cd90-targeted liposomes increase the therapeutic efficacy

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Nano Research & ApplicationsISSN 2471-9838

2016Vol. 2 No. 2:17

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Research Article

DOI: 10.21767/2471-9838.100017

Ayumi Goto1#, Tomomi Akita1#, Shiori Nogami1, Michiko Horiguchi1,2,3, Ryo Abe4 and Chikamasa Yamashita1,2

1 DepartmentofPharmaceuticsandDrugDelivery,FacultyofPharmaceuticalSciences,TokyoUniversityofScience,Japan

2 FusionofRegenerativeMedicinewithDDS,ResearchInstituteforScienceandTechnology,TokyoUniversityofScience,Japan

3 ColumbiaUniversityMedicalCentre,NewYork,USA

4 DivisionofImmunobiology,ResearchInstituteforBiomedicalSciences,TokyoUniversityofScience,Japan

# Theseauthorscontributedequallytothiswork

Corresponding author: ChikamasaYamashita

[email protected]

ChikamasaYamashita,Ph.D.DepartmentofPharmaceuticsandDrugDelivery,FacultyofPharmaceuticalSciences,TokyoUniversityofScience,2641Yamazaki,Noda,Chiba278-8510,Japan.

Tel: +972-4-8359421Fax:+972-3-9211478

Citation: GotoA,AkitaT,YamashitaC,etal.CD90-TargetedLiposomesIncreasetheTherapeuticEfficacyofaRetinoicAcidDerivativeinPulmonaryCarcinoma.NanoResAppl.2016,2:2.

CD90-Targeted Liposomes Increase the Therapeutic Efficacy of a Retinoic Acid

Derivative in Pulmonary Carcinoma

AbstractTitle: ThetherapeuticefficacyofCD90-targetedliposomesinpulmonarycarcinoma.

Background: In refractory lungcancertissue, ithasbeen reported that cellpopulationsexistthatexhibitresistancetoexistingtherapeutics.Thesecellshavecharacteristicssuchashighstemnessandtumorigenicpotential,andtheyarethoughttocontributetorelapseand metastasis. Differentiation inducer 4[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benzoicacid(Am80)hasagrowthinhibitoryeffectonpulmonarycarcinoma cells, but it has been effective only in a high dose that causes damage tonormal cells.We aimed to achieve a dose reduction by specific targeting of anaplasticpulmonarycarcinomacellsandmodificationofantibodiesontheliposomesurfacespecificformembraneproteinofundifferentiatedcellsbecauseundifferentiatedpulmonarycellshaveuniquemembraneproteins.CD90isoneoftheundifferentiatedmarkersexpressedinpulmonarycells.

Methods and Findings: In this study, we examined the usefulness evaluation ofAm80 liposomes conjugated with anti-CD90 antibody using Calu-6, a CD90-positiveundifferentiatedhumanpulmonarycarcinomacellline.

After the preparation of Am80 liposomes, the characterization and cell specificity,therapeuticefficacyof themwere assessed.Am80 liposomes conjugatedwith antibodyselectively combinedwith the surface of Calu-6 and had a significant influence on theinhibitionofcellandtumorgrowth.Thetwice-weeklyintratumoraladministrationofAm80liposomesconjugatedantibody(0.1mg/kgasAm80)inhibitedtumorgrowthto3.64±1.10comparedtoemptyliposomes(5.89±1.61)inrelativetumorvolumeatday28.

Conclusions: WerevealedthatthecellularuptakeofAm80liposomesconjugatedwithanti-CD90antibodybyanaplasticpulmonarycarcinomacellswashighlyeffectiveandefficient.

Keywords:Liposomes;Am80;Anti-CD90;Pulmonarycarcinoma

Abbreviations: Am80: 4[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benzoicacid;APL:AcutePromyelocyticLeukemia;DDS:DrugDeliverySystem;DOX: Doxorubicin; ATRA: All-Trans Retinoic Acid; CYP: Cytochrome P450; RAR: RetinoicAcidReceptor

Received: October26,2016; Accepted: December26,2016; Published: December 28,2016

IntroductionPulmonarycarcinomaisaleadingcauseofcancer-relateddeathsworldwide[1]anditsincidenceisincreasingeventoday[2].Nodrugcancurepulmonarycarcinomacompletely[3]andthus,itisurgentlynecessarytodevelopanoveltherapeuticdrug.Cancerstemcellscanbeconsideredacauseofcancermetastasisandrelapse [4,5].Chemotherapyand radiation therapyare current

treatments for pulmonary carcinoma [6,7] but these therapiestarget cancer cells. It is thought that cancer stemcells surviveandleadtometastasisandrelapsebecauseoftheloweffectofthesetherapiesoncancerstemcells,whichareundifferentiated

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andareresistanttoradiationortheoxidativestressofanticanceragents [8,9]. Therefore, it is necessary to develop a therapythat directly targets cancer stem cells. Differentiation-inducingtherapycanbeconsideredaneffectivetherapyforcancerstemcells that cause cancer metastasis and relapse [10,11]. Thistherapy puts cancer cells on a track towards differentiationofcellsbyusingadifferentiationinducertoreturnthecancercellstonormalcells. Ithasattractedattentionbecauseof thesmallnumber of serious complications produced in comparison tohemorrhageandinfectionwithchemotherapy[12,13].

