使用整合素靶向的PLGA-殼聚糖納米顆粒給藥治療肺癌Chemodrug delivery using integrin-targeted PLGA-Chitosan nanoparticle for lung cancer therapy

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SCIENTIFICREPRTS

OPEN

Received:12September2017

Accepted:16October2017

Chemodrugdeliveryusing

integrin-targetedPLGA-Chitosannanoparticleforlungcancer

therapy

AnishBabu1,6,NarsireddyAmreddy1,6,RanganayakiMuralidharan1,6,GopalPathuri2,5,HariprasadGali2,6,AllshineChen3,6,YanD.Zhao3,6,AnupamaMunshi4,6&

RajagopalRamesh1,6,7

Inthisstudy,wereporttheefcacyofRGD(arginine-glycine-asparticacid)peptide-modifedpolylacticacid-co-glycolicacid(PLGA)-Chitosannanoparticle(CSNP)forintegrinαvβ3receptortargetedpaclitaxel(PTX)deliveryinlungcancercellsanditsimpactonnormalcells.RGDpeptide-modifedchitosanwas

synthesizedandthencoatedontoPTX-PLGAnanoparticlespreparedbyemulsion-solventevaporation.PTX-PLGA-CSNP-RGDdisplayedfavorablephysicochemicalpropertiesforatargeteddrugdelivery

system.ThePTX-PLGA-CSNP-RGDsystemshowedincreaseduptakeviaintegrinreceptormediatedendocytosis,triggeredenhancedapoptosis,andinducedG2/Mcellcyclearrestandmoreoverall

cytotoxicitythanitsnon-targetedcounterpartincancercells.PTX-PLGA-CSNP-RGDshowedless

toxicityinlungfbroblaststhanincancercells,maybeattributedtolowdrugsensitivity,neverthelessthestudyinvitedcloseattentiontotheirtransientoverexpressionofintegrinαvβ3andcautioned

againstcorrespondinguptakeoftoxicdrugs,ifanyatall.Whereas,normalhumanbronchialepithelial(NHBE)cellswithpoorintegrinαvβ3expressionshowednegligibletoxicitytoPTX-PLGA-CSNP-RGD,atequivalentdrugconcentrationsusedincancercells.Further,thenanoparticledemonstrateditscapacityintargeteddeliveryofCisplatin(CDDP),adrughavingphysicochemicalpropertiesdiferenttoPTX.

Takentogether,ourstudydemonstratesthatPLGA-CSNP-RGDisapromisingnanoplatformforintegrintargetedchemotherapeuticdeliverytolungcancer.

Sincemostchemotherapeuticdrugsaretoxictonormalcells,achievingtherelevanttherapeuticdrugconcen-trationincancercellswhilereducingsystemicexposuretothedrugisanimportantgoal

1

–

4

.Tenon-specifc,primarilydose-dependenttoxicityofchemotherapeuticstowardnormalcellsisacontinuingproblem.However,targetednanoparticle-baseddrugdeliveryisahighlypromisingstrategytoovercomethischallenge

5

,

6

.Targeteddrugdeliverysystemsshowhigherafnitytowardtumorcellsoverexpressingspecifcreceptorsthantowardnor-malcells

7

,8

.

Inlungcancers,theoverexpressionofcell-surfacereceptorsisofenexploitedfortargeteddeliveryofthera-peuticswithligand?/antibody-modifednano-drugdeliveryvehicles

9

,

10

.Teintegrin(αvβ3)receptorisofpar-ticularinterest,sinceitsexpressionishighintumorendotheliumandtumorcells

11

,

12

.UsingArg-Gly-Asp(RGD)peptidetotargetintegrin(αvβ3)intumorvascularendotheliumisawell-knownstrategytosuppressangiogen-esisandmetastasis

11

,

13

–

15

.TespecifcafnityofRGDsequenceandintegrin(αvβ3)hasalsobeenharnessed

1DepartmentofPathology,TheUniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,73104,USA.2DepartmentofPharmaceuticalSciences,TheUniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,73104,USA.3DepartmentofBiostatisticsandEpidemiology,TheUniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,73104,USA.4DepartmentofRadiationOncology,TheUniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,73104,USA.5DepartmentofMedicine,TheUniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,73104,USA.6StephensonCancerCenter,TheUniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,73104,USA.7GraduatePrograminBiomedicalSciences,TheUniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,73104,USA.CorrespondenceandrequestsformaterialsshouldbeaddressedtoR.R.(email:

rajagopal-ramesh@

)

