Nanoparticles Effectively Target Rapamycin Delivery to Sites of Experimental Aortic Aneurysm in ...

21 Pages·2016·9.49 MB·English

RESEARCHARTICLE
Nanoparticles Effectively Target Rapamycin
Delivery to Sites of Experimental Aortic
Aneurysm in Rats
TakuroShirasu1,HiroyukiKoyama1,2,3*,YutakaMiura4,5,KatsuyukiHoshina1,
KazunoriKataoka4,5,ToshiakiWatanabe1
1 DivisionofVascularSurgery,DepartmentofSurgery,GraduateSchoolofMedicine,TheUniversityof
Tokyo,Tokyo,Japan,2 TranslationalResearchCenter,TheUniversityofTokyoHospital,Tokyo,Japan,
3 DepartmentofVascularSurgery,SaitamaMedicalCenter,SaitamaMedicalUniversity,Saitama,Japan,
4 DepartmentsofMaterialsEngineeringandBioengineering,GraduateSchoolofEngineering,The
UniversityofTokyo,Tokyo,Japan,5 CenterforDiseaseBiologyandIntegrativeMedicine,GraduateSchool
a11111 ofMedicine,TheUniversityofTokyo,Tokyo,Japan
*[email protected]
Abstract
OPENACCESS Severaldrugstargetingthepathogenesisofaorticaneurysmhaveshownefficacyinmodel
systemsbutnotinclinicaltrials,potentiallyowingtothelackoftargeteddrugdelivery.Here,
Citation:ShirasuT,KoyamaH,MiuraY,HoshinaK,
KataokaK,WatanabeT(2016)Nanoparticles wedesignedanoveldrugdeliverysystemusingnanoparticlestotargetthedisruptedaortic
EffectivelyTargetRapamycinDeliverytoSitesof aneurysmmicro-structure.Wegeneratedpoly(ethyleneglycol)-shellednanoparticlesincor-
ExperimentalAorticAneurysminRats.PLoSONE11
poratingrapamycinthatexhibiteduniformdiameterandlong-termstability.Wheninjected
(6):e0157813.doi:10.1371/journal.pone.0157813
intravenouslyintoaratmodelinwhichabdominalaorticaneurysm(AAA)hadbeeninduced
Editor:HelenaKuivaniemi,StellenboschUniversity
byinfusingelastase,labeledrapamycinnanoparticlesspecificallyaccumulatedintheAAA.
FacultyofMedicineandHealthSciences,SOUTH
AFRICA Microscopicanalysisrevealedthatrapamycinnanoparticlesweremainlydistributedinthe
mediaandadventitiawherethewallstructuresweredamaged.Co-localizationofrapamycin
Received:February17,2016
nanoparticleswithmacrophageswasalsonoted.Rapamycinnanoparticlesinjectedduring
Accepted:June6,2016
theprocessofAAAformationevincedsignificantsuppressionofAAAformationandmural
Published:June23,2016 inflammationat7daysafterelastaseinfusion,ascomparedwithrapamycintreatment
Copyright:©2016Shirasuetal.Thisisanopen alone.Correspondingly,theactivitiesofmatrixmetalloproteinasesandtheexpressionof
accessarticledistributedunderthetermsofthe inflammatorycytokinesweresignificantlysuppressedbyrapamycinnanoparticletreatment.
