadrugisdispersedthroughouttheparticles,whereas nanocapsulesarevesicularsystemsinwhichadrugisconfinedto acavityconsistingofaninnerliquidcoresurroundedbya poly-mericmembrane (Reiset al.,2006).Thisworkis an integrated studythatincludesphysicochemicalcharacterizationand biologi-calanalysisofcompound1-loadedpolymericnanoparticleswhich demonstratesuptake,andeffectonthegrowthofahumanbreast adenocarcinomacellline (MCF-7).In thepresent work,several polymeric nanosystems, nanocapsules and nanospheres, incor-poratingcompound 1 were developed by different techniques: solventdisplacement(SD),emulsification/solventdiffusion(ESD), andemulsification/solventevaporation(ESE),andsome formula-tionfactorswere studiedinorder toobtainnanoparticleswith favorable technologicalcharacteristics. The cytotoxicityof both emptyandloadednanoparticleformulationswasaccessedinthe MCF-7(humanbreastadenocarcinoma)cellline,whichwas criti-calfortheselectionofthemostsuitableformulation.Furthermore, theintracellularuptakeofnanocapsulescontainingafluorescent probe(6-coumarin)wasalsoinvestigatedinthesamecellline.

2. Materialsandmethods

2.1. Materials

3,4-Dihydro-12-hydroxy-2,2-dimethyl-2H,6Hpyrano[3,2-b]xanthen-6-one (1) was obtained by a previously described method (Palmeira et al., 2010) and showed a purity of 98.5% by HPLC-DAD. PLGA 50:50 (MW: 50,000–75,000Da), Pluronic® F-68,glucose, polyvinyl alcohol (PVA),6-coumarin, Tween® ₈₀ and Span® ₈₀ _were _purchased _from _{Sigma–Aldrich} _Química

(Sintra,Portugal)andMygliol®₈₁₂_was_purchased_from_Acofarma (Coimbra,Portugal).HPLCgradereagents methanol,acetonitrile andaceticacidwereobtainedfromCarloErbaReagents,(Valde Reuil,Italy)andultra-puriﬁedwaterwasproducedbyaMillipore Milli-Qsystem(Simplicity®_UV_Ultrapure_Water_System,_Millipore Corporation,Billerica,USA). Alltheotherreagentsand solvents wereofanalyticalorHPLCgrade.

2.2. Apparatusandchromatographicconditions

The HPLC analysiswas performed in a Finnigan Surveyor – Autosampler Plus and LC Pump Plus, Thermo Electron Corpo-ration (Ohio, USA), equipped with a diode array detector TSP UV6000LP,andusingaC-18column(5␮m,250mm×4.6mmI.D.) fromMacherey-Nagel(Düren,Germany).Theinjectedvolumewas 20␮landtheeluentwasmonitoredat254nm.Xcalibur®_2.0_SUR₁ software,ThermoElectronCorporation(Ohio,USA)managed chro-matographicdata.

2.3. Preparationofnanospheres

Nanospheres containing compound 1 were prepared by SD with some modifications to the previously described methods (Fessiet al.,1989;Ziliet al.,2005)(Table1,formulationsI–III). Briefly,anorganicsolutionof1,polymer,andcontainingornot alipophilicsurfactantwaspoured,undermagneticstirringinto 10mlofaqueoussolutionofahydrophilicsurfactant(Pluronic® F-68orTween®_80)._After₅_min_of_stirring,_nanosphere_dispersions were concentrated to 5ml under reduced pressure. Separation ofnon-incorporatedcompound wasperformedfirstbyfiltration (membranewithaporosityof0.45␮m),andthenbycentrifugation at1830rpmfor30min(Sigma1–14,OsterodeamHarz,Germany) aftersolubilizationofacertainamountofglucoseforachievinga5% (w/v)concentration,inordertoavoidaggregationoftheparticles duringthecentrifugationstep.Thesupernatantwasdiscardedand thepelletcontainingthenanosphereswasredispersedinwaterto completetheinitialvolume(5ml).

Thedevelopmentofnanospherescontainingcompound1 pre-pared by ESD was based on a previously described procedure (Quintanar-Guerreroetal.,1996)withsomemodifications(Table1, formulationsIV–V).Briefly,theorganicphasecontainingthe poly-merandthesurfactantwaspouredinto10mloftheaqueousphase, while mixingwithan highspeed homogenizer(20,000rpm for 5min,IKA-T18basic,Ultra Turrax®_,_Germany) _or_by _sonication (130W,90s,VibraCellmodel-75186,Sonics,USA),toformanoilin waternanoemulsion,followedbyevaporationunderreduced pres-sureuntilthefinalvolumeof5mlwasreached.Acertainamount ofglucoseforachievinga10%(w/v)concentrationwassolubilized andtheseparationofnon-incorporatedcompoundwasperformed firstbyfiltration(membranewithaporosityof0.45␮M)andthen