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Figure1.Baselineexpressionofintegrinαvβ3inapaneloflungcancer(H1299,A549,H460,HCC827,

H1437,H1975)andlungfbroblast(MRC9,CCD16,WI38)celllines.Expressionofintegrinαvandactinwasdeterminedfromonegelandintegrinβ3wasdeterminedfromaseparategel.

fortargeteddrugdelivery

16

,

17

anddiagnosticapplicationsusingnanoparticles

18

,

19

.Teexpressionofintegrinsisrelativelyweakinnormalcells.However,transientoverexpressionofintegrinsareobservedinsomenormalcelllinesincludinglungfbroblasts,althoughatvariablelevels

20

,

21

.Terefore,whilehighlightingtheintegrinrecep-tortargeted-nanoparticlebaseddrugdeliveryincancercells,itisalsoimportanttoconsidertheimpactoftar-geteddrugdeliveryinnormalcellsthatexhibithighlevelofintegrinreceptorexpression.BasedonthesereportswehypothesizedthatRGDmodifednanoparticleswillpreferentiallytargetanddeliverchemodrugstointegrinreceptoroverexpressinglungcancercellsandproduceincreasedtherapeuticefciencywhilesparingintegrinnon-expressingnormalcellsfromthedrugtoxicity.

Herein,wedesignedanRGDmodifedpoly-lactic-acid-co-glycolicacid(PLGA)-chitosan-basednanopar-ticlesystem(PLGA-CSNP-RGD)fortargeteddrugdeliveryinnon-smallcelllungcarcinoma(NSCLC)cellshavinghighlevelsofαvβ3integrinexpression.TenanoparticlesystemhasaPLGAcoreloadedwithdrug,andissurface-coatedwithchitosan,towhichlinearRGDpeptide(GRGDSP)isconjugated.Chitosan,abiocom-patiblecationicpolymer,possessesnumerousfunctionalgroupsfortargetingligandmodifcation

22

.Moreover,chitosancoatingenhancestheparticlestabilityandcontrolsdrugrelease

23

.Chitosan’smuco-adhesivepropertycanbeexploitedfortrans-mucosaldeliveryofdrugs,especiallythroughtheintrapulmonaryroute

24

.Inaddition,GRGDSPisalinearpeptidethatpreferentiallyrecognizestheintegrinαvβ3

25

andα5β1receptorsexpressedonthecellsurface

26

.TecelladhesioncapacityofGRGDSPpeptideisseveraltimeshigherthansimilarpeptidesthathaveafnitytowardsfbronectinreceptors

27

.StudiesalsohaveshownthatGRGDSPpeptide-functionalizednano-particlespossessexcellentcell-adhesionpropertiesviaintegrinreceptorsandarebeingusedfortargeteddeliveryofdrugsanddiagnosticagents

28

–

32

.TeseadvantagesofRGDpeptide,chitosanandPLGAnanoparticlehavebeenintegratedinournovelformulationforintegrin-targeteddrugdeliveryinlungcancercells.

WetestedthisPLGA-CSNP-RGDsysteminNSCLCcellsoverexpressingintegrinαvβ3receptors.First,weusedwesternblotanalysisandfowcytometrytoexaminetheintegrinαvβ3expressionlevelsinapanelofNSCLCcellsandnormalcells.Ten,thetargetednanoparticlewasloadedwithpaclitaxel(PTX),apotentanti-cancerdrug,andcell-killingefciencyofthistargetednanoparticlewascomparedwiththatoffreePTXandnon-targetednanoparticles.Apoptosisandcellcycleanalysiswereperformedtoconfrmthetherapeuticactivity.Ten,theefciencyofPLGA-CSNP-RGDwastestedindiferentNSCLCcelllinesandnormalcellswithdiferentlevelsofintegrinexpression.DiferentialtoxicityofPTX-PLGA-CSNP-RGDwasconfrmedinNSCLCandnormallungfbroblasts,whilebroncho-epithelialcellsshowednegligibleresponsetothetoxicityofPTXdeliveredusingPLGA-CSNP-RGD.Finally,weconfrmedthepotentialofPLGA-CSNP-RGDasadeliveryplatformforanalter-nativedrugcisplatin(CDDP),awidely-useddruginlungcancertherapy.

