Cationic drug based self assembled polyelectrolyte complex micelles physicochemical, pharmacokinetic, and anticancer activity analysis

Cellularuptakeanalysis Thecellularuptake of free drugs and drug-loaded PCMwas investigatedinA-549cancercellsusingfluorescence-assistedcell sortingFACS.Briefly,cellswereseededata densityof3

Contents lists available atScienceDirect

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

Thiruganesh Ramasamya, Bijay Kumar Poudela, Himabindu Ruttalaa, Ju Yeon Choia,

Truong Duy Hieua, Kandasamy Umadevib, Yu Seok Younc, Han-Gon Choid,

Chul Soon Yonga,∗, Jong Oh Kima,∗

a College of Pharmacy, Yeungnam University, 214-1 Dae-dong, Gyeongsan 712-749, South Korea

b St Paul’s College of Pharmacy, Osmania University, Hyderabad, Telangana, India

c School of Pharmacy, SungKyunKwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, 440-746, South Korea

d College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-791,

South Korea

a r t i c l e i n f o

Article history:

Received 22 March 2016

Received in revised form 31 May 2016

Accepted 2 June 2016

Available online 5 June 2016

Keywords:

Nanofabrication

Polyelectrolyte complex micelles

Cationic drugs

Pharmacokinetic

Anticancer activity

a b s t r a c t

Nanofabricationofpolymericmicellesthroughself-assemblyofanionicblockcopolymerandoppositely chargedsmallmoleculeshasrecentlyemergedasapromisingmethodofformulatingdeliverysystems Thepresentstudythereforeaimedtoinvestigatetheinteractionofcationicdrugsdoxorubicin(DOX) andmitoxantrone(MTX)withtheanionicblockpolymerpoly(ethyleneoxide)-block-poly(acrylicacid) (PEO-b-PAA)andtostudytheinfluenceoftheseinteractionsonthepharmacokineticstabilityand anti-tumorpotentialoftheformulatedmicellesinclinicallyrelevantanimalmodels.Tothisend,individual DOXandMTX-loadedpolyelectrolytecomplexmicelles(PCM)wereprepared,andtheirphysicochemical propertiesandpH-responsivereleaseprofileswerestudied.MTX-PCMandDOX-PCMexhibiteda differ-entreleaseprofileunderallpHconditionstested.MTX-PCMexhibitedamonophasicreleaseprofilewith

noinitialburst,whileDOX-PCMexhibitedabiphasicrelease.DOX-PCMshowedahighercellularuptake thanthatshownbyMTX-PCMinA-549cancercells.Furthermore,DOX-PCMinducedhigherapoptosis

ofcancercellsthanthatinducedbyMTX-PCM.Importantly,bothMTX-PCMandDOX-PCMshowed pro-longedbloodcirculation.MTX-PCMimprovedtheAUCallofMTX4-foldcomparedtoa3-foldincrease

byDOX-PCMforDOX.WhileadefinitedifferenceinbloodcirculationwasobservedbetweenMTX-PCM andDOX-PCMinthepharmacokineticstudy,bothMTX-PCMandDOX-PCMsuppressedtumorgrowth

tothesamelevelastherespectivefreedrugs,indicatingthepotentialofPEGylatedpolymericmicelles

aseffectivedeliverysystems.Takentogether,ourresultsshowthatthenatureofinteractionsofcationic drugswiththepolyioniccopolymercanhaveatremendousinfluenceonthebiologicalperformanceofa deliverysystem

©2016ElsevierB.V.Allrightsreserved

Conventional chemotherapeuticapproach is themain

treat-ment option for cancer [1] Despite great strides made in

understanding cancer biology, conventional chemotherapeutic

drugsarecharacterizedbynon-specificdistributionandhigh

accu-mulation in healthy cells, leading to dose-limiting side effects

thatseriouslyimpedetheirclinicalapplication [2].Tominimize

sideeffectsandimprovetherapeuticefficacyof

chemotherapeu-∗ Corresponding authors.

E-mail addresses: csyong@ynu.ac.kr (C.S Yong), jongohkim@yu.ac.kr (J.O Kim).

tic drugs, various drug deliverysystems have been developed Among them, block copolymer-based self-assembledpolymeric micelleshave demonstratedpromising potentialin thedelivery

of anticancerdrugs The nanosized micelles offermany advan-tages,includinguniformsizedistribution,core-shellarchitecture, highdrugloading,andphysicalstability[3,4].Polyethyleneglycol (PEG)iswidelyusedtograftthehydrophobicpartofamphiphilic polymersandformtheoutershellofthemicelles.Suchpolymeric micelleshavebeenshowntoincreasethesystemiccirculationtime

ofdrugs and preferentiallyaccumulate in tumorsvia enhanced permeabilityandretention(EPR)effect[5]

Polyelectrolyte complex micelles (PCM), a special class of micellesformedbyelectrostaticinteractionofoppositecharged http://dx.doi.org/10.1016/j.colsurfb.2016.06.004

0927-7765/© 2016 Elsevier B.V All rights reserved.

totumorinterstitialspaces[10]