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Table1

Experimentalconditionsforthepreparationofnanoparticles.a

Nanosphereformulations IandII III IVandV VI

Preparationmethod SD(Zilietal.,2005) SD(Zilietal.,2005) ESD(Quintanar-Guerreroetal., 1998)

ESE(PanyamandLabhasetwar, 2004)

Acetonicsolutionof1(1mg/ml)(0.75ml) Organicphase PLGA(50mg)

CH2Cl2orCH3OH(0.5ml) Acetone(q.s.10ml) PLGA(50mg) CH3OH(0.5ml) Span®₈₀_(16.65_mg) Acetone(q.s.10ml) PLGA(70mg) Pluronic®_F-68_(23.34_mg) EtOAc(3.5ml) PLGA(50mg) CH2Cl2(1ml)

Aqueousphase Pluronic®_F-68_aqueous

solution(0.25%,w/v)(10ml)

Tween®₈₀_aqueous_solution

(0.167%,w/v)(10ml)

Water(10ml) PVAaqueoussolution(2.5%, w/v)(6ml)

Stirringconditions Magneticstirring Highspeedhomogenization (20,000rpm,5min)or sonication(130W,90s)

Sonication(130W,90s)

Nanocapsuleformulations VII VIII IX X

Preparationmethod SD(Bernardietal.,2009) SD(Zilietal.,2005) ESD ESE(PanyamandLabhasetwar, 2004)

Compound1solutionin Organicphase Mygliol®₈₁₂_(3.5_mg/ml)

(0.55ml) PLGA(50mg) Acetone(8.75ml) Mygliol®₈₁₂_(3.5_mg/ml) (0.50ml) PLGA(50mg) Span®₈₀₍₁₀₀_mg) Acetone(8.75ml) Mygliol®₈₁₂_(3.5_mg/ml) (0.45ml) PLGA(144mg) Pluronic®_F-68_(60.12_mg) EtOAc(9ml) Mygliol®₈₁₂_(3.5_mg/ml) (0.40ml) PLGA(50mg) CH3OH(0.4ml) CH2Cl2(1ml)

Aqueousphase Pluronic®_F-68_aqueous

solution(0.385%,w/v)(10ml)

Tween®₈₀_aqueous_solution

(1%,w/v)(10ml)

Water(20ml) PVAaqueoussolution(2.5%, w/v)(6ml)

Stirringconditions Magneticstirring Magneticstirring Sonication(130W,90s)

a_For_each_formulation_I–X,_{corresponding}_empty_formulations_(i–x)_were_also_prepared.

bycentrifugationfor1hat4578rpm(HettichMikro200, Bucking-hamshire,England).Thesupernatantwasdiscardedandthepellet containingthenanosphereswasredispersedinwatertocomplete theinitialvolume(5ml).

AnadditionalformulationwasdevelopedbyESE(Panyamand Labhasetwar,2004andreferencestherein),asdescribedinTable1 (formulationVI).Inbrief,theorganicphasecontainingcompound

1andthepolymerwaspouredinto6mlofanaqueoussolutionof PVA,andmixedbysonication(130W,90s)toformanoilinwater nanoemulsion.Thisnanoemulsionwasstirredatroomtemperature for4htoevaporateorganicsolvent,untiltheﬁnalvolumeof5ml wasreached.Nanoparticleswererecoveredbyultracentrifugation (1hat4578rpm,HettichMikro200,Buckinghamshire,England) andwashedwithMilliQwatertoremoveunentrappedcompound andPVA.

Emptynanospheres(formulationsi–vi)wereprepared accord-ingtothedescribedproceduresbutomittingcompound1inthe organicphase.

2.4. Preparationofnanocapsules

Nanocapsulescontainingcompound1werepreparedbytheSD previouslydescribedmethod(Bernardietal.,2009;Zilietal.,2005), asdescribedinTable1(formulationsVII–VIII).Concisely,an ace-tonicsolutionofPLGAcontainingornotthelipophilicsurfactant Span®₈₀_was_prepared._Compound₁_was_dissolved_in_Mygliol® 812(3.5mg/ml)and0.50or0.55mlofthissolutionwasaddedto thepreviouspreparedacetonicsolution.Thefinalorganicphase waspouredintoanaqueoussolutionofasurfactant(Pluronic® F-68orTween®₈₀₎_under_moderate_stirring_for₅_min._Nanocapsules dispersionfinalvolume(5ml)wasreachedbyevaporationunder reducedpressure.Thenon-encapsulatedcompound1was sepa-ratedby ultrafiltration(centrifugalfilterdevices CentriconK10, Amicon,Millipore)at4000rpmfor30min(BeckmanUL-80 ultra-centrifuge,Albertville,USA),andthevolumecompletedwithMilli Qwater.