ResultsandDiscussion

Baselineexpressionofintegrinαvβ3receptors.Tebaselineexpressionlevelsofintegrinαvβ3recep-torinvariouslungcancercelllinesandlungfbroblastsweredeterminedbyWesternblotting[Fig.

1

,TableST1]andbyfowcytometricanalysis[FigureS1].Amongthelungcancercelllinesthehighestintegrinαvβ3receptorexpressionwasobservedinH1975.Allthelungfbroblastsstudiedshowedhighexpressionslevelsofintegrinαvβ3,amongwhichMRC-9showedthehighestexpression.FromthelungcancerpanelwehavechosenH1975,A549andH1299,andfromthenormallungfbroblastswehaveselectedMRC-9,respectivelybasedontheirinte-grinαvβ3expressionlevels,forourstudies.Webelievedthatitwouldbeofinteresttoknowtheefectofintegrinαvβ3receptortargeteddeliveryofnanoparticlescarryingchemotherapeuticsinselectivityandcytotoxicityinlung

cancerandfbroblastcellswithhighintegrinαvβ3receptorexpressions.

Preparationandcharacterizationofnanoparticles.Preparationofdrug-loadedPLGA-CSNP-RGD

nanoparticleshasthreedistinctivephases:1)synthesisofPTX-loadedPLGAnanoparticlesusingtheemulsion-solventevaporationtechnique,2)synthesisofRGD-peptide-modifiedchitosan,and3)coatingRGD-CSusingthedepositionmethodtopreparedrug-loadedPLGA-CSNP-RGD.PLGAnanoparticlescanencapsulatehydrophobic

33

orhydrophilicdrugs

34

basedonthemethodusedforthepreparation.Particlesizes,zetapotentials,andpolydispersityindexesofthenanoparticleswerediferentineachstageofnanoparticledevelopment.AsshowninTable

1

,theaveragehydrodynamicsize,measuredasintensityversusdiameter,ofthePTX-PLGANPwas175nm,andtheRGD-chitosanmodifcationofPTX-PLGANPresultedin217nmparticles,anaverageincreaseof42nmcontributedbyCS-RGD.Testrongnegativesurfacechargeofacid-terminatedPLGAwasutilizedtodepositpositivelychargedCSasacoatingontoPLGANP.Tus,thenegativezetapotentialofPTX-PLGANPbecomespositiveuponCScoating,whichslightlyreducedinthepresenceofRGDpeptidein

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ParticleSize(d.nm)

PDI

Zetapotential(mV)

PTX-PLGANP

175.25±6.55

0.088±0.006

(?)25.25±3.3

PTX-PLGA-CSNP

212.5±6.02

0.140±0.0232

(+)33.4±0.8

PTX-PLGA-CSNP-RGD

217±13.54

0.133±0.011

(+)29.5±4.04

Table1.Size,chargeanddispersionvaluesofPTX-nanoformulations.*(n=4).

Figure2.PhysicochemicalcharacterizationofPTX-PLGA-CSNP-RGD.(A)TEMimagesofPTX-PLGA-

CSNP-RGD;(B)DrugloadingandencapsulationefcienciesofPTX-PLGA-CSNP-RGD.(n=5);(C)Invitrodrug(PTX)releasefromunmodifednanoparticles(PTX-PLGA-CSNP)andRGDmodifednanoparticles(PTX-PLGA-CSNP-RGD)inPBSpH7.4.

chitosanpolymer.OneofthemajorreasonsthatwehaveusedchitosanasacoatingmaterialforPLGAnanoparti-cleistoexplorethepresenceofnumerousfreeaminogroupsforfunctionalization,forinstanceherewehaveusedthispropertyforRGDpeptidemodifcation.StudiesalsosuggestthatchitosancoatingviaphysicaladsorptionisasuccessfulstrategyusedforcontrolledreleaseofdrugsandenhancecelluptakeofPLGAbasednanoparticles

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–

37

.Moreover,chitosancoatingalsoprovidesacationiclayerthatcanelectrostaticallybindtonegativelychargedther-apeuticmoleculessuchasnucleicacidtherapeutics.Infact,ourpreviousstudyprovedthatsuccessfulco-deliveryofsiRNA/pDNAwithchemotherapeuticsispossibleusingchitosancoatedpoly-lactic-acidnanoparticles

38

.