Well-knownanticancerdrugsdoxorubicin(DOX) and

mitox-antrone(MTX)areanthracyclineswithabroadspectrumofactivity

againstavarietyofcancersincludingbreast,lung,prostate,bone,

andbladdercancers.Theseanticanceragentsactbyintercalating

DNAandinhibitingtopoisomeraseII[11].WhilebothDOXandMTX

areanthracyclinemoieties,theydifferinnumberandsubstitution

statesofaminofunctionalitiespresent.DOXconsistsoffourfused

ringswithasugarmoietycontainingaprimaryaminegroup(-NH2),

whileMTXhasthreefusedringscontainingtwosecondaryamine

groups(-NH-)[6,11].BothDOXandMTXarepositivelychargedat

physiologicalpHwithanaveragepKaof∼8,whichisresponsible

forelectrostaticinteractionswiththecarboxylategroup(pKa∼5)

oftheblockcopolymer[12].Inthepresentstudy,poly(ethylene

oxide)-block-poly(acrylicacid)(PEO-b-PAA)wasusedastheblock

copolymer.ThePCMwereformedbytheelectrostaticinteractionof

theprotonatedaminogroupsofMTXorDOXwiththecarboxylate

moietyofthePAAsegmentofthePEO-b-PAApolymer

Thepresentstudyaimedtoinvestigatetheinteractionsof

differ-entcationicdrugswiththeanionicblockpolymerPEO-b-PAAand

tostudytheinfluenceoftheseinteractionsonthepharmacokinetic

stabilityandantitumorpotentialoftheformedPCMinclinically

relevantanimalmodels.Towardsthispurpose,pH-responsiveness

andreleaseprofilesofindividualdrugsfromdrug-loadedPCMwere

monitored.Inaddition,aninvivopharmacokineticstudyofPCM

(DOX-PCMandMTX-PCM)inratsandanantitumorefficacystudy

inA-549cancercell-xenograftedmousemodelswereperformed

Theeffectofamine-functionalizedanticancerdrugsonthe

physic-ochemicalandbiologicalresponsesofmicellarnanocarrierswas

demonstrated

2.1 Materials

Doxorubicin hydrochloride was supplied by Dong-A

Phar-maceuticalCompany(Yongin,SouthKorea).Mitoxantrone

dihy-drochloride was purchased from Shaanxi Top Pharm Chemical

Co.Ltd(Xi’an,China).Poly(ethyleneoxide)-block-poly(acrylicacid)

(PEO-b-PAA,MWsofPEOandPAAblockswere5000and6800Da,

respectively) wasprocured from PolymerSource, Inc (Quebec,

Canada).Allotherchemicalswereofreagentgradepurityandwere

usedwithoutanyfurthermodifications

2.2 Preparationofdrug-loadedPCM

TheMTXandDOX-loadedPCMwereformedbyasimplemixing

methodaswereportedpreviously[6].Briefly,aqueoussolutions

2.3 Particlesizeand␨-potentialanalysis Particle size (nm), polydispersity index (PDI), and zeta ( ␨)-potential (mV) of MTX-PCM and DOX-PCM were analyzed by dynamiclightscattering(DLS).ZetasizerNanoZS(Malvern Instru-ments, Malvern, UK) equipped with He–Ne laser was used to measuretheparticlesize.Afixedangleof90◦wasselectedandthe laserwasoperatedat635nm.NanoDTSsoftware(version6.34) wasemployedtoanalyzethesize,PDI,andsurfacechargeofthe micelles.Eachmeasurementwasperformedintriplicate

2.4 Morphologicalanalysis Transmissionelectronmicroscopy(TEM)(CM200UT;Philips, Andover,MA,USA)wasusedtocharacterizethemorphologyof drug-loadedPCM.Theparticleswereobservedatanaccelerating voltageof100kV.Briefly,adropofmicellardispersion(R=0.5)was placedinthecarbon-coatedcoppergridandallowedtosettlefor

10min.Excessliquidwassoakedoutwithtissuepaper.Thethin layerofparticleswascounter-stainedby2%phosphotungsticacid (PTA)asanegativestaining.Theparticlesweresubjectedtoinfrared radiationfor5min

2.5 Physicalstatecharacterization TheX-raydiffraction(XRD)patternsoffreeDOX,MTX, DOX-PCM,andMTX-PCMwererecordedusingaverticalgoniometerand X-raydiffractometer(X’PertPROMPDdiffractometer,Almelo,The Netherlands)tomeasureNi-filteredCuK␣radiation(voltage,40kV; current,30mA)scatteredinthecrystallineregionsofthesample Thepatternswererecordedatascanningrateof5◦/minoverthe 10–60◦diffractionangle(2␪)rangeatanambienttemperature 2.6 Invitroreleasestudies

The release profiles of drugs from MTX-PCM or DOX-PCM wereevaluatedbydialysis.Phosphate-bufferedsaline(PBS,pH7.4, 0.14MNaCl)andacetate-bufferedsaline(pH5.0,0.14MNaCl)were usedtosimulatethephysiologicalandtumorpH.Inbrief,1mlof micellardispersion(1mgequivalentofMTXandDOXatR=0.5) wassealedinmembranetubing(Spectra/Por®;3500Dacutoff)and placedat37◦Cat100rpm.Thesampleswerewithdrawnat prede-terminedtimesandreplacedwithequalamountsoffreshmedium Thesampleswerecollected,filtered,andanalyzedusingUV–vis spectrophotometryat609and485nmforMTXandDOX, respec-tively.Theamountofdrugreleasedwasplottedagainsttime.The release kineticswasanalyzed byfittingthedatatoappropriate mathematicalmodels

Fig 1.Schemes illustrating preparation of drug-loaded PCM.