Compound1-loadednanocapsuleswerealsopreparedbyESD by modiﬁcation of a described procedure (Quintanar-Guerrero

etal.,1998)(Table1,formulationIX).Briefly,compound1was dis-solvedinMygliol®₈₁₂_(3.5_mg/ml)_and_0.45_ml_of_this_oily_solution wasaddedtoasolutionofPLGAandPluronic®_F68_in_ethyl_acetate. Thefinalorganicsolutionwaspouredinto20mlofMilliQwaterand submittedtosonication(130W,90s).Thenanocapsulesdispersion wasconcentratedunderreducedpressuretoreachthefinalvolume of5ml.Theamountofnon-encapsulatedcompound1was sepa-ratedbyultrafiltrationusingcentrifugalfilterdevices(Centricon K10,Amicon,Millipore)at4000rpmfor30min(BeckmanUL-80 ultracentrifuge,Albertville,USA).

Finally,adifferentformulationwasdevelopedbyESE,basedon adescribedprocedure(PanyamandLabhasetwar,2004)(Table1, formulation X).In brief,asolutionof PLGA indichloromethane waspreparedandsonicatedwithmethanolandasolutionof com-pound1inMygliol® ₈₁₂_(3.5_mg/ml)._This_organic_solution_was poured into 6ml of an aqueoussolution of PVA and sonicated (130W,90s).Thefinalvolumeofnanocapsulesdispersion(5ml) wasobtainedbystirringfor4h,atroomtemperature.Theamount ofnon-encapsulatedcompound1andresidualPVAwasseparated byultrafiltrationusingcentrifugalfilterdevicesat4000rpmfor 30min(BeckmanUL-80ultracentrifuge,Albertville,USA).

Empty nanocapsules (vii–x) were prepared accordingto the sameprocedures,usingthesameamountofoil,butwithout com-pound1.

2.5. Physicochemicalcharacterization 2.5.1. Particlesizeandzetapotential

Particle size analysis of nanoparticles was performed by dynamic lightscattering(DLS).Zetapotentialwasevaluatedby laser Doppleranemometry(LDA). In both determinations, sam-pleswereanalyzedfollowingappropriatedilutionwithultrapure water, using a Brookhaven, BI-MAS90Plus (Brokhaven Instru-ments,NewYork,USA).For nanospheres,thedilutionusedwas 1:2(nanospheres:water),andfornanocapsules1:100and1:200 (nanocapsules:water).Valuespresentedarethemean±standard deviation(SD)ofatleastthreedifferentbatchesofeach nanopar-ticleformulation.

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2.5.2. Quantificationofcompound1contentinnanoparticles Quantificationofthe nanoparticlesofcompound 1 was per-formedbyaHPLCvalidatedmethod(unpublishedwork).Sample solutions were prepared by dissolving an aliquot of the dihy-dropyranoxanthone1nanosphereornanocapsuledispersionsin acetonitrile(correspondingtoadilutionof1/50and1/500, respec-tively)andsubjectedtoHPLCanalysis.Consideringanentrapment ofcompound1intonanoparticlesof100%,theobtainedsample solutionshadamaximumtheoreticalconcentrationof3mg/mlin nanospheresandrangingfrom0.56to0.77mg/mlinnanocapsules, dependingontheprocedureused.Allanalyseswereperformedin triplicateandtheresultspresentedarethemean±SD. Incorpora-tionefficiency(IE)wascalculatedasfollows:

IE(%)=A B×100

whereAisthecompound1concentration(␮g/ml)inthe nanopar-ticledispersionsandBisthetheoreticalcompound1concentration (␮g/ml).

2.6. Scanningelectronmicroscopy(SEM)

Scanningelectronmicroscopy(SEM)wasperformedtoevaluate thesurfacemorphologyofnanoparticlesusingaSEMequipment (JEOLJSM6301F),atCEMUP(CentrodeMateriaisdaUniversidade doPorto).Nanoparticlessamplesweredriedfor24hbeforethe analysis.Asmallamountofthedriednanoparticleswasapplied directlyonametallicsurfacestandwithoutcoating.

2.7. EffectonthegrowthofMCF-7humantumorcellline

Cells(5.0×103_cells/well) _were_plated _in _96-well _plates _and allowedtoadherefor 24h. Cellswere then treated withserial dilutionsofcompound1alone(from18.75␮Mto150.00␮M), com-pound1-incorporatedintonanocapsules(from0.62to50.00␮M) orempty nanocapsules (usingequal volumestothe onesused forthecompound1-incorporated intonanocapsules).Following 48hincubation,theeffectofthesetreatmentsincellgrowthwas analyzedwiththesulforhodamineB(SRB)assayaccordingtothe procedureadopted by theNational CancerInstitute (NCI, USA) (Queirozetal.,2010;Vazetal.,2010;VichaiandKirtikara,2006). Brieﬂy,afterwashingwithPBS,cellswereﬁxedinsituwith10% trichloroaceticacid,stainedwithSRBandwashedwith1%acetic acid.Thebounddyewasthensolubilizedwith10mMTrisBaseand absorbancewasmeasuredat510nminamicroplatereader(Biotek InstrumentsInc.SynergyXS,Winooski,USA).ADMSOcontrol(the vehicleofcompound1)wasalsoincludedintheexperiments. 2.8. Internalizationstudies