WeusedmaleimidecrosslinkingchemistrytoconjugateGRGDSPpeptidewithchitosanpolymer.TemultiplestepsinvolvedinthesynthesisofRGD-CSaredepictedinFigureS2.TePEGpolymerpresentinthemaleimidelinkermightbeusefulinpreventingrapidbio-clearanceofnanoparticles

39

.TePTX-PLGA-CSNP-RGDparticleswerewelldispersedandhadsphericalmorphology,asshownintherepresentativeTEMimage[Fig.

2A

].Tefnalparticles(PTX-PLGA-CSNP-RGD)werestableinaqueoussolutionin4°Cforatleasttwoweeks,withoutanyvisiblesedimentationbutwithlessthan5%increaseinparticlesizeasmeasuredbyDLS[FigureS3].However,whenweincubatedthenanoparticleswithdiferentserumconcentrationsslighttosignifcantincreaseinparticlesizesandchangeinzetapotentialswereobserved,dependingonserumconcentrationsandtimeofincubation[TableST2].Tisindicatestheformationofbiocoronainnanoparticles.WhenA549cellsweretreatedwiththeseseraincubatednanoparticles(FluTax-PLGA-CSNP-RGD),weobservedthatcellaccumulationofthefuorescentpaclitaxeldependedonserumconcentrations[FigureS4].Typically,lowserumconcentrations(2%and5%)didnothaveanyimpactoncelluptakewhile10%serumreducedthecelluptakeofFluTax-PLGA-CSNP-RGD(p<0.05).Whileitisknownthatproteincoronaformationisinfuencedbyphysicochemicalpropertiesofthenanoparticles,reportssuggestthatthebiologicalfateofnanoparticleishoweverdeterminedbytheidentityoftheproteincoronaratherthanthechangesinphysicochemicalpropertiesinducedbycoronaformation

40

,

41

.

Further,PLGA-CSNP-RGDshowedanaveragePTXencapsulationefciencyof93.7%anddrugloadingof6.5%[Fig.

2B

].Tedrugloadingefciencyofournanoparticleseemsreasonableandisconsistentwithrangeofdrugloadingfoundinliterature,from1%wt/wt

42

to10.46%wt/wt

43

orhigher,thathaveshownsuccessfulther-apeuticefciency.

Next,wemeasuredthedrugreleaseproflefromRGDmodifed(PTX-PLGA-CSNP-RGD)andunmodi-fed(PTX-PLGA-CSNP)nanoparticlesinPBS(pH7.4)[Fig.

2C

].At1hanaverage4%ofPTXwasreleasedfromRGDunmodifiedsystem(PTX-PLGA-CSNP)comparedonly1%fromRGDmodifiednanoparticles(PTX-PLGA-CSNP-RGD;p<0.05).Inthefrst6h,around13%PTXwasreleasedfromPTX-PLGA-CSNP-RGDcomparedto19.9%fromPTX-PLGA-CSNP,suggestingamoderateburstrelease.Attheearlytimepoints,drugreleaseratemighthaveinfuencedbyinitialthrustinthewaterpenetrationthroughthenanoparticlematrix

44

.However,thereleaseofPTXwassloweddownovertime;asobservedover24hthrough96hthedrugreleasereached24.8%and45.9%,respectivelyforPTX-PLGA-CSNP-RGD.TePTXreleaseinPTX-PLGA-CSNPalsofollowedasimilarpatternobservedwithPTX-PLGA-CSNP-RGD(P>0.05),showingnosignifcantdiferencein24h(28.7%)though96h(49.4%).Terefore,ourresultsclearlyindicatethatthedrugreleasepatternissim-ilarforbothunmodifedandmodifednanoparticles.TecorePLGAmatrixwithCSorCS-RGDmodifcationmighthavecontrolledandsustainedthedifusionofPTXovertime.However,therearemanycomplexvariablesthatinfuencethedrugreleasepatternsuchasdrugpropertiesandafnityofdrugtowardsthepolymermatrix,