2.7 Cellculture

A-549smalllungcancercellsweregrowninRPMI1640medium

supplementedwith10%(v/v)fetalbovineserum(FBS)inthe

pres-ence of penicillin and streptomycin (100U/mL and 0.1mg/mL,

respectively).Thecellsweremaintainedunderambientconditions

(37◦C containing5% CO2) ina T-75 flask andperiodically

sub-cultured

2.8 Cellularuptakeanalysis

Thecellularuptake of free drugs and drug-loaded PCMwas

investigatedinA-549cancercellsusingfluorescence-assistedcell

sorting(FACS).Briefly,cellswereseededata densityof3×105

cells/wellina6-wellplateandincubatedovernight.Thecellswere

treated withfree DOX,free MTX, DOX-PCM,and MTX-PCM (in

equivalentconcentrationsof10␮g/mL)andincubatedforthe

indi-catedperiods.ThecellswerewashedtwicewithPBSandharvested

Thecellswereresuspendedin1mLofPBSandanalyzedinaflow

cytometer(FACSCalibur,BDBiosciences,SanJose,CA,USA)

2.9 Apoptosisanalysis

Thecellswereseededatadensityof3×105 cells/wellina

6-wellplateandincubatedovernight.Thecellsweretreatedwith

freeDOX,freeMTX,DOX-PCM,andMTX-PCM(inequivalent

con-centrationsof5␮g/mL)andincubatedfor24h.Nextday,cellswere

washed,trypsinized,harvested,andwashedagainwithcoldPBS

Thepelletwastreatedwith2.5␮LofAnnexinV-FITCand2.5␮Lof

7-AADfor15minatroomtemperature.Thepercentageof

apop-toticcellswasanalyzedusingaflowcytometer(FACSCalibur,BD

Biosciences,SanJose,CA,USA)

2.10 Pharmacokineticanalysis

The in vivo pharmacokinetic study was performed in male

Sprague-Dawleyrats(220±10g).Theexperimentalprotocolsand

animalcarewereinaccordancewiththeprotocolslaidby

Insti-tutionalAnimalEthicalCommittee,YeungnamUniversity,South

Korea.Theratsweredividedintofourgroupswith4ratsineach group

2.11 Administrationandbloodcollection Theratswereheldinasupineposition.Therightfemoralartery wascannulatedtocollectthebloodsamples,whiletheleftfemoral arterywascannulatedtoadministertheindividualDOXandMTX formulationsasasingledose(5mg/kg).300␮LofpreparedPCM formulationswereadministeredtoeachratviaatailvein injec-tion.The micelles formulated ata feeding ratioof R=0.5 were employed.Bloodsamples(200␮L)werecollectedatdesignated intervals(0.25,0.5,1,2,4,6,8,10,12,and24h).Thesurgical open-ingswereimmediatelysealedwithsurgicalsuturestoeasepainand increasethelengthofthestudyperiod.Afterbloodwaswithdrawn,

itwasimmediatelycentrifuged(Eppendorf,Hauppauge,NY,USA)

at13000rpmfor10minsothatplasmacouldbeseparatedand extractedforfurtheranalysis

2.12 PreparationandevaluationofplasmasamplesbyHPLC

150␮L of plasma was mixed with 150␮L of methanol and vortex-mixed for 30min The mixture was centrifugedat high speed;supernatantwasseparatedandsubjectedtovacuum evap-oration The evaporated residue wasreconstitutedwithmobile phase and injected into the HPLC column (20␮L) Two differ-entmobilephaseswereused:sodiumformate(80nM)/methanol (80/20;pH2.9)forMTXandmethanol/water/aceticacid(50/49/1;

pH3)forDOX.Flowrateofthemobilephasewas1ml/minand effluentsweremeasuredat254and480nmfor MTXandDOX, respectively

2.13 Pharmacokineticparameters

A non-compartmental model was used to plot the plasma concentration–timevaluesusingWinNonlinsoftware(professional edition,version2.1;Pharsight Corporation,MountainView, CA, USA).Pharmacokineticparametersincludedtheeliminationrate (K ),half-life(t ),maximumplasmaconcentration(Cmax),time

Aftertheappropriatetumorvolume wasattained,formulations

wereinjectedviatailvein(3timeswithagapof3daysbetween

eachinjection).Thetumorvolumewasmeasured(V=0.5×longest

diameter×shortestdiameter)usinga Verniercaliper.The body

weightofmicewasmonitoredinordertoobservethesafety

pro-fileoftheformulations.Attheendoftheexperiment,micewere

sacrificedaccordingtotheinstitutionalethicalguidelines.Tumors

weresurgicallyremovedandweighedindividually

2.15 Statisticalanalysis

Student’s t-test was used to evaluate the statistical

signifi-canceofdifferencesbetweenformulations.Valueswerereported

asmean±standarddeviation(SD)andthedatawereconsidered

statisticallysignificantatp<0.05

3.1 Preparationofdrug-loadedPCM

Thepresentstudyaimedatinvestigatingtheinteractionsof

dif-ferentcationicdrugswiththeanionicblockpolymerPEO-b-PAA

TheprimaryaminogroupofDOX(-NH2)andtwosecondaryamino

groups(-NH-)ofMTXareresponsibleforelectrostaticinteractions

withtheionizedcarboxylgroup(pKa∼5)ofPEO-b-PAA[11]