Recently, itwasreportedthatall-transretinoicacid(ATRA)haseffects on differentiation induction pathologically and on anti-proliferation of pulmonary carcinoma cells [14,15]. BecauseATRA, which is an active metabolite of vitamin A, can highlyinduce cell differentiation, it hasbeenusedas therapyagainstacute promyelocytic leukemia (APL), which represents themostsuccessfulexampleofdifferentiation-inductiontherapyinclinicaloncology[16].However,lowmetabolicstabilityhasbeenpointed out as a problem of ATRA [17,18]. There are severalpossibleexplanationsforthisproblem,suchasadecreasedbloodconcentration of ATRA caused by an increase in cytochromeP450(CYP)oroxidativeenzymeactivityorinducedexpressionofcellularretinoicacidbindingprotein,anagentinvolvedinATRAmetabolism[19].Therefore,wefocusedon4[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carbamoyl] benzoic acid(Am80),whichisaderivativeofATRA.Am80isakindofsyntheticretinoidthatwasdevelopedtoimprovedrugresistanceagainstAPLandreducesideeffects.AlthoughsimilartoATRA,Am80isan agonist of the retinoic acid receptor (RAR), and it has highselectivity in RAR receptors.While ATRA binds to all subtypesofRAR(RARα,RARβ,andRARγ),Am80bindsonlytoRARαandRARβ[20].Furthermore,Am80hasmoreactivitythanATRAandthe differentiation ability against HL-60 human promyelocyticleukemiacellsorNB-4cellsofAm80isapproximately10timeshigherthanthatofATRA[21].Therefore,Am80hasbeenusedasatreatmentforrelapsedorrefractoryAPLpost-ATRAremission.Inaddition,itishighlystableagainstlightandheat,isnoteasilyoxidized,andhashighmetabolicstability.Forexample,regardingCYP,althoughAm80ismetabolizedbyCYP3A4andATRA,Am80isnotmetabolizedbyCYP26,whichisinvolvedinthemetabolismofretinoicacid[22].ItisreportedthatAm80islesssusceptibletometabolismthanATRAbecauseofitsloweraffinityforcellularretinoicacidbindingproteinthanATRA[23].

We reported that Am80 has an antiproliferative effect againstpulmonarycarcinomacellsatahighdose[24]andsystemicside

effectssuchasretinoicacidsyndromecanbeaconcerninclinicalapplication. Retinoic acid syndrome is a fatal complication inwhichincreasednumbersofmaturecellscausedbysimultaneouscell differentiation invade the lung and cause cellular damagesuch as that to the vascular endothelium, which leads tohypoxemia,heartfailure,andotherproblems[25,26].Therefore,thetargetingtospecificcellisneeded.

Recently, the nanocarriers have been developed in the drugdeliverytothecancercells[27].Inthepresentstudy,weaimedtoreducetheamountofAm80deliveredbyusingwithliposomestoreducethesideeffectsofAm80.Liposomeisaclosedendoplasmicreticulumcomprisingalipidbilayerthatconsistsofcomponentssimilar to a biological membrane, such as phospholipid,cholesterol,protein,andglycolipid. It iseasy toadjust thesizeof thesmallvesiclesandthecomponentsofphospholipid,andit is possible to enclose a water-soluble drug, lipophilic drug,polymer,orothermaterials.Furthermore,liposomescandelivertheencloseddrugstotumortissueviatheenhancedpermeationand retention effect. This effect is a phenomenon in which ananocarrier such as liposome can deliver the enclosed drugsto tumor tissue efficiently via blood vessels around the tumortissue, which has many gaps in comparison to normal tissue[28]. Inaddition, liposomes leakingout frombloodvesselscanremain in tumor tissue for a long time because the lymphatictissuesurroundingtumortissueisimmatureandcannotexcludeforeignsubstancesintissue[29].Liposomesarecarrierssuitablefor localized drug delivery that make the reduction of sideeffectsortheslowreleaseofadrugpossible.Doxorubicin(DOX)encapsulatedinliposomesisanexamplewherebyareductionofsideeffectsisachievedbyusingliposomes.InanevaluationofthetherapeuticeffectofDOX,which isarepresentativeanticancerdrug encapsulated in liposomes, on Meth-A-sarcoma-bearingmice,themicetreatedwithDOXencapsulatedinliposomesdidnotdie,whereas themice treatedwithnon-encapsulatedDOXdid [30]. This result showed that the serious cardiotoxic sideeffectsofDOXwerereduced.Inthisway,liposomeshavebeenapplied to a drug delivery system (DDS) as low-toxicity drugcapsuleswithastructuresimilartothatofcells[31].