CreativeCommonsAttributionLicense,whichpermits
Ourfindingssuggestthatthenanoparticle-baseddeliverysystemachievesspecificdelivery
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare ofrapamycintotheratAAAandmightcontributetoestablishingadrugtherapyapproach
credited. targetingaorticaneurysm.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:ThisstudywassupportedbytheFunding
ProgramforWorld-LeadingInnovativeR&Don
Introduction
ScienceandTechnology(FIRSTProgram,nospecific
number)fromtheJapanSocietyforthePromotionof
Aorticaneurysmisacommonandlethaldisease,whichspontaneouslyexpandsandcarriesthe
Science(JSPS)andGrants-in-AidforScientific
riskofrupture[1,2].Thecurrentmainstreamtreatmentsforaorticaneurysminvolveinvasive
Research(No.23591859toK.H.)fromtheMinistryof
therapiesincludingsurgicalreplacementwithavascularprosthesisandendovascularimplanta-
Education,Culture,Sports,ScienceandTechnology
ofJapan. tionofstentgrafts;however,fewdrugtherapieshavebeenappliedclinicallytosuppressaortic
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CompetingInterests:Theauthorshavedeclared aneurysmformation[3].Accumulatedevidencehasdemonstratedthattheaneurysmalforma-
thatnocompetinginterestsexist. tionisassociatedwithchronicinfiltrationofinflammatorycellsanddegradationoftheextra-
cellularmatrix(ECM)intheaorticwall[4].Onthebasisofthisfinding,severaldrugsagainst
suchpathogenesishavebeentestedinanimalmodelsofaorticaneurysmandhavesuccessfully
shownsignificanteffectstowardthesuppressionofaneurysmalformation[5–10].However,
thesepromisingdrugshaveexhibitedonlylimitedtherapeuticefficaciesinclinicaltrials[11–
16],potentiallybecausenomethodisavailabletospecificallydeliverthedrugstotheaneurys-
malwall.Specifictargetingtotheaneurysmalwallmightallowthedeliveryofsufficientdoses
ofthedrugforclinicaleffectwithoutadverseeffectstootherorgans,leadingtotheestablish-
mentofadrugtherapymechanismforaorticaneurysm.
Inthepresentstudy,weattemptedtodevelopaspecificdrugdeliverysystemtargeting
aorticaneurysmbyutilizingnanoparticlesasadrugcarrier.Previoushistopathologicalstud-
iesofaneurysmalspecimensrevealedconsiderabledisruptionanddegenerationofthewall
structureofaorticaneurysms,indicatingtheabundanceofstructuralmicro-defects[17–20].
Wethereforeconsideredthatnanoparticlesinthebloodstreammightinfiltrateandbedis-
tributedtotheaneurysmalwallviathesedefects,whichrepresentsapossibilityforrealizinga
specificdeliverysystemforaorticaneurysm.Toverifytheapproachutilizingnanoparticles,
wefirstincorporatedrapamycinintothenanoparticle(rapamycinnanoparticle),asrapamy-
cinwaspreviouslyreportedtosuppressaneurysmalformationbyitsanti-inflammatory
effects[21].Subsequently,wesystemicallyadministeredtherapamycinnanoparticlestoarat
modelofabdominalaorticaneurysm(AAA)andevaluatedtheirdistributionandtherapeutic
effectsagainstAAA.
MaterialsandMethods
Preparationofrapamycinnanoparticles(Fig1)
Poly(ethyleneglycol)-b-poly(γ-benzylL-glutamate)(PEG-b-PBLG;number-averagemolecular
weight=21,000,polydispersity=1.04,degreeofpolymerizationinPBLGsegment=41)wassyn-
thesizedbasedonourdevelopedmethods[22–25].Forfluorescenceanalysis,PEG-b-PBLGwas
labeledbyconjugatingtheAlexa647succinimidylesters(ThermoFisherScientific,Carlsbad,CA,
USA)totheω-aminogroupofthepolymer.Inordertopreparerapamycinnanoparticles,PEG-
b-PBLG(25mg)andrapamycin(25mg)weredissolvedinN,N-dimethylformamide(DMF,1.0
mL,WakoPureChemicalIndustries,Tokyo,Japan)andstirredfor24h.TheDMFsolutionwas
thenaddedtovigorouslystirredwater(49mL).Theresultingsuspensionwaspurifiedbydialysis
withacellophanetube(Spectra/Pro6membrane:MWCO,3500,GEHealthcare,Uppsala,Swe-
den)againstwater.Thepurifiedsolutionwasconcentratedbyultrafiltrationusingapolyethersul-
fonemembrane(Vivaspin6;MWCO,300,000,GEHealthcare,Chalfont,UK)andfilteredbefore
use.Toestimatetherapamycinconcentrationintheobtainedrapamycinnanoparticles,a1.0mL
sampleofthepurifiedsolutionwasfreeze-driedandanalyzedby1Hnuclearmagneticresonance
(1HNMR)spectroscopy(solvent,d-DMSO;r.t.;JEOLLNM-ECS400,JEOL,Tokyo,Japan).The
diameterandpolydispersityindex(PDI)oftherapamycinnanoparticlesweremeasuredviaa
dynamiclightscatteringmethodusingaZetasizer(MalvernInstruments,Worcestershire,UK).