There-fore,pH-responsivenessofindividualdrugsandtheirabilitytoform

drug-loadedPCMwerestudiedindetail.ThePCMwereformedat

twodifferentchargeratios(R=0.25and0.5)ofMTXandDOXto

carboxylategroups(R=[drug]/[COO−]).Theschematicillustration

offormationofPCMispresentedinFig.1

AsshowninFig.2,thePCMwerepreparedwithbothdrugsat

variouspHconditions.ThePCMwereformedbytheimmobilization

ofweaklybasicdrugs(MTXandDOX)intothecoresofPEO-b-PAA,

aweakpolyacid,viaastrongelectrostaticinteraction.Asexpected,

weobservedpH-sensitivebehaviorofPCM.ComparedtoPCM

par-ticlesizeatpH6,particlesizereducedremarkablywiththeincrease

inpH(DOX-PCM).Consistently,␨-potentialofPCMdecreasedas

pHincreased,indicatingthathigherpHfavorstheionizationofthe

polymerblockresultingincomplexationofdrugs.The␨-potential

decreasedintheentirepHrangestudied.Apossibleinterpretation

isthatatlowerpHvalues,owingtopartialorinsufficient

ioniza-tionofthePAAblock,thedrug-polymerphysicalinteractionforms

alooseaggregateresultinginlargerparticlesize.Uponincreasein

thepHofthemedium,PAAattainsmaximumionizationresulting

inefficientcomplexationofdrugs.Itisworthnotingthatparticle

sizemarkedlydecreasedwhenthechargeratiowasincreasedfrom

R=0.25toR=0.5,indicatingtheneutralizationofthePAAsegments

duetotheelectrostaticinteractionofMTXandDOXinthePCM.As

expectedathigherchargeratios,greaterneutralizationofnegative

Fig 2.Effect of pH on(A)hydrodynamic particle size and(B)␨-potential at different feeding ratios (R = 0.25 and 0.5).

chargeofthePAAblockresultsinstablePCMwithhighly hydropho-biccoreandhighpayloadcapacity[13].Appreciablehydrophobic coreandPEGshellonthesurfacestabilizethePCMinsystemic conditions.Specifically,MTX-PCMexhibitedasmallerparticlesize comparedtothatofDOX-PCM.Thisdifferenceinparticlesizesof cationicdrug-basedPCMcanbeattributedtothebindingstrength

ofindividualdrugstothepolymerblock.Overall,thesizeof MTX-PCMandDOX-PCMwaslessthan100nm,makingthesePCMideal fortumordrugdelivery.Ithasbeenreportedthatparticlesmaller than200nmcanpreferentiallyaccumulate intumortissuesvia diffusion-mediatedpassivetransport(EPReffect),whereas parti-clessmallerthan100nmcanpenetratedeepintheleakytumor vasculature(typicalporesize50–100nm)andarenotlimitedto vascularsurfaceonly[14,15]

Fig 3.TEM images of(A)MTX-PCM and(B)DOX-PCM.

3.2 Morphologicalandphysicalstateanalysis

Regardlessofthenatureof bindingof individualdrugs, both

MTX-PCMandDOX-PCMexhibiteddistinctsphericalshaped

par-ticlesuniformlydispersedontheTEMgrid(Fig.3A, B).Core-shell

architecturewasnotobservedduetothelongitudinalassembly

ofpolymerchains,butanelectron-densedarkcorerepresenting

thedrug-polymercomplexwasseen.Theparticlesizeobserved

intheTEMexperimentwassmallerthansizeobservedusingDLS

Thisdiscrepancyinobservedsizescanbeattributedtothefactthat

DLSmeasuresthehydrodynamicmicellesizewhileTEMcaptures

thedriedstate.Thephysicalstateoffreedrugsanddrug-loaded

micelleswasstudiedusingX-raydiffractionpatterns.Asshownin

Fig S1,freeDOXshowednumeroussharpandintensepeaksat

sev-eral2␪scatteredangles(12.5◦,16.2◦,17.3◦,21.2◦,22.5◦,25.1◦,and

26.2◦)andfreeMTXshowedpeaksbetween22.5−25.5◦reflecting

itshighcrystallinity.Allcharacteristicpeakswereabsentin

DOX-PCMaswellasMTX-PCM,indicatingthecompleteincorporationof

drugs.Theseresultssuggestthepresenceofdrugsintheamorphous

ormolecularlydispersedstate[16]