Furthermore,toaddtargetingfunctionstocellsandtissue,theactivetargetingtypeof liposome,whichhasanantibodyonitssurface, is gathering attention as a next-generation DDS drug[32]. Because cancer cells have specific membrane proteins,the targeting of these proteins by anticancer drugs has beenconducted.Inotherwords,byspecificallytargetingcancercells,

liposome anti-CD90antibody Am80

CD90

Figure 1 Agraphicalpresentationofthenanocarriers.


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3© Under License of Creative Commons Attribution 3.0 License

areductionintheamountofdrugnecessaryhasbeenachievedby modifying a specific antibody on the surface of liposomesagainst specific membrane proteins of the cancer cells, thusmakingtheliposomessuitableforlocalizeddrugdelivery[33,34].It is reported that alveolar epithelial progenitor cells, whichhave the ability to differentiate into alveolar epithelial cells,were identifiedfromnormalhumanpulmonarytissueandthatmembrane protein CD90 is expressed on their surface [35].Moreover,CD90isreportedasacandidatemarkeroflungcancerstemcellsaswellasCD133andsoon[36-40].Fromthesereports,we expect that that CD90 is also expressed on the surface ofanaplasticpulmonarycarcinomacellssuchasCalu-6cells,andwehavetargetedCD90asaproteinofpulmonarycancerstemcells.

In this study, we examine the usefulness evaluation and thepreparation of Am80 liposomes conjugated with anti-CD90antibodyusingCalu-6.

Materials and MethodsEthics statementAlltheanimalexperimentswereconductedundertheapprovalof the TokyoUniversityof Scienceethics committee (ApprovalNumber Y15042). The administration of Am80 was stoppedwhentheaveragetumorvolumereachedover600mm3orwhennecrosis was induced in the tumor. Then, to ameliorate thepainanddistressassociatedwithtumorformation,theanimalswereeuthanized,and the tumorswereenucleatedat thenextscheduleddateofadministration.

MaterialsAm80waskindlyprovidedbyProf.ShudooftheItsuuLaboratory(Tokyo, Japan). Am80 was dissolved in ethanol and storedat -30°C until use. Cholesterol was purchased from TokyoChemical Industry Co., Ltd. (Tokyo, Japan). Hydrogenated soyphosphatidyl choline (HSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-m- [methoxy (polyethyleneglycol)-2000](mPEG2000-DSPE)werekindlyprovidedbyNipponFineChemicalCo., Ltd. (Osaka, Japan). DSPE-PEG-MAL (SUNBRIGHT DSPE-020MA)waskindlyprovidedbytheNOFCORPORATION(Tokyo,Japan). O, O’-dietradecanoyl-N-(α-trimethyl ammonioacetyl)diethanolamine (DC-6-14) was kindly provided by SogoPharmaceuticalCo.,Ltd.(Tokyo,Japan).Ratanti-mouseCD90.2was kindly provided by Prof. Abe of the Tokyo University ofScience. Goat anti-human CD90 (Thy-1) polyclonal antibodywaspurchasedfromSantaCruzBiotechnologyInc.(SantaCruz,CA,USA). Donkey anti-goat IgG-fluorescein isothiocyanatewaspurchased from Millipore (CA, USA). Donkey anti-rabbit IgGH&L(AlexaFluor594)waspurchasedfromabcam(Cambridge,UK).DAPI(4’,6-diamidino-2-phenylindole)waspurchasedfromRocheDiagnostics (Mannheim,Germany),andcoumarin-6waspurchasedfromTokyoChemicalIndustryCo.,Ltd.(Tokyo,Japan).

Cell lineCalu-6 cells, an established human pulmonary carcinoma cellline(#HTB-56),werepurchasedfromtheAmericantypeculturecollection(Rockville,MD,USA).

Cell cultureCalu-6cellswerecultured inEagle’sminimalessentialmedium(EMEM; Wako Pure Chemical Industries, Ltd., Osaka, Japan)supplementedwith10%fetalbovineserum(FBS),non-essentialaminoacids,and1mMsodiumpyruvateat37°Cinahumidified5% CO2 atmosphere. The established human pulmonarycarcinoma cell line A549 was provided by Prof. Nishioka ofTokushimaUniversityGraduateSchool.A549cellswereculturedin Dulbecco’s Modified Eagle’s Medium (DMEM; Wako PureChemicalIndustries,Ltd.,Osaka,Japan)supplementedwith10%FBSat37°Cinahumidified5%CO2atmosphere.

Expression of CD90 in Calu-6Immunostaining: Cellswereseededonto8-wellchamberslides(6×103cells/cm2)andwashedtwicewithPBSandfixedby4%paraformaldehydeinPBSfor15min.ThecellswerewashedthreetimeswithPBS,blockedusing10%bovineserumalbuminatroomtemperaturefor60minutesandthenincubatedwiththeprimaryantibodyagainstCD90ata1:200dilutionat4°Covernight.Afterincubation at room temperature for 2 h, the samples werewashedthreetimeswithPBScontaining0.1%polysorbate80andthen incubatedwithAlexa Fluor 594donkey anti-rabbit IgG at1:200dilutionandDAPI,whichisusedforthestainingofnuclei,atroomtemperaturefor2h.SlideswerewashedthreetimeswithPBScontaining0.1%polysorbate80,andaftermountingofthesections,theslideswereobservedusingaBZ-9000fluorescencemicroscope(Keyence,Osaka,Japan).

