Lipid nanocapsule functionalization by lipopeptides derived from human papillomavirus type-16 capsid for nucleic acid delivery into cancer cells

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InternationalJournalofPharmaceuticsxxx (2013) xxx–xxx

ContentslistsavailableatSciVerseScienceDirect

International Journal of Pharmaceutics

jou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / i j p h a r m

Lipid nanocapsule functionalization by lipopeptides derived from human papillomavirus type-16 capsid for nucleic acid delivery into cancer cells

M. Weyland

^a,b,1

, A. Griveau

^a,b,1

, J. Bejaud

^a,b

, J-P. Benoit

^a,b

, P. Coursaget

^c

, E. Garcion

^a,b,∗

aInsermU1066,Microetnanomédecinesbiomimétiques,F-49933Angers,France

bLUNAMUniversité,F-49933Angers,France

cFacultédesSciencesPharmaceutiques,31AvenueMonge,F-37200Tours,France

a r t i c l e i n f o

Articlehistory:

Received28February2013 Receivedinrevisedform6June2013 Accepted10June2013

Available online xxx

Keywords:

Papillomavirusderivedpeptide Nanoparticles

DNA siRNA Vectorization Cancer

a b s t r a c t

PlasmidDNA(pDNA)andsmallinterferingRNAs(siRNAs)areveryusefultoolsforthetreatmentofcancer.

However,pDNAandsiRNAsefﬁcacyisrestrictedbytheirnegativechargeandsusceptibilitytodegradation byendonucleasesthatpreventthempenetratingtissueandcellularbarrierssuchastheplasmaand endolysosomalmembranes.Viralvectorshavesomeadvantagesbuttheiruseislargelylimitedbytheir immunogenicity.Ontheotherhand,syntheticnanoparticleshaveadvantageofbeingrelativelynon- immunogenicbuttheirabilitytodelivernucleicacidsremainslessefﬁcientthantheirviralcounterparts.

Thepresentstudyisfocussedonthedevelopmentandevaluationofbiomimeticlipidnanocapsules (LNCs)functionalizedwithaL1papillomavirustype-16capsid-derivedlipopeptideontheirsurface,for transfectionofU87MGgliomacellsandCaco-2colorectaladenocarcinomacellswithpDNAorsiRNAs.

SincetheL1-peptidehasbeendescribedasanuclearlocalizationsignalabletocomplexwithnucleicacids andbindtoheparansulfateonthecellsurface,thestructureandfunctionofL1-peptideboundtoLNCs (L1-LNCs)wereinvestigated.AlthoughL1-LNCswereshowntocomplexwithbothpDNAandsiRNAs,the pDNA-L1-LNCcomplexesshowedonlyweaktransfectionefﬁciency.Incontrast,siRNA-L1-LNCcomplexes appearedaseffectiverepressorsoftargetedmessengers.

1. Introduction

Sincecancerdevelopmentreliesontheacquiredorinherited geneticandepigenetic activationofoncogenesand ontheinac- tivationoftumour suppressorgenes,substituting alteredgenes by healthy plasmidDNA (pDNA) copies may beused for ther- apy(OrtizandEndy,2012).Geneinsertionintocancercellsusing pDNAmayalsobeusefultorenderthemmoresensitivetoradio- chemotherapyortointroducediscriminatorysuicidegenes(Zhang etal.,2008).pDNA-basedtherapywouldalsobebeneﬁcialforvac- cinationby generatingadaptive immunityagainst tumour cells notably through MHC class I restricted cytolytic T-lymphocyte responses(Liu, 2011).As wellaspDNA,small interfering RNAs (siRNAs), 20–25-nucleotide long double-stranded RNA (dsRNA) fragments,havedemonstratedtheircapacitytomediatespeciﬁc silencingofhomologousgenes(Elbashiret al.,2001;Fireetal.,

∗Correspondingauthorat:InsermU1066,IBS-CHUAngers,4rueLarrey,49933 Angerscedex9,France.Tel.:+33244688543;fax:+33244688546.

E-mailaddress:[email protected](E.Garcion).

1 Theseauthorscontributedequallytothiswork.

1998;WiannyandZernicka-Goetz,2000).Byinteractingwiththe RNA-induced silencing complex(RISC) locatedin thecell cyto- plasm,siRNAsleadtothedegradationofcomplementarymRNA sequencesandthusspeciﬁcallydownregulatetheproductionof theencodedprotein.ThetherapeuticpotentialofsiRNAshasbeen investigated withsomesuccess, leading toclinical trials(Davis etal.,2010).