3.3 Drugloadingandinvitroreleasestudy

BoththeDOX-PCMandMTX-PCMexhibitedahighentrapment

efficiencyofmorethan90%withanactivedrugloadingof∼45%

w/wforMTX(MTX-PCM)and∼70%forDOX(DOX-PCM)atR=0.5

ThereleasestudyofMTXandDOXfromMTX-PCMandDOX-PCM

wasperformedinphosphate-bufferedsaline(pH7.4)and

acetate-bufferedsaline(pH5.0)tosimulatethephysiologicalandtumor

pHconditions AsevidentfromFig.4,MTX-PCMand DOX-PCM

exhibiteddifferentreleaseprofilesat bothpHconditions

MTX-PCMexhibiteda sustainedrelease profilethroughoutthestudy

periodwithnoinitialburst.DOX-PCM,ontheotherhand,exhibited

abiphasicreleasepattern.DOX-PCMexhibitedafasterrelease

pro-fileduringtheinitialtimeinterval(10–12h),butshowedaslower

releaselateron(48h).Forinstance,inthecaseofR=0.5,∼9%of

thedrugwasreleasedfromMTX-PCMafter12handapproximately

25%ofthedrugwasreleasedbytheendof48hatpH7.4.In

con-trast,∼30%ofthedrugwasreleasedfromDOX-PCMduringthe

first12h,whilethetotalreleasewas∼38%attheendofthestudy

period.AsimilartrendwasobservedinreleasemediaatpH5.0

whereinMTXwasreleased in a continuousfashion

(monopha-sic),whileDOXwasreleasedinabiphasicmanner.Thedifference

inreleasepatternscouldbeattributedtothechargedensityand

bindingaffinityofindividualdrugstowardstheanionicPAAblock

Fig 4.Release profiles of(A)MTX and(B)DOX from MTX-PCM and DOX-PCM at

pH 5.0 and pH 7.4 MTX-PCM and DOX-PCM were prepared at pH 7.0 The study was carried out in phosphate-buffered saline (pH 7.4) and acetate-buffered saline (pH 5.0) at 37 ◦ C.

Fig 5 (A)In vitro cellular uptake of (a) MTX and MTX-PCM and (b) DOX and DOX-PCM in SCC-7 cancer cells The cellular uptake experiment was performed by incubating the formulations at different time points.(B)Annexin V-FITC/PI based apoptosis assay in SCC-7 cancer cells The drug treated cells were stained with Annexin V-FITC and PI and evaluated using flow cytometry.

MTX,withtwosecondaryaminogroupscouldbeexpectedtohave

strongerbindingaffinityforthepolymerthanDOXwithasingle

primaryaminogroup

AnothersignificantobservationwasthatMTXandDOXwere

releasedfasterinacidicpH(pH5.0)thaninphysiologicalpH(pH

7.4).Forexample,approximately55%ofMTXwasreleasedfrom

MTX-PCMatacidicpH,whileonly∼25%ofthedrugwasreleased

atphysiologicalpHafter48hatafeedingratioofR=0.5.Asimilar

trendwasobservedinthecaseofDOX-PCM,wherein∼62%ofthe

drugwasreleasedinacidicmediacomparingto∼38%DOXrelease

after48hinbasicmedia.Theacceleratedreleaseofdrugsatacidic

pHcanbeattributedtotheprotonationofcarboxylicgroupsofthe

PAAblockinthemicelles[11]

Ingeneral,it isinteresting tonotethattherelease ratewas

higherfromPCMpreparedatafeedingratioofR=0.5thanfrom

those prepared with R=0.25 For example,∼14% of MTX was

releasedfromMTX-PCMwithR=0.25and∼21%wasreleasedfrom

MTX-PCMwithR=0.5duringthefirst8hatpH5.0.Asimilartrend

wasobservedatpH7.4:∼17%ofDOXwasreleasedfromDOX-PCM

withR=0.25and∼23%wasreleasedfromPCMwithR=0.5

dur-ingthefirst8h.Thisdifferenceinreleasecanbeattributedtothe

bindingandlocalizationofthedrugsinthecoreofPCM.Ahigh

loadingcapacityofPCMatR=0.5accountsforthelargerpresence

ofdrugsatthecore-shellinterfacefromwherethedrugscanrapidly

diffuseintothereleasemedium[17].Thegreaternumberofdrug

moleculestrappedinthecoreofPCMpreparedwithR=0.5hasa greaterchancetoreleasequicklyinthemediathanthefewerdrug moleculesfromthePCMpreparedwithR=0.25.Furthermore,at thelowfeedingratio(R=0.25),considerablenegativechargesare stillavailableonthePAAchainthatwillfurtherinducestrong elec-trostaticinteractions betweendrugsandthepolymerleadingto slowerreleaserates

3.4 CellularuptakepatternsofDOX-PCMandMTX-PCM Thecellularuptakebehavioroffreedrugsanddrug-loadedPCM wasinvestigatedinSCC-7cancercellsusingFACS[18,19].Asshown

inFig.5A,freeDOXandMTXshowedahighercellularuptake com-paredtodrug-loadedPCM.Thehighercellularuptakeoffreedrugs wasattributedtothesimplediffusionofdrugstotheintracellular environment,whereasmicellarnanocarrierscouldonlybe inter-nalizedby thecells throughendocytosis.The meanfluorescent intensity(MFI)offreeDOXwasgreatercomparedtoDOX-PCMand similarly,MFIofMTXwasgreatercomparedtothatofMTX-PCM after60-minincubationinSCC-7cancercells.Consistently, DOX-PCMandMTX-PCMshowedatypicaltime-dependentbehaviordue

tothepresenceofanendocytosisprocesswithinthesystem(Fig.