Accordingly,therapamycinnanoparticlesweresuspendedin150mMNaClsolution(pH=7.4)
andthelightscatteringintensitiesofthesuspensionweremeasuredsequentiallybytheZetasizer.
ElastaseinducedmodelofAAAinrats
AllprocedureswereperformedasrecommendedbytheGuidefortheCareandUseofLabora-
toryAnimalspublishedbytheUSNationalInstitutesofHealth(NIHPublication,8thedition,
2011).AllprotocolswereapprovedbytheInstitutionalAnimalCareandUseCommitteeof
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Fig1.Structureandpropertiesofrapamycin-incorporatednanoparticles.(A)Illustrationshowingthatrapamycin-incorporatednanoparticles
(rapamycinnanoparticles)aresynthesizedbymixinganequalmassofrapamycinandpoly(ethyleneglycol)-b-poly(γ-benzylL-glutamate)(PEG-b-PBLG).
RapamycinisincorporatedintothecoreofthePEG-shellednanoparticle.(B)ThediameterofnanoparticlesmeasuredbyaZetasizer.Thesizeofthe
nanoparticlesformedsolelywithPEG-b-PBLGwas42nm(dottedline),whereasthatoftherapamycinnanoparticleswas106nm(solidline).(C)Time-
courseofthescatteredlightintensityofrapamycinnanoparticlesunderphysiologicalconditions.Rapamycinnanoparticlesarestableoverthefirst2days.
Datarepresentthemeans±standarddeviation(s.d.).
doi:10.1371/journal.pone.0157813.g001
theUniversityofTokyo(PermitNumber:M-P09-017)andwereperformedinaccordancewith
theinstitutionalguidelinesasstatedbytheUniversityofTokyo.MaleSprague-Dawleyrats(7–
8weeks,300–350g;CharlesRiverLaboratories,Yokohama,Japan),fedanormaldiet,were
usedintheexperiments.Theanimalswerekeptinanair-conditioned(21°C±1°C)andspe-
cific-pathogenfreeenvironmentwitha12hlight-darkcycle.Inanisolationracktherewere
2–3companionsatmost,andtheyhadfreeaccessfoodandwater.Thetotalnumberofrats
usedinthisexperimentwas62.AAAwasinducedaccordingtothemethodofAnidjarand
Dobrinwithmodifications,asdescribed[26,27].Inbrief,ratswereanesthetizedwith
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inhalationof1–2%isoflurane,withconcentrationadjustmentsifslightmovementoftherats
wasdetected.Therefore,theheartrateandrespiratoryratewerestableduringtheentireproce-
dure.A10-mmsegmentoftheinfrarenalaortawascompletelyisolatedunderamid-linelapa-
rotomy.Themaximumdiameteroftheisolatedaortawasmeasuredwithelectricmicro-
calipersinsystole.Then,aPE-10tubewasintroducedintotheinfrarenalaortafromtheright
saphenousartery,andtheproximalanddistalpartsoftheisolatedaortawereclampedby
meansoftemporaryligation.ThroughthePE-10tube,porcinepancreaticelastase(0.27units,
TypeI;Sigma-Aldrich,St.Louis,MO,USA)in0.27mLphosphatebufferedsaline(PBS)was
continuouslyinfusedover0.9hoursusingasyringepump.Aftertheinfusion,theaortawas
declamped,thePE-10tubewasremoved,andthemaximumdiameteroftheisolatedaortawas
measuredinthesamemanner.