MCF-7cells(3.5×105_cells/well)_were_seeded_on_glass cover-slips(in24wellplates)andallowedtoadherefor24h.Cellswere thenexposedtonanocapsulesincorporating10␮Mof6-coumarin (fluorescentcompound)ortoemptynanocapsules(equalvolume tothe usedfor the nanocapsules incorporating coumarin). The internalizationofthenanocapsuleswasanalyzedatdifferent time-pointsupto48h.ThiswaspossiblebywashingthecellswithPBS, fixingwith4%paraformaldehyde(inPBS)andmountingthe cover-slipsin Vectashield® _with_DAPI₍₄_{-6-diamidino-2-phenylindole,} Vector Laboratories). Cells were observed with a fluorescence microscope(LeicaDMIRE2000).

2.9. Invitroreleasestudies

Invitroreleasestudiesofcompound1,forthemostpromising nanocapsuleformulationdeveloped(formulationX)–thatshowed

thelowestcytotoxiceffectagainstMCF-7cells–,werecarriedout at37◦C,bythebulk equilibriumreversedialysisbag technique (LevyandBenita,1990).Avolumeofnanocapsuledispersion corre-spondingto10%ofthemaximumtheoreticalaqueoussolubilityof compound1(1.6␮g/ml),inphosphatebuffersaline0.1M,pH7.4 (PBS)at37◦C,wasplaceddirectlyinto200mlofPBS.Tothis solu-tion,eightdialysisbags(cellulosemembraneMwcut-off10,000Da, Sigma–Aldrich,Sintra,Portugal)containing1mlofPBS,were pre-viouslyimmersed,andsubmittedtomechanicalstirringat37◦C. Atgiventime intervals,adialysisbag waswithdrawnfromthe releasemediumandthecompound1contentwasdirectlyassayed byHPLC.Calibrationsolutionsovertherangeof0.5–3.0␮g/mlwere preparedbydilutingcompound1stocksolutioninacetonitrilewith PBS.Valuesreportedarethemean±SDobtainedforthreedifferent batchesofthereferredformulations.

2.10. Calculationsandstatistics

IBMSPSSStatistics-19®_was_applied_for_statistic_calculations_(t testandftest).

3. Results

3.1. Characterizationofcompound1-loadednanoparticles

Sixdifferentformulationsofnanosphereswereprepared(I–VI, Table 1) and their particle size, polidispersity index (PI), and zeta potential determined (Table 2). Compound 1-loaded and emptynanosphere dispersions presentedmacroscopic homoge-neousaspect,withabluishopalescentappearanceduetoTyndall effect.

Overall,theresultsshowedlowersizeswithSD(<150nm)when comparedwithESDandESE(<400nm)(Table2).Whencomparing themeanparticlesizevaluesbetweenloadedandempty formu-lations,onlyinformulationII(compound1-loadednanospheres prepared by SD, with methanol as co-solvent) no signiﬁcantly differences (P>0.05) to empty nanospheres were observed. Regardingcompound1-loadednanospheres,theuseofmethanol ordichloromethane,asco-solvents,hadnoinﬂuenceinthemean particlesizeobtained(P>0.05).UsingSpan®_80/Tween®₈₀ (formu-lationIII)insteadofPluronic® _F-68_{(formulations}_I_and_II)_led_to nanospheredispersionswithlowervaluesof meanparticlesize (∼96nm,P<0.05).NanospherespreparedusingSDandESD tech-nique(formulationsI–V)exhibitednegativesurfacechargewith zetapotentialvalueslower than−28mV.Thehighestvalues of meanparticlesizewerefoundforthenanospherespreparedwith theESEtechnique(formulationVI);zetapotentialvalueswerealso thelowest,whatcouldforeshadowlowstabilityforthis formula-tionssincemorepronouncedzetapotentialvalues(beingpositive ornegative)tendtostabilizeparticlesuspension.Inthis formula-tion,thepresenceofPVAenhancedafasterresuspensionafterthe washingstep.

Theincorporationefficiencyvaluesofcompound1intoPLGA nanospheres(formulationsI–VI)werealsoinvestigated(Table3). IntheSDmethod,theuseofmethanolordichloromethaneas co-solvents,didnotsignificantlyaffecttheincorporationefficiency (P>0.05)(formulationsIandII).Theoverallresultsrevealedthe ESEasthemostsuitablemethodforthepreparationofnanospheres containingcompound 1(formulationVI),presenting thehighest incorporationefficiencyvalues, although,theincorporation effi-ciencyachievedwaslowerthan40%.