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Figure3.Celluptakestudies.(A)Fluorescentmicroscopyimagesand,(B)graphicalrepresentationof

fuorescenceintensityper10000cellsofuptakeofFreeFlutax(FluorescentTaxol-OregonGreen488),and

FluTax-PLGA-CSNPwithorwithoutRGDmodifcationinA549,H1299,H1975andMRC-9cellsfor24h.Testudiesforeachcelllinewereperformedseparatelyandthephotosassembledtorepresentthetreatmentgroupforeachcellline.Magnifcation60X.

polymercomposition,polymerdegradationanderosionrate,temperature,pH,releasemediumtypeand/oracombinationoftheseprocesses

44

,

45

.Nevertheless,ourresultsareconsistentwiththepreviousreportssuggestingthatthereleaseofhydrophobicdrugsfromPLAorPLGAnanoparticlesoccursslowlyandoverseveraldays

46

.

Anotherimportantpointisthatwemeasuredthedrugremainedinthenanoparticlesinthedialysisapparatus(retaineddrug)ratherthaninthereleasingmedium

47

.However,theretaineddrugquantitywascalculatedforallthetimepointsofstudyinthreeseparateexperiments.Tismethodwasfollowedtoavoidtheanalyticallimita-tionthatwehadwiththeHPLCmeasurement.Further,ourstudywasfocusedtounderstandwhetherthedrugreleasecouldbecontrolledandreleasedovertimeusingournanoparticles,whichwecouldobservebyfollowingdrugreleaseupto96h.However,basedonthereleaseprofleobservedthereleasemightcontinueforseveraldaysafer96h.Tedrugreleaseisthuspredictedtobeinthesamefashioncontinuedforalongerperiodasreportedinsimilarsystemsintheliterature

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,

49

.

OptimizationofRGDconcentrationforefcientcelluptakeofnanoparticles.Forenhancednan-

oparticleuptakebyreceptor-overexpressingcancercells,nanoparticlesshouldpresentanoptimalligandconcen-trationontheirsurfaces

50

.WeinvestigatedthetargetingpotentialofRhodamineB-encapsulated-PLGA-CSNPmodifedwithvariousconcentrationsofRGDinA549lungcancercells.Tenanoparticleswereincubatedwithcellsfor6hand24h.FigureS5AandBshowsthefuorescentmicroscopyimagesandfuorescenceintensityplotrespectivelyfromtheEnvisionmicroplatereadingexperiment.TehighestcelluptakewasobservedwithRB-PLGA-CSNPwith0.34μMRGDconcentration.At6hand24htheaveragefuorescenceintensitiesof0.34μMRGDgroupwas96942.67(a.u.)and226756.66(a.u.)respectively.TesevaluesweresignifcantlyhigherthanuntreatedcontrolandothergroupswithdiferentRGDconcentrations,especiallyat6h(p<0.01).Tisobserva-tionfromfuorescenceintensitymeasurementswassupportedbymicroscopyimagesthatshowedtheelevatedRBfuorescenceat6hand24h,obtainedfromRB-PLGA-CSNP-treatedgroups.Henceforth,allourexperimentswereconductedwiththisRGD(0.34μM)concentrationinPLGA-CSNP.

Celluptakeoftargetedversusnon-targetednanoparticles.Integrinαvβ3expressionlevelsaredifer-

entfordiferentlungcancercelllines

51

.Terefore,theRGDpeptide-basedselectiveuptakeofnanoparticlesmaydependonintegrinαvβ3expressionlevels.Tounderstandthecelluptakeofnanoparticlesinlungcancercellsandnormalcells,weincubatedthecellswithfuorescentpaclitaxelOregongreen488(FluTax)-PLGA-CSNP,withorwithoutRGDmodifcation.Figures

3A,B

andS6showsthefuorescentimagesandgraphicalrepresentationoffuorescenceintensitycorrespondingtoFluTaxobtainedfromFreeFlutaxandFluTax-PLGA-CSNP-(RGD+/?)treatedcells.Teresultsclearlyshowthattargeted(RGD-modifed)nanoparticlesshowedenhancedFluTaxdeliverycomparedwithnon-targeted(unmodifed)nanoparticlesandFreeFluTax.Notably,FluTaxfuorescence