Western blot analysis: CellswerewashedwithPBS, and lysedin2×samplebuffer[0.5MTris(hydroxymethyl )aminomethanehydrochloride (Tris-HCl ), pH 6.8, with 10% sodium dodecylsulfate (SDS), 2-mercaptoethanol, glycerol, and bromophenolblue]anddenaturedat95°Cfor10min.ProteinconcentrationsincelllysatesweredeterminedbytheBradfordassay(Bio-Rad).Equivalent total protein amountswere loaded onto each laneofanSDS-18%polyacrylamidegelelectrophoresisgel,followedby transfer to a polyvinylidenefluoride (PVDF)membraneandblocking in 5% nonfat dry milk containing 0.1% polysorbate20 at room temperature for 1 h. The blotted membrane wasincubatedwithappropriateprimaryantibodyagainstCD90(Goatanti-humanCD90antibody)ata1:500dilutioninCanGetSignalImmunoreaction Enhancer Solution (TOYOBO Co., Ltd., Osaka,Japan)atroomtemperaturefor2handthenat4°Covernight.After six washes with PBS containing 0.1% polysorbate 20,the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (donkey anti-goat IgG-HRP) ata 1:1000 dilution in can get signal immunoreaction enhancersolution(TOYOBOCo.,Ltd.,Osaka,Japan)atroomtemperaturefor2h.AftersixwasheswithPBScontaining0.1%polysorbate20, the immunoreactions were visualized by immobilonwesternchemiluminescentHRPSubstrate(MerckMilliporeCo.,Darmstadt, Germany) with an ImageQuantLAS4000EPUVmini(FUJIFILM,Tokyo,Japan).



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antibody(Goatanti-mouseIgGH&L)ata1:100000dilutionincangetsignalimmunoreactionenhancersolution(TOYOBOCo.,Ltd.,Osaka, Japan) at room temperature for 2 h. After twowasheswithPBScontaining0.1%polysorbate20,theimmunoreactionswere visualized by immobilon western chemiluminescent HRPSubstrate (Merck Millipore Co., Darmstadt, Germany) usingImageQuantLAS4000EPUVmini(FUJIFILM,Tokyo,Japan).

Stability of Am80 liposomes conjugated with anti-CD90 antibodyToassess the stabilityof theAm80 liposomes conjugatedwithanti-CD90antibodyinHBSbufferandculturemedium(10%FBS/EMEM),weaddedAm80 liposomes (preparedasdescribed) to4°CHBSand37°Cculturemedium,respectively.After0,12,18,24,36,and48hofincubation,theaveragesizeoftheliposomeswas determined by dynamic light scattering with an ELSZ2Panalyzer.

Cellular uptake of liposomesCalu-6(6×103cells/cm2)wasseededonto8-wellchamberslidesand overnight pre-cultured. Coumarin-6 liposomes conjugatedwith anti-CD90 antibody and coumarin-6 liposomes withoutconjugation(500μM,100μL/well)wereadded,respectively,andweretreatedfor16min.Aftertreatmentwiththeliposomes,cellswerewashedtwicewithPBSandfixedby4%paraformaldehydeinPBSfor15min.ThecellswerewashedthreetimeswithPBS,blockedusing10%bovineserumalbuminatroomtemperaturefor60min,andthenincubatedwiththeprimaryantibodyagainstCD90 at a 1:200 dilution at 4°C overnight. After incubation atroomtemperaturefor2h,thesampleswerewashedthreetimeswithPBScontaining0.1%polysorbate80andthenincubatedwithAlexaFluor594donkeyanti-rabbitIgGat1:200dilutionandDAPI,for staining of the nuclei, at room temperature for 2 h. SlideswerewashedthreetimeswithPBScontaining0.1%polysorbate80,andaftermountingofthesections,theslideswereobservedwithaBZ-9000fluorescencemicroscope(Keyence,Osaka,Japan).

Cell specificity of liposomesCoumarin-6liposomesconjugatedwithanti-CD90antibody(500μM,100μL/well)wereaddedtoCalu-6cellsseededonto8-wellchamberslidesandovernightpre-cultured,respectively.After0,2,4,8,16,32,and64minofincubation,cellswerewashedtwicewithPBSandfixedby4%paraformaldehydeinPBSfor15min.ThecellswerewashedthreetimeswithPBSandthenincubatedwithDAPIat room temperature for2h.Cellmembraneswerelabeled by Cell Light Plasma Membrane-RFP, BacMam 2.0according to thekitmanufacturer’sprotocol (LifeTechnologiesJapanLtd.,Tokyo,Japan).SlideswerewashedthreetimeswithPBScontaining0.1%polysorbate80,andaftermountingofthesections,theslideswereobservedwithaBZ-9000fluorescencemicroscopeat400nm(forDAPI),495nm(forcoumarin-6),and565nm(forcellmembranes).