DistributionofrapamycinnanoparticlesintheAAAratmodel
RatsweresubjectedtoelastaseinfusiontoinduceAAA,and7dayslater,500μgAlexa647-la-
beledrapamycinnanoparticlesweresuspendedin500μLPBS,andthesuspensionwasinjected
viathetailveinoftherats(n=19,Fig2A).Afterbloodsampling,theanimalswerekilledbyan
anesthesiaoverdoseat1,4,8,16,or24hoursafterinjection(at1,4,8,24hours,n=4;at16
hours,n=3)afterbloodsampling.Subsequently,thethoracicaorta,abdominalaorta,and
bilateraliliacarterieswereharvestedtogether,andweregentlyrinsedwithPBStowashoutthe
remainingblood.Theaorto-iliacspecimensweresetflatwiththeanteriorsidefacingupwards,
andthedistributionofAlexa647-labeledrapamycinnanoparticleswasfirstevaluatedwithan
IVIS1imagingsystem(excitationfilter:640nm,emissionfilter:680nm,PerkinElmer,Hop-
kinton,MA,USA).Asacontroltoeliminatetheintrinsicfluorescenceoftherataortictissue,
anotherrat(n=1)wassubjectedtoelastaseinfusionandsacrificed7dayslaterwithoutinjec-
tionofAlexa647-labeledrapamycinnanoparticles,followedbysamplecollectioninthesame
manner.
Forquantitativeevaluation,plasmawasseparatedfromeachbloodsample,andthefluores-
cenceintensitiesoftheplasmasamplesweremeasuredat680nm.Thedistributionhalf-lifeand
eliminationhalf-lifewereestimated,andtheresidualquantitiesofrapamycinnanoparticlesin
theplasma(residualratio)werecalculatedasdescribedpreviously[28].ThesegmentofAAA
andthethoracicaortawerecutoutindividuallyfromeachaorto-iliacspecimenaftertheevalua-
tionbyIVIS1.Eachsegmentwasweighed(W [g])andlysedin1mLPassivelysisbuffer
sample
(E194A,Promega,Fitchburg,WI,USA).Theabsorbanceofeachlysateat680nm(A )was
sample
measuredusinganInfinite1microplatereader(Tecan,Männedorf,Switzerland).Theweight
andabsorbanceofthecontrolsamplewerealsomeasured(W [g]andA ).A data
control control sample
wereadjustedasvaluesper1gsampleweightasfollows:
Asample Acontrol
Adjustedabsorbancevalue¼ þ
Wsample Wcontrol
HistologyofrapamycinnanoparticlesdistributedtotheAAA
ToanalyzethemicroscopicdistributionofrapamycinnanoparticlesintheAAAwall,another
setofrats(n=3)weresubjectedtoelastaseinfusionandtreatedwithinjectionofAlexa647-la-
beledrapamycinnanoparticlesatday7inthesamemanner.ThemiddlesegmentsoftheAAAs
wereexcisedfromeachratat1dayaftertheparticleinjectionandembeddedinOCTcom-
pound(SakuraFinetek,Tokyo,Japan).Transversecrosssections(10μm)werestainedby
Hoechst33342(ThermoScientific,Waltham,MA,USA),andthesectionwasanalyzedbycon-
focallaserscanningmicroscopy(LSM780,CarlZeiss,Oberkochen,Germany).
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Fig2.Flow-chartsofthestudiestoanalyzedistributionandtherapeuticeffectofrapamycin
nanoparticles.(A)Flow-chartofthestudyonthedistributionofrapamycinnanoparticlestoabdominalaortic
aneurysm(AAA).(B)Flow-chartofthestudyonthetherapeuticeffectofrapamycinnanoparticlestosuppress
theformationofAAA.