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Figure4.ComparativecellkillingefciencyofPTXformulationsinNSCLCcells.Cellviability(%)ofH1299

andA549cellswhentreatedwithfreePTX,PTX-PLGA-CSNP,andPTX-PLGA-CSNP-RGDfor24hand48hmeasuredbytrypanblueexclusionassay.(n=3);*p<0.05;**p<0.01;NS,non-signifcant.

correspondedtointegrinαvβ3expressionlevelsinlungcancercells,whenconsideringthenanoparticledeliverygroups.FreeFluTax,irrespectiveofcelllinesandtimepoints,showedcelluptakewhichiseithercomparableorlesserthannon-targetednanoparticlegroups(FluTax-PLGA-CSNP),butsignifcantlylesseruptakethantargetednanoparticlegroups(FluTax-PLGA-CSNP-RGD)(p<0.01).Basedonthefuorescenceintensityobtainedfromfuorescentpaclitaxel(FluTax),2nMFluTaxequivalenttreatmentofA549cellsusingFluTax-PLGA-CSNP-RGDresultedinanuptakeof28pg/1×105cellsin24h.IncontrastfreePTXuptakewas15.5pg/1×105cells,andfornon-targetednanoparticlesitwas20pg/1×105cells.TiswascalculatedfromfuorescenceintensityofknownFluTaxconcentration(2nM,100ul),keptaspositivecontrol.

BindingofRGDpeptide-integrinαvβ3receptorsafnitymediatestheendocytoticuptakeofRGD-modifednanoparticle

52

.AmongNSCLCcells,H1975cellsdisplayedthehighestuptakeofRGD-modifednanoparticles,anexpectedresultbasedontheirhigherintegrinαvβ3expression.Basedontheirexpressionofintegrinαvβ3,weanticipatedthatMRC-9cellswoulddisplayenhanceduptakeofRGD-modifednanoparticles,asobservedinthepresentstudy.However,normalbronchialepithelialcells(NHBE),withnegligibleintegrinαvβ3expression,showedtheleastuptakeofRGD-modifednanoparticles[FigureS7AandB].TisfndingclearlysuggeststhatuptakeofFluTax-PLGA-CSNP-RGDwasdependentonintegrinαvβ3expressionlevelsincells,whichtookupmoreFluTax-PLGA-CSNP-RGDthanunmodifedFluTaX-PLGA-CSNP.However,unmodifednanoparticleswithpositivechargemayefcientlyinteractwithnegativelychargedcellmembrane;asaresultcellularentryispossiblebycharge-mediatedadsorptiveendocytosis

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.Suchacellularentrymechanismforcationicparticlesisnon-specifcthatdoesnotdiferentiatebetweencancercellsandnormalcells.Interestingly,decorationofnan-oparticleusingpositivelychargedchitosanitselfwasreportedtohaveselectivitytowardscancercellscomparedtonormalcells,sincecancercellsshowedastrongernet-negativechargeonthecellmembrane

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.Terefore,acombinedefectofchargebasedinteractionandRGDbasedspecifctargetingmighthavecontributedtotheobservedcellaccumulationFluTax-PLGA-CSNP-RGD.NeverthelessthecelluptakestudyhighlightsthatRGDconjugationselectivelyincreasedthetargetingofthePTX-PLGA-CSNPtolungcancercells,butnottonormalbroncho-epithelialcells.SinceMRC-9cellsshowedselectiveuptakeofFluTax-PLGA-CSNP-RGD,wefurtherwantedtodeterminewhetherthiscelluptaketranslatedtocytotoxicityofdelivereddrug.

CellkillingefciencyofPTXformulationscomparedwithfreePTXinNSCLCcells.Toevaluate

thecellkillingefciencyoffree-PTXandPTXformulations,cellviabilityexperimentswereconductedfor24hand48hpost-incubation.TeIC50valuesinA549(~12.5nM)andH1299(~28.0nM)obtainedfromastandardlogarithmicplot[FigureS8]werechosenasthePTXdosesforourcellviabilityexperimenttocomparefreePTXandPTX-PGA-CSNP-(RGD+/?)formulations.AsshowninFig.