Cell viability assayThe cellular viability of Calu-6 cells after Am80 treatmentwasassessed using a CellTiter-Glo Luminescent Cell Viability Assay

Preparation of Am80 liposomes conjugated with anti-CD90 antibodyThecompositionoftheliposomeswasasinapreviouslyreportedmethod [41]. After dissolving lipid (HSPC:cholesterol:mPEG2000-DSPE: DC-6-14:DSPE-PEG-MAL = 2:1:0.1:2:0.1) and Am80 in100%EtOH,weinjectedthesolutionintoHEPES-bufferedsaline(HBS) with a syringe and prepared the liposomes. After thecentrifugation for 40 min at 64200 rpm, anti-CD90 antibodythat had been subjected to a reduction treatment (rat anti-mouse CD90.2, 1mg/mL) was added to the solution, and theantibody-modified liposome surfacewas formedby incubationfor16hoursatroomtemperature.Finally,anyunboundantibodywasremovedbycentrifugationtopreparetheAm80liposomesconjugatedwithanti-CD90antibody.Andwepreparedtheanti-CD90EmptyliposomebysamemethodwithoutAm80.

Particle size and zeta potential of liposomesTheparticlesizedistributionandzetapotentialoftheliposomesweredeterminedbydynamiclightscatteringtechniquewithanELSZ2Panalyzer(OtsukaElectronicsCo.,Ltd.,Osaka,Japan).

Percentage entrapment of Am80 in the liposomesPercentagedrugencapsulationintheliposomeswasdeterminedby centrifugation. The solution of Am80 liposome conjugatedwithanti-CD90antibodyand2%Triton-X(WakoPureChemicalIndustries,Ltd.,Osaka,Japan)weremixedeachinequalquantityandthencentrifugedat20,000rpmfor15min.Thesupernatantswere collected, and the Am80 concentration was measuredby high-performance liquid chromatography (HPLC). TheentrapmentefficiencyofAm80intheliposomeswasestimatedas:E=(WEncapsulated/WFirstadded)×100%,whereEistheencapsulationefficiency of liposomes,Wencapsulated is themeasured amount ofAm80intheliposomesuspensionsaftercentrifugation,andWfirst

addedisthemeasuredamountofAm80atfirstaddition.

Am80 was quantified by a COSMOSIL 5CN-R Packed Column(NACALAI TESQUE, INC., 250×4.6 mm) and detected by HPLC,whichwasperformedwithanLC-20ADsolventdeliverysystemequippedwith an SPD-20AVUV/VIS detector (both, Shimadzu,Kyoto, Japan). The mobile phase was composed of 1) 1%ammoniumacetatesolutionand2)acetonitrile,usingagradientelutionandperformedat40°Cwithaflowrateof1.0mL/min.TheeluatewasmonitoredbyaUVdetectorat285nm.

Percentage of antibody modifiedThe solution of Am80 liposome conjugated with anti-CD90antibody was centrifuged at 64,200 rpm for 20 min. Thesupernatants were removed, and then 2% Triton-X 10 μLwasaddedtocreateblottingsamples.PVDFmembranewasincubatedwith100%ethanolfor15s,thenwithultrapurewaterfor2min,andsubsequentlywithtransferbufferfor5min.Afterdryingthesurfaceofmembrane,the2μLblottingsampleswereblottedtothe membrane. The blotted membrane was dried completelyandwashedoncewithPBScontaining0.1%polysorbate20andincubated with horseradish peroxidase-conjugated secondary


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(Promega KK, Tokyo, Japan) according to the manufacturer’sprotocol.Cells(625cells/cm2)wereseededin100μLofmediumin 96-well flat-bottomplates and incubated overnight at 37°C.After exposure to drugs for 2-6 days, the plateswere assayedusing an Envision Plate Reader (PerkinElmer Japan Co. Ltd.Kanagawa,Japan).ThemediumcontainingAm80wasexchangedevery48h.

In vivo tumorigenesis assayFive-week-oldmalecongenitalathymicBALB/Cnude(nu/nu)micewerepurchased fromSankyoLaboServiceCorporation(Tokyo,Japan).Atotalof1.3×106Calu-6cellsweresuspendedin100µLofPBS(50%Matrigel;BectonDickinson&Co.,SanJose,CA)andinjectedsubcutaneouslyintoboththeleftandrightflanksofthenudemice.Whenthetumorsreachedover50mm3 in volume, the miceweredividedrandomlyintothreegroups(HBS,anti-CD90-Empty liposome, and anti-CD90-Am80-liposome). The twice-weeklyintratumoraladministrationofAm80liposomes(0.1mg/kgasAm80andliposomeswaspreparedinHBSat0.1mg/mLastheliposomeconcentration),dissolvedinasalinesolutionwithupto1%DMSO,wasthen initiated(day0).ThedoseofAm80wasselectedbasedonpreviousreports[42,43]andourpreviouspreliminaryexperiments.Themicewereweighed,andthetumorvolumewasdeterminedusingthestandardformulaL×W2×0.5,whereListhelongestdiameterandWistheshortestdiameter,aspreviouslydescribed[44].