doi:10.1371/journal.pone.0157813.g002
Othersectionswereimmuno-stainedforCD68orα-smoothmuscleactin(αSMA)toevalu-
atetheco-localizationofrapamycinnanoparticleswithmacrophagesandsmoothmusclecells
(SMCs),respectively.Afterblocking(Blockingone,NacalaiTesque,Inc.,Kyoto,Japan),a
monoclonalantibodyagainstCD68(1:100;T-3003,BMABiomedicals,Augst,Switzerland)or
againstαSMA(1:100;M0851,Dako,Glostrup,Denmark)wasapplied,andthesectionswere
incubatedovernightat4°C.Then,thesectionsweretreatedwithAlexaFluor1555-labeled
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secondaryantibody(1:300;ab150118,Abcam,Cambridge,UK)andanalyzedbyconfocallaser
scanningmicroscopy.
Therapeuticeffectsofrapamycinnanoparticles
Ratswererandomlyassignedtofivetreatmentgroups,andelastasewasinfusedintotheinfra-
renalaortaofeachanimalasdescribed.Measurementofthemaximumdiameteroftheisolated
aortawasconductedineachanimalbeforeandafterelastaseinfusionasdescribedabove(Fig
2B).Immediatelyafterandat2,4,and6daysaftertheelastaseinfusion,theratsofeachgroup
respectivelyreceivedintravenousinjectionsofthefollowingsuspensionorsolutionviathetail
vein(i.e.,injectionsfourtimesineachgroup):i)1mL/kgPBSalone(PBS,n=6),ii)0.1mg/kg
freerapamycinin1mL/kgspecialsolvent(free/RAP-0.1,n=6),iii)1mg/kgfreerapamycinin
1mL/kgspecialsolvent(free/RAP-1,n=6),iv)0.1mg/kgrapamycinnanoparticlesin1mL/kg
PBS(RAP/nano-0.1,n=6),orv)1mg/kgrapamycinnanoparticlesin1mL/kgPBS(RAP/
nano-1,n=6).Thespecialsolventforfreerapamycinconsistedof5%Tween80,5%polyethyl-
eneglycol400,and5%ethanolindistilledwater[29].At7daysaftertheelastaseinfusion,the
inducedAAAofeachanimalwasexposed,andthemaximumdiameterofeachAAAwasmea-
suredinsystole.Thediameterratioisexpressedforeachratastheratioofthediameterafter
elastaseinfusiontothediameterbeforeelastaseinfusion.Subsequently,allratsweresacrificed
andsubjectedtoperfusionfixationwith4%phosphatebufferedparaformaldehyde(0.1molL
PO ,pH7.3)at120mmHg.ThesegmentofAAAwasexcisedandimmersedinthesamefixa-
4
tivefor1hour.ThesamplesegmentwasexcisedfromthemiddlepartoftheAAAandembed-
dedinparaffin.
HistologicalanalysesoftheAAAwall
Transversecrosssections(5μm)oftheAAAwallwerecutfromparaffin-embeddedsamples.
Thesectionswerestainedbyhematoxylin-eosin(HE)orElasticavanGieson(EVG)staining.
Othersectionswereimmuno-stainedforCD68orαSMA.Afterhydrogenperoxidetreatment,
blocking,andincubationwithprimaryantibodiesasdescribed,thesectionsweretreatedwith
biotin-labeledsecondaryantibody(1:500;sc-2039,SantaCruzBiotechnology,Dallas,TX,
USA)andvisualizedusinganavidin-biotincomplexdetectionkit(ABCElitekit,VectorLabo-
ratories,Burlingame,CA,USA).Photomicrographsweretaken,andtheareaoftheaneurysmal
wallwasmeasuredineachsectionusingImageJsoftware(NIH,Bethesda,MD,USA).The
CD68-positivecellsintheaneurysmalwallwerecounted,andtheCD68-positivecelldensity
wascalculatedasfollows:
numberof CD68(cid:2)positivecells
CD68(cid:2)positivecelldensity¼
specimenarea
Gelatinaseactivityandexpressionofinflammatorycytokines
Anothersetofratswasassignedtothreetreatmentgroups,andelastasewasinfusedintotheir
infarenalaortas.Accordingtothesameprotocolasdescribed,theratsofeachgroupreceived
injectionsofPBS(n=3),free/RAP-1(n=3),orRAP/nano-1(n=3)immediatelyafterandat
2,4,and6daysaftertheelastaseinfusion.At7daysafterelastaseinfusion,thesegmentof
AAAwasexcisedfromeachanimalandlysedin200μLPassivelysisbuffer.Gelatinaseactivity
ofthetissuelysateswasdeterminedusingagelatin-zymographykit(AK47,PrimaryCellCo,
Sapporo,Japan)accordingtothemanufacturer’sinstruction.Equaltotalprotein(50μg)
amountsofeachlysatewereseparatedonSDS-PAGEcontaining1%gelatin,andthe
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SDS-PAGEgelwasincubatedinincubationbufferfor10hoursat37°C.Matrixmetalloprotei-
nase(MMP)marker(includedinthegelatin-zymographykit,seesupplier’sdatasheet:http://
search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/PMC_/AK47COS_E.20160418.