S2).We haveobservedthatthenanocarriersprimarily accumu-lateinthecytoplasmicregionwherethedrugisliberatedafterthe

internalizationofPCMinthiscellline

3.5 Apoptosisassay

Theexternalizationofphosphatidylserineduringapoptosisof

cancercellswasevaluatedbyannexin-V/PIstaining(Fig.5B).The

resultsshowedthatDOXinducedasignificantincreaseinAnnexin

V-positive and Annexin V+PI-positive cells, corresponding to

earlyand late apoptotic cells, respectively The MTX, however,

inducedapoptosisaswellasnecrosisofcancercells.Importantly,

drug-loadedPCMinducedhigherapoptosisrates ofcancercells

compared tofree drugs For example, MTX-PCMinduced∼25%

ofcellapoptosiscompared to∼15%inducedbyfree MTX

Simi-larly,DOX-PCMinduced∼34%ofcellapoptosiscomparedto∼25%

inducedbyfreeDOX.Highercellularapoptosisratesinducedby

PCMcouldbeattributedtothesustainedrelease oftherapeutic

cargointheintracellularenvironment.Itshouldbenotedthat

DOX-basedtherapywasmoreeffectiveininducinganticanceractivity

thanMTX-basedtherapy

3.6 Pharmacokineticanalysis

Theplasmaconcentration-timeprofilesoffreedrugs,MTX-PCM,

andDOX-PCMfollowingsingledoseadministrationarepresented

inFig.6 Asshown,free MTXand freeDOX wereclearedfrom

thesystemic compartment within 4–6hof intravenous

admin-istration.LinearpharmacokineticprofilesofMTXandDOXwere

consistentwithpreviousreports.Asexpected,PCMformulations

significantlyenhancedthebloodcirculationofbothMTXandDOX

Bothanticancerdrugsmaintainedsignificantlyhigherplasma

con-centrationsfor 24h Importantly,MTX-PCM showedprolonged

bloodcirculation,comparedtoDOX-PCM.After12h,theplasma

concentrationof MTXfromPCM was1.824±0.801␮g/ml

com-pared toplasma concentration of only 0.576±0.389␮g/mL for

DOX.ThefinalconcentrationsofMTXandDOXreleasedfromPCM

were1.258±0.392␮g/mLand0.176±0.151␮g/mL,respectively

Therespectivepharmacokineticparametersofdifferent

formu-lationsarepresentedinTable1.Consistentwithpreviousreports,

freedrugsexhibitedshortt1/2,highKel,andlowAUCall.Although

both PCM formulations improved thesystemic performance of

drugs,theymarkedlydifferamongthemselves.Forinstance,

MTX-PCM improvedthe AUCall of MTX 4-foldcompared to a 3-fold

increasebyDOX-PCMforDOX.Similarly,MTX-PCMhadan

approxi-mately5-fold(14.79±4.89h)highert1/2thanMTX,comparedwith

2.5-foldhighert1/2ofDOX-PCM(4.82±0.83h)inrelationtoDOX

Notably,Kelofthefreedrugswasreduced5-foldbyMTX-PCMand

2-foldbyDOX-PCM.Withregardtoallpharmacokinetic

parame-ters,MTX-PCMshowed2-foldhigherperformancethanDOX-PCM

Thesefindingsindicatetheremarkablebloodcirculationpotential

ofMTX-PCMcomparedtothatofDOX-PCM.Thedifferenceinthe

circulatoryperformanceofthetwoPCMformulationsisattributed

totheirphysiologicalstability[20].Previously,wehaveshownthat

DOX-PCMhavelowersaltstabilitythanMTX-PCM.Twosecondary

aminogroupsconferonMTXastrongerbindingaffinityforthe

polymer,comparedtoDOXwithitssingleprimaryaminogroup

[11,21]

Manyinferencescanbedrawnfromthisexperiment.First,the

bindingaffinityofthecationicdrugtothepolymerdeterminesits

bloodcirculationpotential;second,basedonthebindingstrength,

thereleaseofthedrugwillbesustainedorfaster;third,thegreater

thebindingstrength,thegreatertheinvivoperformanceof

drug-loadednanocarriersinthephysiologicalenvironment[22–25]

Fig 6.Plasma concentration-time profiles of MTX and DOX after intravenous administration of free drugs or drug-loaded PCM to rats at a dose of 5 mg/kg Each value represents the mean ± SD (n = 4) Drug-loaded PCM were prepared at R = 0.5 and pH 7.0.

3.7 Invivoantitumorefficacy The prolongedblood circulation and controlledrelease pro-filesof drug-loaded PCM were expected to contribute to their superiorantitumorefficacy.Theantitumorefficacyofindividual formulationswasinvestigatedinA-549cancercellsxenografted

onBALB/cnudemice.FreeMTX,freeDOX,MTX-PCM,and DOX-PCMwereintravenouslyinjectedintothetumorbearingmiceat

afixeddoseof5mg/kg.AsshowninFig.7A,tumorsrapidlygrew

intheuntreatedcontrolgroup,buttheirgrowthwassignificantly suppressedin groups treated with free drugs as wellas drug-loadedPCM.Notably,bothMTX-PCMandDOX-PCMsignificantly suppressedtumorgrowth.Invitrocytotoxicityassaysrevealedthe

IC50valuesforindividualformulations.TheIC50valuesofMTXand MTX-PCMwerefoundtobe0.85␮g/mland 0.94␮g/ml,

respec-Fig 7.Effect of drug-loaded PCM on(A)tumor growth and(B)body weight in A-549

xenograft-bearing female BALB/c nude mice (n = 6 per group) Each formulation was

administered three times at three day intervals Drug-loaded PCM were prepared at

R = 0.5 and pH 7.0.

tively,whiletheIC50valuesofDOXandDOX-PCMwere1.68␮g/ml

and2.13␮g/ml,respectively.Whileadefinitedifferenceinblood

circulationwasobservedbetweenMTX-PCMandDOX-PCMinthe

pharmacokineticstudy,nosignificantdifferenceinantitumor

effi-cacycouldbedetected.BothMTX-PCMandDOX-PCMinhibited

tumorgrowthtothesamelevelthroughoutthestudyperiod.These

whichresultedinoverallbodyweaknessthataffectedthetumor tissueaswell.TheenhancedtumorregressioncausedbyPCM for-mulationscanbeattributedtotheprolongedhalf-lifeofanticancer drugs,reducedeliminationofindividualdrugs,andmost impor-tantlytothepreferentialaccumulationofnanocarriersinthetumor tissueduetotheEPReffect[26–28]