StatisticsNormallydistributeddataarepresentedasthemean±standarddeviation(SD).TheStudentt-testwasusedforthecomparisonsof two groups. P values <0.01 or <0.05 were considered to

indicatestatisticalsignificance.

ResultsExpression of CD90 in Calu-6Toidentifytheanaplasticpulmonarycellprotein,wetestedtheexpression of CD90 as a marker of human alveolar epithelialprogenitor cells in Calu-6. Calu-6 (reported as an anaplasticpulmonary carcinoma cell) and A549 (reported as a maturepulmonary carcinoma cell) were evaluated by immunostainingandWestern blot analysis. Assessment of CD90 expression byfluorescencemicroscopy revealed that Calu-6 expressed CD90moregreatly than itexpressedA549 (Figure 2A).Westernblotanalysis showed the expression level of CD90 in Calu-6 to besignificantlyhigherthanthatofA549(Figure 2B).

Characterization and stability of Am80 liposomes conjugated with anti-CD90 antibodyToevaluatethecharacterizationofAm80liposomesconjugatedwithanti-CD90antibody,weconductedexperimentsonparticlesize, zeta potential, percentage of drug entrapment, andpercentageofantibodymodification(Table 1).TheparticlesizeandzetapotentialoftheAm80liposomesconjugatedwithanti-CD90antibodywere116.4nmand -5.97mV, respectively.Theparticlesizewasincreasedbyabout10nmandthezetapotentialwas decreased by 2 mV compared with the pre-modificationvalues.Thepercentageofdrugentrapmentwas15.5%andthatofantibodymodificationwas21.7%.

AfterAm80liposomesconjugatedwithanti-CD90antibodywererespectivelyincubatedin4°CHBSand37°Cculturemedium,we

(A) (B)CD90 DAPI Merge

CD90

A549 Calu-6

23kDa

43kDaβ-ACTIN

2.5

2

1.5

1

0.5

0

*

A549 Calu-6

Cal

u-6

A54

9

Rel

ativ

e pr

otei

n le

vels

CD

90/β

-AC

TIN

Figure 2 ExpressionofCD90inCalu-6andA549cells.(A)TheexpressionofCD90inCalu-6andA549cellsasindicatedbyimmunostaining.(B)TheexpressionlevelsofCD90inCalu-6andA549cellsasindicatedbyWesternblotting.Dataaregivenasmean±SDfromn=3.**p<0.01comparedwithA549.



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evaluated the stability of the liposomes bymeasuring particlesize over time. The particle size of the liposomes was almostunchanged in both the 4°C HBS and 37°C culture medium,indicatingthattheliposomeswerestableoveraperiodof48h(Figure 3).

Cellular uptake of liposomes conjugated with anti-CD90 antibodyWe compared the cellular uptake of liposomes in pulmonarycarcinomacellsbetweentheconditionsofbeforeandafterCD90-antibodymodification.Calu-6wasseeded,andliposomesolutionwasaddedat200µMasafinalconcentration.Afterthesolutionwas incubated for 16min,we observed the cellular uptake ofliposomesinCalu-6.Theuptakeoftheliposomesconjugatedwithanti-CD90antibodywasgreaterthanthatofthenon-conjugatedliposomes (Figure 4).Weobserved thatmany liposomeswerecoheringtothecellsurface.

Cell specificity of liposomes conjugated with anti-CD90 antibodyToassessthespecificityoftheliposomesconjugatedwithanti-CD90antibodyagainstCD90-positivecells,Calu-6andA549cellswereseededand liposomesolutionwasaddedat200µMasafinal concentration After incubation, we observed the cellularuptakeoftheliposomesovertimeinbothcelllines.TheuptakeintheCalu-6cellswasincreasedoverthatintheA549cells(Figure 5).WeobservedthatcomparedwiththeA549cells,manymoreliposomeswerecoheringtothemembraneoftheCalu-6cells.

Cell growth inhibition of Am80 liposomes conjugated with anti-CD90 antibody against pulmonary carcinoma cellsToassesscellgrowthinhibitionoftheAm80liposomesconjugatedwithanti-CD90antibodyagainstCalu-6,weevaluatedcellviabilityafter Am80 liposome treatment. Calu-6 was seeded and wasexposedtoAm80andtoAm80liposomesconjugatedwithanti-CD90antibodyfor6days,respectively.Theresultsshowedthat10µMAm80inhibitedcellgrowthbyabout11%,whereas10µMAm80 liposomes conjugatedwith anti-CD90antibody inhibitedcellgrowthbyabout87%,indicatingthatAm80encapsulatedinliposomesinhibitedcellgrowthsignificantlymorethanthenon-encapsulatedAm80did(Figure 6).

We then evaluated the effect of Am80 liposomes conjugatedwithanti-CD90antibodyoncellgrowthinhibitionovertime.After2doftreatment,10µMAm80liposomesconjugatedwithanti-CD90antibodysignificantly inhibitedcellgrowthbyabout38%compared with the non-encapsulated Am80 (Figure 7). TheseresultsshowedthatAm80liposomesconjugatedwithanti-CD90antibodyhaveaneffectofgrowthinhibitionagainstCalu-6.