pdf)containingMMP-2,thelatentformMMP-2(pro-MMP-2),andthelatentformMMP-9
(pro-MMP-9)wereappliedinseparatelanes.Thegelwasscannedusingatransmissionmodel
scanner(ESPER-SCANNER1,SeikoEpson,Nagano,Japan)forzymographyanalysis,andthe
densitometricanalysiswasperformedusingImageJsoftware.Inaddition,theexpressionof
theinflammation-relatedcytokinesandchemokinesinthetissuelysateswasevaluatedusinga
ProteomeProfilerArraykit(ratcytokinearraypanelA,R&DSystems,Minneapolis,MN,
USA)accordingtothemanufacturer’sinstruction[30].Thiskitenablestheprofilingoftherel-
ativelevelsofthefollowingcytokinesandchemokinesbetweentissuelysates:cytokine-induced
neutrophilchemoattractant(CINC)-1,CINC-2α/β,CINC-3,interleukin(IL)-1α,IL-1β,IL-6,
IL-10,IP-10,macrophageinflammatoryprotein(MIP)-1α,MIP-3α,regulatedonactivation,
normalTcellexpressedandsecreted(RANTES),andtumornecrosisfactor(TNF)-α.Eachtis-
suelysatewith400μgtotalproteinwasappliedtothearray.Theresultantarraymembrane
wasexposedtoX-rayfilmandthepixeldensitiesonthedevelopedX-rayfilmwerecollected
usingatransmissionmodelscanner.TheacquireddatawereanalyzedbyImageJsoftware.
Statisticalanalyses
Thevaluesareexpressedasthemeans±standarddeviation.Dunnett’stestwasappliedto
determinethesignificanceofthedifferencescomparedtothecontrolgroup.Thedifferences
betweentwogroupswereanalyzedbyanunpairedStudent’st-test.Apvalue<0.05wascon-
sideredtobesignificant.
Results
Characteristicsofrapamycinnanoparticles
Rapamycin-incorporatednanoparticlesweresimplypreparedbymixinganequalmassofrapa-
mycinandPEG-b-PBLG(Fig1A)[31].Theyieldofrapamycinincorporationwasmeasuredby
1HNMRandestimatedtobe82%.TheanalysesusingtheZetasizerrevealedthatthediameter
oftherapamycinnanoparticleswas106nmwithamoderatePDI(i.e.,0.170),whilethatofpar-
ticlesformedsolelywithPEG-b-PBLGwas42nm(Fig1B).Underphysiologicalconditions
(pH=7.4,150mMNaCl,37°C),thescatteredlightintensitiesoftherapamycinnanoparticles
werestableduringtheinitial2days.Thereafter,thescatteredlightintensitiesdecreasedgradu-
ally,althoughthevalueatday6remainedat78%ofthatatthestartofmeasurement(Fig1C).