Thefourdifferentnanocapsulesformulationsdeveloped (for-mulations VII–X) were also investigated for their particlesize, PI, and zeta potential (Table 2). The maximum amount of oil core (Mygliol® ₈₁₂₎ _that _allows _the _preparation _of _stable

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Table2

Physicochemicalpropertiesoftheobtainednanoparticles.

Method Formulation Diameter(nm) PI Zetapotential(mV)

Nanospheres SD I 171.48±8.57 0.20±0.06 −33.33±0.27 i 131.80±3.70 0.14±0.03 −32.07±0.75 II 149.22±13.49 0.08±0.02 −33.72±1.38 ii 109.53±1.45 0.13±0.01 −35.97±0.42 III 95.67±2.32 0.20±0.02 −42.72±0.83 iii 139.80±3.95 0.13±0.01 −33.63±2.53 ESD IV 194.48±25.67 0.14±0.02 −33.99±2.71 iv 361.68±33.16 0.37±0.02 −27.58±0.27 V 166.67±7.48 0.15±0.02 −41.41±1.87 v 158.23±0.83 0.10±0.01 −38.50±3.65 ESE VI_vi 400.53_238.53±_±8.83_12.91 _0.0760.19_±±0.02_0.01 −33.23_−9.18_±±3.33_1.95 Nanocapsules SD VII 219.3±3.3 0.15±0.04 −24.57±4.3 vii 213.3±4.3 0.16±0.05 −35.51±3.1 VIII 319.07±21.8 0.20±0.01 −39.34±1.0 viii 368.72±39.2 0.26±0.01 −35.22 ±3.4 ESD IX_ix 210.27_241.47±_±0.7_2.8 _0.060.09±_±0.002_0.01 _−40.50−38.07_±±1.2_1.2 ESE X 283.93±12.18 0.093±0.006 −15.20±0.64 x 238.53±0.09 0.097±0.025 −14.37±0.49

nanocapsuledispersions was determined for every preparation

techniqueemployed.FornanocapsulespreparedbySD,

formula-tionscontaining0.40,0.50and0.55mlofMygliol®₈₁₂_showed_a

smallpelletatthebottomoftheﬂask(attributedtonanospheres)

andacreamlayeratthesurfacecorrespondingtonanocapsules;

no free oilcould be detected in these formulations indicating

thattheoilwascompletelycoated bythepolymer. The

formu-lationsincluding0.60mlofMygliol® ₈₁₂_showed_a_free_oil_layer

atthesurface of thedispersionsupon centrifugation indicating

poorstability. Therefore theamountof oilselectedtobeused,

which allowed the preparation of stable nanocapsule

formula-tionswas0.55ml.Then, fornanocapsulepreparation 0.55mlof

compound1solutioninMygliol®₈₁₂_was_used._Nanocapsules

for-mulationspresentedmacroscopichomogeneousaspects,withan

opalescentmilky-likeappearance.Forallformulationsmean

parti-clesizerangedfrom∼210to370nm.Themeanparticlesizeofthe

formulationsdevelopedwasnotaffectedbytheincorporationof

compound1(P>0.05).WhenusingPluronic® _F-68_as_surfactant

(formulationVIIand IX)insteadofTween®_80/Span®₈₀

(formu-lationVIII),nanocapsulesdispersionsshowedlowerparticlesize

values(P<0.05).Zetapotentialvaluesshowedthat,forbothempty

andcompound 1-loadednanocapsules,negativesurfacecharges

were achieved, rangingfrom −14.37±0.49 to −40.50±1.2mV,

whichindicatesthatstableformulationswereproducedwiththis

method.

Incorporationefﬁciencyvaluesofcompound1inPLGA

nanocap-sules were also determined (Table 3). With the SD technique,

usingTween-80®_/Span-80®_(formulation_VIII)_instead_of_Pluronic F-68® _(formulation _VII) _as _surfactants, _the _final _{concentration} andincorporationefficiencyvalues,raisedfrom196.18±24.16to 323.57±2.67␮g/ml,respectively (∼30% increased). Overall,the incorporation efficiency for nanocapsules was better than the one achieved for nanospheres (Table 3) with formulation VIII presenting an incorporation efficiency of compound 1 of 84%. Moreover,thedevelopednanocapsuledispersionsshowedhigher concentrationsthantherespectivetheoreticalaqueoussolutionof compound1(16␮g/ml,ACD/Labsprogram):20foldforformulation VIII,15foldforIXand13foldforX,respectively.