In vivo therapeutic efficacy of Am80 liposomes conjugated with anti-CD90 antibodyWe evaluated the therapeutic efficacy of Am80 liposomesconjugatedwithanti-CD90antibodyinBALB/cnude(nu/nu)micebearingCalu-6cellsubcutaneoustumors.Afterthetumorswerepalpated (50 mm3), HBS and anti-CD90-Empty liposome wereadministeredintratumorallyintothemiceinthecontrolgroups

Liposome formulation Particle size (nm), mean ± SD

Zeta potentials (mV)

Drug encapsulation efficiency (%), mean ± SD

Coupling efficiency (%), mean ± SD

Am80liposome(withoutanti-CD90TTantibody) 106.7±24.9 -5.97 - -anti-CD90-Am80liposome 116.4±30.1 -8.18 15.5±1.5 21.7±1.27

Table 1CharacterizationofAm80liposomesconjugatedwithorwithoutanti-CD90antibody(n=3).

120

100

80

60

40

20

00 12 24 36 48

Size

(nm

)

Time (h)

120

100

80

60

40

20

00 12 24 36 48

Size

(nm

)

Time (h)

(A) (B)4ºC HBS 37ºC culture medium

Figure 3 Stabilityofliposomesinbuffer.SizeoftheAm80liposomesmeasuredfor48-hincubationinat4°CHBS(A)and37°Cculturemedium(B).Dataaregivenasmean±SDfromn=3.


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Coumarin-6 CD90 DAPI Merge

norm

allip

osom

elip

osom

ew

ith a

ntib

ody

Figure 4 Immunofluorescenceanalysisofcellularuptake inCalu-6cells. ImmunofluorescenceanalysisofCalu-6cells showsnucleus,coumarin-labeled liposomes,CD90,andmerged imagesbyfluorescencemicroscopy.The intracellularuptakeofcoumarin-6liposomesconjugatedwithorwithoutanti-CD90antibodywasobservedwithinCalu-6andA549cellsafter16minofincubation.

A549Calu-6

Coumarin-6Cell

membrane DAPI Merge Coumarin-6Cell

membrane DAPI Merge

64 m

in

32

min

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6 mi

n

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Figure 5 Cellspecificityof liposomesbyfluorescencemicroscopyanalysis inCalu-6andA549cells.Fluorescencemicroscopyanalysisshowsnucleus,coumarin-6-labeledliposomes,cellmembrane,andmergedimages.TheintracellularuptakeofliposomeswascomparedinCalu-6(CD90-positive)andA549(CD90-negative)cellsafter2-64minofincubation.



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Figure 6 CellgrowthinhibitionwithAm80liposomesconjugatedwithanti-CD90antibody.Cellsweretreatedwith10µM(0.351µg)Am80oranti-CD90-Am80-liposomefor6days.Dataaregivenasmean±SDfromn=3.**p<0.01comparedwithAm80treatmentgroup.Forplotsofcellsurvivalrate,themeanbasalvalue(control)ofeachgroupissetat100%.

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Figure 7 Time-dependentcellgrowthinhibitionwithAm80liposomesconjugatedwithanti-CD90antibody.Cellsweretreatedwith10µM(0.351µg)Am80oranti-CD90-Am80-liposomefor2,4,6days.Dataaregivenasmean±SDfromn=3.*p<0.05,**p<0.01comparedwiththeAm80treatmentgroup.Forplotsofcellsurvivalrate,themeanbasalvalue(day0)ofeachgroupissetat100%.

twice a week. Anti-CD90-Am80-liposome (Am80 liposomesconjugated with anti-CD90 antibody) was administeredintratumorally in the treatment group twice a week. Tumorgrowthwas significantly inhibited in anti-CD90-Am80-liposometreatmentgroupcomparedwiththecontrolgroupfromday10(Figure 8A).However,wecouldnotconfirmtheweightchangeofthemicebetweenthecontrolandtreatmentgroup(Figure 8B).

TheresultsshowedthatAm80liposomesconjugatedwithanti-CD90antibodyhad an antitumoreffect against thepulmonarycarcinomacellssubcutaneouslytransplantedinmice.

DiscussionIn this study, we found that the CD90-targeted liposomes


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Figure 8 TherapeuticefficacyinthemicebearingCalu-6cellsubcutaneoustumors.(A)Averagetumorgrowthcurvesfromarepresentativeexperiment.MicereceivedintratumoraladministrationofAm80liposomesconjugatedwithanti-CD90antibodyatanequivalentdoseof0.1mg/kgondays7,10,14,17,21,24,and28.(B)Bodyweightchangesofthemiceovertheexperimentalperiod.Dataaregivenasmean±SDfromn=10-12.**p<0.01comparedwiththeanti-CD90Emptyliposomegroup.

increased the therapeutic efficacy of Am80 in Calu-6 cells andtumors. These findings support the idea that CD90-targetedliposomeswouldbeanovelantitumorDDSforthetreatmentofpulmonary carcinoma (Figure 1).