Distributionandplasmaclearanceofrapamycinnanoparticles
Toassesstheinvivodistributionoftherapamycinnanoparticles,Alexa647-labeledrapamycin
nanoparticleswereintravenouslyinjectedintoratsinwhichAAAhadbeeninducedbyinfus-
ingelastaseintotheabdominalaorta.Theanimalswereeuthanizedbyoverdoseanesthesiaat
1,4,8,16,or24hoursafterinjection(at1,4,8,24hours,n=4;at16hours,n=3)afterblood
sampling.Intheaorto-iliacspecimensharvestedat1hourafterinjection,IVIS1imaging
showedabundantfluorescencesignalovertheentirespecimens,andastrongintensityoffluo-
rescenceontheaorticarchascomparedwithotherpartsofthespecimens(Fig3A).At4hours
afterinjection,evidentfluorescencesignalwasdetectedonthesegmentsoftheaorticarchwith
AAA,andthentheevidentfluorescencesignalwasrestrictedtoonlytheAAAsegmentat8,16,
and24hoursafterinjection.
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Fig3.AccumulationofAlexa647-labeledrapamycinnanoparticlesintheAAAratmodel.(A)Representative
imagesofmacroscopicdistributionofAlexa647intheaorto-iliacspecimensasimagedusingtheIVIS1imaging
system.AbundantsignalsofAlexa647werespecificallydistributedintheAAAat4,8,16,and24hoursafter
injectionofAlexa647-labeledrapamycinnanoparticles(red-staining,at1,4,8,and24hours,n=4;at16hours,
n=3).(B)Theplasmaclearanceofrapamycinnanoparticlesintheratmodel.Theresidualratioofrapamycin
nanoparticlesintheplasmawashighat1hourafterinjection.(C)FluorescenceintensitiesofthelysatesofAAA
andthethoracicaortaareshownasadjustedabsorbancevalues.ThevaluesofAAAweresignificantlyhigherthan
thoseofthethoracicaortaat8,16,and24hoursafterinjection.Datarepresentthemeans±s.d.N.C.indicates
negativecontrol.*p<0.05,†p<0.01(unpairedStudent’st-test).
doi:10.1371/journal.pone.0157813.g003
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Beforeharvestingthespecimensat1,4,8,16,or24hoursafterinjection,aplasmasample
wascollectedfromeachanimal.Then,thefluorescenceintensitiesoftheplasmasampleswere
measured,andtheclearanceoftherapamycinnanoparticleswascalculated.Thedistribution
half-lifeandeliminationhalf-lifewereestimatedas2.1hoursand15.8hours,respectively(Fig
3B).Theresidualratioofrapamycinnanoparticlesintheplasmawasconsiderablyelevatedat1
houraftertheinjectionofAlexa647-labeledrapamycinnanoparticles,althoughthevalue
decreasedto15%at4hoursaftertheinjection.
ThesegmentsofAAAandthoracicaortawereexcisedfromtheaorto-iliacspecimensafter
IVIS1analyses,andtheadjustedabsorbancevaluesoftheirlysateswerecalculated.Inthese
values,werejectedthedataat1hourafteraninjection,sincethevaluesat1hourwere
markedlybiasedbythehighintensityofplasma(Fig3C).Theadjustedabsorbancevaluesof
AAAexhibitedadelayedpeakat8hoursafterinjection,whereasthevaluesofthethoracic
aortadiminishedandbecamezeroby8hours.ThevaluesofAAAweresignificantlyhigher
thanthoseofthethoracicaortaat8,16,and24hoursafterinjection.
HistologyofrapamycinnanoparticlesdistributedtotheAAA
Alexa647-labelledrapamycinnanoparticleswereinjectedintotheratAAAmodelinthesame
manner,andthemicroscopicdistributionofthenanoparticleswasanalyzed.Confocallaser
scanningmicroscopydetectedtheAlexa647-labelledrapamycinnanoparticlesinthesections
ofAAAassmallfluorescentdots,andalsodepictedtheintrinsicfluorescenceofthemedial
elasticlaminaeoftheAAAwall,whichprovidedinformationconcerningitsstructuraldestruc-
tion.Therewasnootherauto-fluorescencesuchasthatofredbloodcells.Inallsectionsofthe
AAAsamples,thesmallfluorescentdotsweredistributedtothemediaandadventitiawithpro-
gressivedestructionofthewallstructure(Fig4A).Sectionsimmuno-stainedforCD68revealed
thatthemajorityofthesmallfluorescentdotswereco-localizedwithCD68-positivecells(Fig
4B,S1Fig).Incontrast,immuno-stainingforαSMAshowedlittleco-localizationofthefluores-
centdotsandαSMA-positivecells(Fig4C).