The toxicityof formulations wasevaluatedusing micebody weight(Fig.7B).Asshown,freeMTXcausedanapproximately30% decreaseinbodyweightindicatingitsseveredrug-relatedtoxicity MTX-PCM,however,greatlyreducedMTXtoxicityinsystemic cir-culation.ThiscouldbeduetothefactthatencapsulationofMTXin thePCMreducedtherandomexposureofnormaltissuestoitand increasedMTX’spassiveaccumulationintumortissues,thereby reducing theundesirablesideeffects [29,30].DOX-PCMdidnot exhibitanybodyweightreduction

In summary, cationic drugs-loadedPCM were prepared and evaluatedintermsofphysicochemicalandinvivoparameters.Both MTX-PCMandDOX-PCMdisplayedsphericalnanosizedparticles withuniformdispersityindices.MTX-PCMandDOX-PCMexhibited differentreleaseprofilesunderallpHconditionsstudied.MTX-PCM exhibitedamonophasicreleasepatternwithnoinitialburst,while DOX-PCMexhibitedabiphasicreleasepattern.Interestingly,drug releaserateswerehigherfromPCMpreparedatafeedingratioof

R=0.5thanfromthosepreparedwithR=0.25.DOX-PCMshowed

ahighercellularuptakecomparedtoMTX-PCMinSCC-7cancer cells;consistentlyDOX-PCMinducedhigherapoptosisratesof can-cercellsthanMTX-PCM.Incontrast,MTX-PCMshowedprolonged bloodcirculationcomparedtoDOX-PCM.MTX-PCMimprovedthe AUCallofMTX4-foldcomparedtoa3-foldincreasebyDOX-PCMfor DOX.Similarly,MTX-PCMhada5-foldhighert1/2thanMTX,while DOX-PCMincreasedtheDOXt1/22.5-fold.However,both MTX-PCMandDOX-PCMsuppressedtumorgrowthtothesamelevelsas theirrespectivefreedrugs.Takentogether,ourresultsshowthat natureofinteractionsofcationicdrugswiththepolyionic copoly-mercanhaveatremendousinfluenceonthebiologicalperformance

ofdeliverysystems

Theauthorsdeclarenoconflictofinterestinthiswork

Acknowledgements

Thisworkwassupportedbythe2015Yeungnam University ResearchGrant

Appendix A Supplementary data

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.colsurfb.2016.06

004

References

[1] M.M Gottesman, T Fojo, S.E Bates, Multidrug resistance in cancer: role of

ATP dependent transporters, Nat Rev Cancer 2 (2002) 48.

[2] A.T Fojo, K Ueda, D.J Slamon, D.G Poplack, M.M Gottesman, I Pastan,

Expression of a multidrug-resistance gene in human-tumors and tissues, Proc.

Natl Acad Sci U S A 84 (1987) 265.

[3] G Riess, Micellization of block copolymers, Prog Polym Sci 28 (2003)

1107–1170.

[4] J.F Cohy, Block copolymer micelles, Adv Polym Sci 190 (2005) 65–136.

[5] H Maeda, The enhanced permeability and retention (EPR) effect in tumor

vasculature: the key role of tumor-selective macromolecular drug targeting,

Adv Enzyme Regul 41 (2001) 189–207.

[6] J.O Kim, A.V Kabanov, T.K Bronich, Polymer micelles with cross-linked

polyanion core for delivery of a cationic drug doxorubicin, J Control Rel 138

(2009) 197–204.

[7] J.O Kim, T Ramasamy, C.S Yong, N.V Nukolova, T.K Bronich, A.V Kabanov,

Cross-linked polymeric micelles based on block ionomer complexes,

Mendeleev Commun 23 (2013) 179–186.

[8] A.V Kabanov, T.K Bronich, V.A Kabanov, K Yu, A Eisenberg, Soluble

stoichiometric complexes from poly(N-ethyl-4-vinylpyridinium) cations and

poly(ethylene oxide)-block-polymethacrylate anions, Macromolecules 29

(1996) 6797–6802.

[9] T.K Bronich, A.M Popov, A Eisenberg, V.A Kabanov, A.V Kabanov, Effects of

block length and structure of surfactant on self-assembly and solution

behavior of block ionomer complexes, Langmuir 16 (2000) 481–489.

[10] T Ramasamy, T.H Tran, H.J Cho, J.H Kim, Y.I Kim, J.Y Jeon, H.G Choi, C.S.

Yong, J.O Kim, Chitosan-based polyelectrolyte complexes as potential

nanoparticulate carriers: physicochemical and biological characterization,

Pharm Res 31 (2014) 1302–1314.

[11] T Ramasamy, J.H Kim, H.G Choi, C.S Yong, J.O Kim, Novel dual drug-loaded

block ionomer complex micelles for enhancing the efficacy of chemotherapy

treatments, J Biomed Nanotech 10 (2014) 1304–1312.

[12] B Manocha, A Margaritis, Controlled release of doxorubicin from

doxorubicin/-polyglutamic acid ionic complex, J Nanomat 2010 (2010)

(Article ID 780171).