WefoundthatCD90wasexpressedsignificantlymoregreatlyinCalu-6thaninA549cells(Figure 2).CD90isreportedlyexpressedin human alveolar epithelial progenitor cells [35]. Therefore,CD90wasexpressed inCalu-6anaplasticpulmonarycarcinomacellsaswellasinundifferentiatedcells,whereastheexpressionof CD90was low inA549,which is a differentiatedpulmonarycarcinomacellline.

We observed increased uptake of the liposomes conjugatedwith anti-CD90 antibody compared with that of the non-conjugated liposomes by evaluating cellular uptake (Figure 4) and that theuptake inCalu-6was increasedover that inA549cells by evaluating the cell specificity of liposomes (Figure 5).Liposomes conjugated with anti-transferrin receptor antibody,which encapsulates DOX, are reported to specifically combinewithtransferrinreceptoronthecellsurfacedependentlyagainstaDOX-resistantsublineofhumanleukemiaK562cellsofK562/ADM,andthentheDOXisincorporatedefficientlyintothecellviaendocytosis[45].BecauseourresultsshowedincreaseduptakeofliposomesinCalu-6,asindicatedbythehighexpressionofCD90inCalu-6versusA549webelievethattheliposomesconjugatedwithantibodycombinedwiththesurfaceoftheCalu-6cellsandendocytosisoccurred.

We evaluated cell growth inhibition of Am80 liposomesconjugated with antibody against pulmonary carcinoma cells.Am80 encapsulated in liposomes significantly inhibited cell

growthatalowdoseandwithhigheffectcomparedwiththenon-encapsulatedAm80(Figure 6),suggestingtheusefulnessofAm80liposomesconjugatedwithantibody.Ourresultssuggestedthatthe liposomeswere internalized into cellsbyendocytosis afterbindingspecifically toCD90,which isexpressedon thesurfaceofCalu-6cells,andthenAm80wasreleasedintothecytoplasm.Therefore,itissuggestedthatAm80encapsulatedinliposomeswaseffectivelyinternalizedintothecellscomparedwiththenon-encapsulatedAm80,thusproducingabettereffect.

Taking these results together, we then investigated thetherapeutic efficacy of Am80 liposomes conjugated with anti-CD90antibody in vivo. In the treatmentgroup receivingAm80liposomes conjugatedwith anti-CD90 antibody, tumor volumewas significantly reduced compared with that in the controlgroup(Figure 8).Inapreviousstudy,aftertreatmentwith0.1mg/kgbufalinfor16daysinmicebearingmelanomasubcutaneoustumors, thebufalin liposomesanchored toanti-CD40antibodysignificantly reduced tumor volume to 70 mm3 indicating anantitumor effect [46]. Likewise, we similarly revealed theusefulnessofliposomesconjugatedwithanti-CD90antibody.

By encapsulating differentiation-inducing drugs againstanaplastic carcinoma into targeting liposomes conjugatedwiththe appropriate antibody, the differentiation inducer can bedeliveredatahighdosespecificallytotheanaplasticcarcinomacellsandnottonormalcells.Thesestudiesprovidethepossibilityoflighteningtheburdenonpatientsbypreventingthesystemicside effects of drugs and contributing to the realization ofpulmonary carcinoma therapy without metastasis or relapse.Inaddition,bychangingtheantibodythatisconjugatedonthesurfaceoftheliposomeandthedrugthatisencapsulatedwithin



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liposome,itwillbepossibletoapplytherapytoothercancersinadditiontopulmonarycarcinoma.

ConclusionsAm80liposomeconjugatedwithanti-CD90antibodysignificantlyinfluenced the inhibition of the cell growth in a pulmonarycarcinomacellline.WerevealedthatthecellularuptakeofAm80liposomes conjugated with anti-CD90 antibody by anaplasticpulmonary carcinoma cells was highly effective and efficientcomparedwiththatofnon-encapsulatedAm80.

AcknowledgementAm80 was kindly provided by Dr. Koichi Shudo (JapanPharmaceutical Information Center). HSPC and mPEG2000-DSPEwere kindly provided byNippon Fine Chemical Co., LTD.

(Osaka,Japan).DSPE-PEG-MALwaskindlyprovidedbytheNOFCORPORATION (Tokyo, Japan).DC-6-14waskindlyprovidedbySogoPharmaceuticalCo.,Ltd.(Tokyo,Japan),andratanti-mouseCD90.2waskindlyprovidedbyDr.RyoAbe(AbelaboratoryintheTokyoUniversityofScience).

FundingThis study was partially supported by the Grant-in-Aid forKAKENHI(ChikasamaYamashita.15K07901)andNagaiMemorialResearch Scholarship for the Pharmaceutical Society of japan(TomoniAkita)

Competing InterestsTheauthorsdeclarenoconflictofinterest


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cd90-targeted liposomes increase the therapeutic efficacy

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