InhibitionofAAAenlargementbyrapamycinnanoparticles
DuringtheprocessofAAAformationafterelastaseinfusion,ratsreceivedintravenousinjec-
tionsofthefollowingsuspensionorsolution;i)PBS,ii)0.1mg/kgfreerapamycin(free/RAP-
0.1),iii)1mg/kgfreerapamycin(free/RAP-1),iv)0.1mg/kgrapamycinnanoparticles(RAP/
nano-1),orv)1mg/kgrapamycinnanoparticles(RAP/nano-1).Allanimalssurvivedthis
experiment.Immediatelyafterelastaseinfusion,nosignificantdifferencewasdetectedinthe
sizesoftheabdominalaortasbetweenthefiveexperimentalgroups(Fig5).At7daysafterelas-
taseinfusion,thesizesoftheinducedAAAsafterinjectionsoffree/RAP-1,RAP/nano-0.1,and
RAP/nano-1weresignificantlysmallerthanthoseafterPBSinjections(Fig5).Furthermore,
RAP/nano-0.1andRAP/nano-1enhancedtheinhibitoryeffectonaneurysmalgrowthcom-
paredwithfree/RAP-0.1andfree/RAP-1,respectively(Fig5).Bloodsampleswerealsocol-
lectedfromallanimalsatday7,andbiochemicalexaminationofthemshowednosignificant
adverseeffectsafterthetreatmentofRAP/nano-0.1andRAP/nano-1(S1Table).
Histologyaftertreatmentwithrapamycinnanoparticles
TheAAAsamplesobtainedduringtheaboveexperimentwerehistologicallyinvestigatedby
HEandEVGstaining.Inlow-powerfields,thecross-sectionalareasoftheAAAsafterinjec-
tionsofRAP/nano-1weresmallestamongthoseofallexperimentalgroups,andtheareasof
theAAAsafterinjectionsofRAP/nano-0.1andfree/RAP-1wereslightlylarger(Fig6A–6C).
Incontrast,theareasoftheAAAsafterinjectionsofPBSandfree/RAP-0.1weremarkedly
PLOSONE|DOI:10.1371/journal.pone.0157813 June23,2016 9/21
NanoparticlesTargetRapamycinDeliverytoAAA
Fig4.MicroscopicdistributionofAlexa647-labeledrapamycinnanoparticlesintheratAAA.(A)
ConfocallaserscanningmicrographoftheratAAA7-dayspostinductionand24hoursafterinjectionofthe
Alexa647-labeledrapamycinnanoparticles.NotetheabundantaccumulationofAlexa647-labeledrapamycin
nanoparticles(reddots)distributedinthemediaandadventitiawithprogressivedestructionofthewall
structure.NucleiarestainedbyHoechst33342(bluedots),andelasticlaminaeofthemediaarevisualizedby
intrinsicfluorescence(green).Lindicatesthelumen,scalebar=500μm.(B)Micrographofthecrosssections
stainedforCD68(green).Co-localizationwithAlexa647-labelledrapamycinnanoparticles(reddots)appears
asayellowcolor.Themajorityofnanoparticledotswereco-localizedwithCD68-positivecells(S1AFig).
Scalebar=10μm.(C)MicrographofthecrosssectionsstainedforαSMA(green).Thereislittleco-
localizationwithAlexa647-labeledrapamycinnanoparticles(reddots,S1BFig).Scalebar=10μm.
doi:10.1371/journal.pone.0157813.g004
PLOSONE|DOI:10.1371/journal.pone.0157813 June23,2016 10/21