[13] T Ramasamy, Z.S Haidar, T.H Tran, J.Y Choi, J.H Jeong, B.S Shin, H.G Choi,

C.S Yong, J.O Kim, Layer-by-layer assembly of liposomal nanoparticles with

PEGylated polyelectrolytes enhances systemic delivery of multiple anticancer

drugs, Acta Biomat 10 (2014) 5116–5127.

[14] D.W Kim, T Ramasamy, J.Y Choi, J.H Kim, C.S Yong, J.O Kim, H.G Choi, The

influence of bile salt on the chemotherapeutic response of docetaxel loaded

thermosensitive nanomicelles, Int J Nanomed 9 (2014) 3815–3824.

[15] B Gupta, B.K Poudel, T.H Tran, R Pradhan, H.J Cho, J.H Jeong, B.S Shin, H.G.

Choi, C.S Yong, J.O Kim, Modulation of pharmacokinetic and cytotoxicity

profile of imatinib base by employing optimized nanostructured lipid carriers,

Pharm Res 32 (2015) 2912–2927.

[16] B Gupta, B.K Poudel, S Pathak, J.W Tak, H.H Lee, J.H Jeong, H.G Choi, C.S Yong, J.O Kim, Effects of formulation variables on the particle size and drug encapsulation of imatinib-loaded solid lipid nanoparticles, AAPS

PharmSciTech (2015).

[17] T Ramasamy, J.H Kim, J.Y Choi, T.H Tran, H.G Choi, C.S Yong, J.O Kim, pH sensitive polyelectrolyte complex micelles for highly effective combination chemotherapy, J Mater Chem B 2 (2014) 6324.

[18] X Qi, Y Chen, Z Sun, X Ma, W Guo, Y Lu, Duan, Cytotoxicity and cellular uptake evaluation of mitoxantrone-loaded poly(lactic acid-co-lysine) arginine–glycine–aspartic acid nanoparticles, J Appl Polym Sci 119 (2011) 1011–1015.

[19] R Zeng, A.M Morgenstern, Nyström, Nanoparticle-directed sub-cellular localization of doxorubicin and the sensitization breast cancer cells by circumventing GST-mediated drug resistance, Biomaterials 35 (2014) 1227–1239.

[20] H.B Ruttala, Y.T Ko, Liposome encapsulated albumin-paclitaxel nanoparticle for enhanced antitumor efficacy, Pharm Res 32 (2015) 1002–1016 [21] J.O Kim, H.S Oberoi, S Desale, A.V Kabanov, T.K Bronich, Polypeptide nanogels with hydrophobic moieties in the cross-linked ionic cores: synthesis, characterization and implications for anticancer drug delivery, J Drug Target 21 (2013) 981–993.

[22] T.H Tran, T Ramasamy, D.H Truong, B.S Shin, H.G Choi, C.S Yong, J.O Kim, Development of vorinostat-loaded solid lipid nanoparticles to enhance pharmacokinetics and efficacy against multidrug-resistant cancer cells, Pharm Res 31 (2014) 1978–1988.

[23] T.H Tran, J.Y Choi, T Ramasamy, D.H Truong, C.N Nguyen, H.G Choi, C.S Yong, J.O Kim, Hyaluronic acid-coated solid lipid nanoparticles for targeted delivery of vorinostat to CD44 overexpressing cancer cells, Carbohydr Polym.

114 (2014) 407–415.

[24] T Ramasamy, Z.S Haidar, Formulation, characterization and cytocompatibility evaluation of novel core–shell solid lipid nanoparticles for the controlled and tunable delivery of a model protein, J Bionanosci 5 (2011) 143–154 [25] T Ramasamy, T.H Tran, J.Y Choi, H.J Cho, J.H Kim, J.H Choi, C.S Yong, J.O Kim, Layer-by-layer coated lipid?polymer hybrid nanoparticles designed for use in anticancer drug delivery, Carb Polym 102 (2014) 653–661.

[26] J.U Assumpc¸ ao, M.L Campos, M.A Ferraz Nogueira Filho, K.C Pestana, H.M Baldan, T.P Formariz Pilon, et al., Biocompatible microemulsion modifies the pharmacokinetic profile and cardiotoxicity of doxorubicin, J Pharm Sci 102 (2013) 289.

[27] D.S Alberts, Y.M Peng, G.T Bowden, W.S Dalton, C Mackel, Pharmacology of mitoxantrone: mode of action and pharmacokinetics, Invest New Drugs 3 (1985) 101–107.

[28] J.H Kim, Y.S Kim, K Park, S Lee, H.Y Nam, K.H Min, et al., Antitumor efficacy

of cisplatin-loaded glycol chitosan nanoparticles in tumor-bearing mice, J Control Rel 127 (2008) 41–49.

[29] T Ramasamy, H.B Ruttala, J.Y Choi, T.H Tran, J.H Kim, S.K Ku, H.G Choi, C.S Yong, J.O Kim, Engineering of a lipid-polymer nanoarchitectural platform for highly effective combination therapy of doxorubicin and irinotecan, Chem Commun 51 (2015) 5758.

[30] T Ramasamy, J.Y Choi, H.J Cho, S.K Umadevi, B.S Shin, H.G Choi, C.S Yong, J.O Kim, Polypeptide-based micelles for delivery of irinotecan:

physicochemical and in vivo characterization, Pharm Res 32 (2015) 1947–1956.