4

,freePTXshowedthehighestcyto-toxicityinA549andH1299celllinesatbothtimepoints.TecellkillingefciencyofPTX-PLGA-CSNPwasmoderateatequivalentPTXdosescomparedwithfreePTX.Tistrendwasexpected,asthePTXreleasefromnanoparticlesisslowandcontrolledovertime.Weobservedthattheaverage(%)cellviabilityoffreePTXandPTX-PLGA-CSNP-RGDtreatmentgroupsinA549cellswascomparableat24h(50.6%and54.7%respectively,p>0.05)and48h(29.7%and35.9%respectively,p>0.05),whereasfreePTXshowedsignifcanttoxicitycom-paredwithPTX-PLGA-CSNP-RGDinH1299cells(p<0.05).

However,PTX-PLGA-CSNP-RGDshowedreducedcellviabilityinbothcelllines,comparedwithPTX-PLGA-CSNP.Forinstance,themarginofcellviability(%)betweenPTX-PLGA-CSNP-treatedA549cellsandPTX-PLGA-CSNP-RGD-treatedA549cellswassignifcantat24h(65.9%and54.7%,respectively)(p<0.01)and48h(49.1%and35.9%)(p<0.01)indicatingaclearenhancedcellkillingefciencyofPTX-PLGA-CSNP-RGDtreatment.H1299cellsalsoshowedasimilartrendincellviabilityefcacyofPTX-PLGA-CSNP-RGDcom-paredwithPTX-PLGA-CSNP,especiallyat24h.ThecellviabilityforPTX-PLGA-CSNPgroupwas70.1%,

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whichissignificantlyhigherthanPTX-PLGA-CSNP-RGDgroupwith61.6%(p<0.01).However,thisdif-ferencewasnotsignifcantat48h.TecellviabilityforPTX-PLGA-CSNPgroupwas45.7%,whichissignif-cantlyhigherthanPTX-PLGA-CSNP-RGDgroupwith35.2%(p<0.01).TeoverallreductionincellviabilityinPTX-PLGA-CSNP-RGDindicatesthattheaccumulationofPTXisincreasedwithintegrintargeteddeliverycomparedtonon-targeteddelivery,resultingincorrespondingtoxicitylevels.

PTX-PLGA-CSNP-RGDinducesG2/McellcyclearrestandsignificantapoptosisinNSCLC

cells.TheeffectofPTX,PTX-PLGA-CSNP,andPTX-PLGA-CSNP-RGDincellcyclewasanalyzedinH1299andA549cellsafer24hand48hoftreatment.Figure

5A

showsthegraphicalrepresentationofcellcyclephases(%)comparedwiththeuntreatedcontrols.WeobservedthatthefreePTXandPTX-nanoparticlesinducedcellcyclearrestattheG2/Mcellcycle.NosignifcantdiferenceswereobservedbetweenfreePTXandPTX-PLGA-CSNP-RGDformulationsineithercelllineat24hand48h.Acomparativelysmallercellpopu-lationshowedG2/McellcyclearrestwithPTX-PLGA-CSNPtreatment.InA549cellsat24h,theG2/Mpop-ulationswere54.1%,45%,and52.9%,respectivelyforcellstreatedwithfreePTX,PTX-PLGA-CSNP,andPTX-PLGA-CSNP-RGD.Further,atime-dependentincreaseinG2/MarrestwasobservedinA549cellsat48h:63.6%,53.9%,and68.5%,forfreePTX,PTX-PLGA-CSNP,andPTX-PLGA-CSNP-RGDtreatmentgroupsrespectively.TeresultsshowedasignifcantenhancementinG2/MpopulationsbecauseofPTXtreatmentirre-spectiveoftheformulationscomparedtountreatedcontrols(p<0.01).Similarly,inH1299cellsat24h,theG2/Mpopulationswere46.8%,36.9%,and47.4%,respectivelyforcellstreatedwithfreePTX,PTX-PLGA-CSNP,andPTX-PLGA-CSNP-RGD.TePTX-PLGA-CSNP-RGDgroupinducedsignifcantlyhigherG2/Marrestcom-paredtoPTX-PLGA-CSNPgroup(p<0.01).At48hH1299cellsshowed60.1%,53.5%,and64.6%G2/Mpop-ulations(%),forfreePTX,PTX-PLGA-CSNP,andPTX-PLGA-CSNP-RGDtreatmentgroupsrespectively.TePTX-PLGA-CSNP-RGDtreatedgro