3.2. EffectonthegrowthofMCF-7humantumorcellline

Basedonthetechnologicalparameters,ﬁveofthedeveloped formulationswereselectedforfurtherevaluationinthehuman breastadenocarcinomacelllineMCF-7,regardingthecellgrowth inhibitory effect. The chosen formulations were: nanospheres developedbySDandESE(formulationsIIIandVI)andnanocapsules developedbySD,ESD,andESE(formulationsVIII,IX,and formula-tionX).Byanalysingtheeffectofthedifferentemptynanoparticles ontheMCF-7cellgrowth(withtheSRBassay),itwaspossibleto observethatonlytheemptyformulationofnanocapsules devel-opedbyESE(formulationx),didnotpresent majorcytotoxicity tothiscelllineattheconcentrationsanalyzed(Fig.2).Theother

Fig.2. Effectofemptyandcompound1-loadedformulationXinthecellgrowthof MCF-7cells.Cellsweretreatedfor48hwithincreasingconcentrationofcompound

1,compound1incorporatedintonanocapsules(compound1loadedformulationX) orwithequalvolumesofemptynanocapsules(emptyformulationx)andanalyzed withtheSRBassay.Resultsarerepresentedas%ofcellgrowth,consideringthe valuesforuntreatedcellsas100%.Resultsarethemean±standarderrorofﬁve independentexperiments.

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Table3

Incorporationefﬁciencyofcompound1innanoparticles.

Method Formulation Compound1theoretical concentration(␮g/ml) Compound1ﬁnal concentration(␮g/ml) Incorporation efﬁciency(%) Nanospheres SD I 150 37.77±6.07 25.18±1.85 II 39.12±4.69 26.08±3.12 III 43.04±1.08 28.69±0.72 ESD IV_V 28.23_33.98±_±0.78_3.66 18.82_22.65±_±0.39_2.44 ESE VI 58.17±7.13 38.78±4.75 Nanocapsules SD VIIVIII 385350 196.18323.57±±24.162.67 56.0584.04±±6.900.69 ESD IX 315 242.97±2.85 77.99±0.90 ESE X 280 209.56±32.35 77.85±11.55

investigatedemptyformulationspresentedcytotoxicity(datanot

shown),whichmaybeexplainedbytheamountofexcipientsused

intheirdevelopment(Table4).

Whencomparingthecellgrowthinhibitoryeffect(inMCF-7 cells)ofthefreecompound1withtheeffectofformulationX,the GI50values (concentrationthatinhibitscellgrowthby50%) sig-niﬁcantlydecreasedfrom46.8±1.8␮Mto16.3±2.1␮M(P<0.05) (Fig.2).Moreover,noapparentcellulartoxicitywasobserved fol-lowing treatmentwiththeempty nanocapsules, at thevolume correspondingtothedeterminedGI50(Fig.2).

3.3. Internalizationstudies

Agreenﬂuorescentcompound(6-coumarin)wasincorporated into formulation x and following treatmentwith nanocapsules incorporating6-coumarin,cellswereobservedatdifferent time-points.Greenﬂuorescence(indicatingthepresenceofcoumarin) wasevidentinthecellcytosolimmediatelyafterthenanocapsules incorporating6-coumarinwereaddedtothecells(Fig.3).

Althoughtheinitialgreen ﬂuorescencewasweak,it became strongeranddiffusedthroughoutthecellcytoplasmattheother time-pointsanalyzed.Theintracellularconcentrationwas partic-ularlystrong6hfollowingtreatmentandtheintensityreducedat 48hfollowingtreatment,indicatingthatthecoumarinwas proba-blymetabolizedoreliminatedbythecells.

3.4. Scanningelectronmicroscopy(SEM)

SEMwasusedtoinvestigatethemorphologyof formulation X (Fig. 4A). Nanocapsules displayed a spherical shape with a smoothsurfaceandnoaggregationwasobserved.Nodifference wasobservedinthemorphologicalpropertiesofnanocapsulesdue topresenceofthedrug.Infact,SEManalysisconﬁrmedthe nano-metricsizeofformulationXdeterminedbyDLS(Fig.4A).

3.5. Invitrorelease

Invitroreleasestudiesofcompound1-loadednanocapsules for-mulationwereperformedunder“sinkconditions”(LevyandBenita, 1990),toavoidtheinterferenceofcompound1solubilityinthese experiments(Fig.4B).AsobservedinFig.4B,animportantrelease ofcompound1fromnanocapsules(formulationX)duringtheﬁrst 2h(>80%release)followedbyaslowreleaseuptotheendofthe assay(24h)wasobserved.Thekinetic processisprobably gov-ernedbytheoil–waterpartitioncoefﬁcientasdescribedforother nanocapsuleformulations(Teixeiraetal.,2005a).

4. Discussion

Thisstudyaimedtodevelopsuitablepolymericnanoparticles incorporatingthexanthone1,aninhibitorofp53–MDM2 interac-tion.Thisapproachwaspreviouslydemonstratedtobeefﬁcient inimprovingtheNOproductioninhibitoryeffectofsimple oxy-genatedxanthones(Teixeiraetal.,2005b).Toachieveasuitable formulation,severaltechniquesandexcipientswereusedinthe preparationofpolymericnanoparticlesandinvestigatedfortheir cytotoxicityinahumanbreastadenocarcinomacellline(MCF-7).

Sixdifferentformulationsofnanospheresandfourdifferent for-mulationsofnanocapsulesweredevelopedandtheirtechnological parametersanalyzed.Theoverallresultsindicatesthatthedifferent techniquesemployedwereappropriateinachievingstable poly-mericformulationsshowingzetapotentialvaluesnear−30mV, revealingtobestableinsuspension,asthesurfacechargeprevents aggregationoftheparticles(MohanrajandChen,2006).

It is alwaysa challengetoformulatenanoparticleswiththe smallestsizepossiblebutwithmaximumstability. Smaller par-ticles have larger surface area, which meansthat most of the compound associated would beat or nearthe particlesurface, leadingtoafastdrugrelease, alsohavinggreaterriskof aggre-gation during storage and transportation (Mohanraj and Chen, 2006);whereas,largerparticleshavelargecoreswhichallowmore Table4

Amountofexcipientsdeliveredtothecells.a

Formulations [Compound1nanoparticles]b_(␮M) _Dilution

factor PLGA (mg/ml) PVA (mg/ml) Tween®_80/Span® 80(mg/ml) Pluronic® (mg/ml) Mygliol®₈₁₂_(ml/ml) III 134 N.A. 10.00 – 20.00 – – VI 291 1.94 5.15 15.46 – – – VIII 1108 7.40 1.35 – 10.31 – 13.51 IX 703 4.70 2.98 – – 0.99 19.15 X 697 4.60 2.17 6.52 – – 17.39

a_For_each_formulation,_{corresponding}_empty_formulations_were_also_{investigated.}

b _[1]_corresponds_to_the_{concentration}_of_loaded_{nanoparticles}_and_was_used_to_determine_the_amount_of_{nanoparticles}_suspension_to_be_tested,_based_on_the_maximum

concentrationofcompound1tested(150␮M).

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Fig.3. Uptakeofnanocapsules(formulationX)incorporating6-coumarinintotheMCF-7cells,observedbyfluorescencemicroscopy.Cellswerefixedfollowingincubation withtheemptynanocapsules(at0h,20minor60min)orwiththenanocapsulesincorporatingcoumarin(at0h,20min,60min,6hor48h).Cellnucleiwerestainedwith DAPIandthecytoplasmicgreenfluorescenceresultedfromcoumarin.

compoundtobeencapsulatedandslowlydiffuseout.Ingeneral,the nanospheresobtainedfurnishedformulationswithsmaller parti-clesizebutlowerincorporationefﬁciency,whencomparingwith nanocapsules.Theseresultswerepredictable,sincethe dihydropy-ranoxanthone1islipophilicandcouldthereforebebetterdissolved intheoilcoreofthenanocapsules.

Theincorporationofcompound1intonanosystemsaimsmainly atovercomingproblemsrelatedtothecompoundwithlowwater solubility.Foralltheconditionsstudied,theﬁnalconcentrationof compound1-loadednanoparticleswashigherthanthecalculated aqueoussolubilityconcentration(16␮g/ml).Basedonthesedata, twoformulationsofnanospheres(IIIandVI)andthree formula-tionsofnanocapsules(VIII–X),allwithfavorablephysicochemical properties,wereselectedforfurtherinvestigations.

Inthepresentstudy,thecytotoxicityevaluationinMCF-7cell lineofbothemptyandloadedformulationslimitedthefutureuse offouroutofﬁvedevelopednanoparticles.Consideringtheeffect ofTween®₈₀_and_Span®_80,_although_they_have_been_described_as beingnontoxicandbeingallowedforintravenousadministration

(Roweetal.,2009),theamountsusedforthepreparationofabove describednanoparticleswerefoundtoinﬂuencethecytotoxicity of formulationsIIIand VIII(Table4)which couldberelated to theircellpermeabilizationeffects(Olivier,2005);similar conclu-sionscouldbedrawnforPluronic® _(formulation_IX,_Table_4)._In contrast,thedispersingoilMygliol®_was_also_not_responsible_for theobservedtoxicity,sincenanocapsulesXusedhigheramounts ofoil(whencomparedwithnanocapsulesVIII)withoutcytotoxicity atcompound1GI50value,independentlyoftheirzetapotentials (Table2).InwhatconcernsPLGA,fromtheobtainedresults,one mayhypothesizethatthetoxicityobservedisnotdirectlyrelated tothisexcipientinaccordancetopreviouslydescribed toxicologi-calstudies(Semeteetal.,2010).Indeed,whileforthenanocapsules VIII,theamountofPLGAwas1.35mg/ml,andthisformulationwas toxic,fornanocapsulesX,theamountusedwashigher(2.17mg/ml) andnocytotoxicitywasobservedatcompound1GI50values.PVA isalsoknowntohavelowtoxicity(DeMerlisandSchoneker,2003) butduetoseveralvariablesinformulationsVIandXno extrapola-tionscanbedrawn.Sincethetoxicpotentialofnanoparticleshas

Fig.4.(A)Scanningelectronmicrographsofcompound1-loadednanocapsules(formulationX)withconﬁrmationofnanocapsulessizes.(B)Invitroreleaseproﬁleoffree compound1fromnanocapsules(formulationX)followedbyitsdiffusionthroughthedialysisbag.Eachpointrepresentsthemean±SDvaluesobtainedfromthreedifferent batches.

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beenreportedtobestronglydependentontheirsurface proper-ties(JongandBorm,2008)andparticularlyontheircharge(Mura etal.,2011),thelowestzetapotentialvaluesobtainedforemptyand loadedformulationsX(−14.37±0.49and−15.20±0.64mV)could hadsignificantinfluenceinthelowestinvitrotoxicityobserved. Consequently,theamountand typeofexcipientspresentinthe finalformulationofnanoparticlesmayplayacriticalroleinthe futuretherapeuticapplicationofthistechnology.Theevaluation ofthecytotoxicityofemptynanoparticlesistherebyanimportant issuewhendevelopingnewnanoparticleformulationsandthe cor-relationofthesurfacepropertiesofpolymericnanoparticleswith theircytotoxicitydeservesfurtherattention.

Fromtheoverallresultsoncellgrowtheffects,itcanbeinferred that, the cytotoxic effect of formulation X, nanoparticles with amountsof PLGAand PVA below 2.17and6.52mg/ml,maybe onlyduetotheeffectoftheincorporatedcompound(Fig.2).At thetimethisstudywasbeingcarriedout,anotherstudyonsolid dispersionsof␣-mangostin(Fig.1)inpolyvinylpyrrolidone(PVP) revealedtheenhancementof␣-mangostinsolubilityand intracel-lulardelivery,although,noenhancementof␣-mangostincytotoxic effect wasobserved withthis formulation (Aisha et al., 2012). Herein,this studyfurnishedaformulation ofthehitcompound withathree-foldimprovementintheGI50valuesinMCF-7cellline. Additionally,anefficientinternalizationof6-coumarin nanocap-suleswasachieved.Entrapmentofafluorescentprobewithinthe nanocapsulesenabledustoconfirmthattheywereinternalizedas intactnanocapsules,releasingthefluorescentcompoundonlyin thecellcytoplasm.

Drugreleasestudiesfromthenanoparticlesaregenerally per-formedtounderstand therateand mechanism of drug release ratherthanasaroutinequalitycontrolmethodasusedinthecase ofconventionaldosageforms(PanyamandLabhasetwar,2004). Invitrostudiesindicatedthatthepresenceofthepolymerdidnot affectthereleaseofcompound1,beingthepartitionbetweenthe oilycoreandtheexternalaqueousmediumthemainfactor gover-ningtheprocess,asalreadyreportedforotherdrugs(Mora-Huertas etal.,2010;Santos-Magalhãesetal.,2000;Teixeiraetal.,2005a).

5. Conclusions

Inthepresentintegrativework,severaltechniqueshavebeen employedforthedevelopmentofpolymericnanoparticle formula-tionsofapoorlywater-solubledihydropyranoxanthone,inhibitor ofp53–MDM2interaction(compound1).Thisallowedtoenhance compound1concentrationinaqueoussolutionsbya minimum oftwo-foldinnanospheresto13-foldinnanocapsules.Fromthe ﬁveselectedformulations,onlyonepreparedbyESEwithPVAas surfactantshowednosigniﬁcanttoxicityinthecelllinestudies. Thedevelopedformulationwithfavorabletechnological parame-tersledtothree-foldimprovementintheGI50valuesofcompound

1andcouldbeavaluablestrategyaspharmaceuticalcarriersof xanthones.

Acknowledgements

To FCT – Fundação para a Ciência e a Tecnologia -PEst-OE/SAU/UI4040/2011 and PTDC/SAU-FAR/110848/2009 under the project CEQUIMED, and REEQ/1062/CTM/2005 and REDE/1512/RME/2005 – CEMUP; FEDER through the COMPETE programundertheprojectsFCOMP-01-0124-FEDER-011057and FCOMP-01-0124-FEDER-015752;andtoU.Porto/SantanderTotta for financialsupport. ToSara Cravofor technical support.A.M. Paiva (PTDC/SAU-FCT/100930/2008; Liga Portuguesa Contra o Cancro/Pfizer)andR.T.Lima(SFRH/BPD/68787/2010)arethankful toFCTandLigaPortuguesaContraoCancro/Pfizerfortheirgrants.

IPATIMUPisanAssociateLaboratoryofthePortugueseMinistry of Science, Technology and Higher Education and is partially supportedbyFCT.

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