However,theuseofpDNAorsiRNAsfortherapeuticpurposes copiesishinderedbyefﬁcacy,safetyanddeliveryconcernsthat limittheirclinicalapplication.Inordertohavetheintendedeffect these nucleicacids need to reachtheirsubcellular targets: the nucleusforpDNAandtheRISCcomplexinthecytoplasmforsiRNAs.

Hence,multipletissueandcellularbarriersneedtobeovercome, includingintracellularuptake,largelypreventedbecauseoftheir negativecharge andhydrophilicity (Lametal., 2012), ability to escape endolysosomalmaturation(ElOuahabiet al.,1997)and susceptibilitytodegradationbynucleases(Kawabataetal.,1995).

Inthiscontext,nanomedicine,representedbyliposomes,poly- mericnanospheres, lipidnanoparticlesor nano-emulsions, may provideasolution.Whenencapsulatedorcomplexed,thenucleic acidisnolongerdependentonlyonitsintrinsic propertiesbut alsoonthoseofitscarrier.Substantialbeneﬁtswouldbeobtained 0378-5173/$–seefrontmatter© 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.ijpharm.2013.06.013

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2 M.Weylandetal./InternationalJournalofPharmaceuticsxxx (2013) xxx–xxx

inthecrossingofbiologicalbarriers,thekineticsofnucleicacid release,bioavailabilityandefficacyinthetargetorgansandcells, preventingside effects and permittingreduction of doses(Peer etal.,2007;Torchilin,2009).However,althoughrelativelynon- toxic or non-immunogenic, non-viral vectorstodeliver nucleic acidswithonlyweaktransfectionefficacy(Perez-Martinezetal., 2011).Incontrast,viralvectorsareknownfortheirhighefficacy oftransfectionnotablyduetotheirwell-adaptedendolysosomal escapesignals(Choetal.,2003),butfacesmajorlimitationsdueto theirimmunogenicityandtheriskoftriggeringserioussideseffects (Hacein-Bey-Abinaetal.,2008).

Onepossibilitywouldbe,therefore,tocombinedtheadvantages ofeachofthesestrategiesbyfunctionalizingnanoparticleswith biomimeticpeptides(Maetal.,2012).Indeed,followingthedis- coveryoftheﬁrstcellpenetratingpeptidederivedfromDrosophila antennapediahomeodomain(Derossietal.,1994),theywereinves- tigatedfortheirabilitytodeliveractivemoleculesintocells(Poillot andDeWaard,2011).Furthermore,forpotentialtherapeuticappli- cationstheywerecombinedwithnanoparticles(Torchilin,2009).

Biomimeticpeptidesmaybeusedasactiveentitiesontheirown orastargeting moieties capabletorecognizespecific biological componentsor receptors (Kim and Huang, 2012; Pearce et al., 2012; Ruoslahti, 2012; Su et al., 2012). In this respect, it was recentlyestablishedthatanL1motifsequence(TSTTAKRKKRKLK) derivedfrompapillomavirustype16capsidproteinwassufficient totriggerheparan sulfate recognition,endocytosisand efficient genetransferintotarget cells(Bousarghinetal.,2003a,b,2004, 2009).

Thus,byfocusingonlipidnanocapsules(LNCs)thathavebeen conﬁrmedascarriersforconventionalanticancerdrugs(Garcion etal.,2006;Lacoeuilleetal.,2007;Weylandetal.,2011)andradio- pharmaceutics(Vanpouille-Boxetal.,2011), whilealsoeliciting beneﬁcialadjuvanteffectsuchasinhibitionofmultidrugresistance (Garcionetal.,2006)orendolysosomalescape(Paillardetal.,2010;

Rogeretal.,2010),theaimofthepresentstudywastodevelopLNCs combinedwiththeL1-peptideandtoinvestigatetheiraptitudefor thedeliveryofpDNAandsiRNAstocancercells.

2. Materialsandmethods

2.1. PreparationofcontrolLNCsandNileRed-loadedLNCs (NR-LNCs)

50nmLNCswerepreparedaccordingtotheprocessdescribed elsewhere(Garcionetal.,2006),usingaphaseinversionprocess followingtheformationofanoil/watermicroemulsion contain- inganoily/fattyphase(caprylic-capricacidtriglycerides:Labrafac^® WL1349;Gattefossé,Saint-Priest,France),anon-ionichydrophilic surfactant(polyethyleneglycolhydroxystearate:Solutol^® HS15;

Basf,Ludwigshafen,Germany)andalipophilicsurfactant(soybean lecithincontainingat least69%ofphosphatidylcholine:Lipoïd^® S75-3;LipoïdGmbH,Ludwigshafen,Germany). Briefly, Solutol^® HS15,Lipoid^® S75-3,Labrafac^® WL 1349,NaClandwater (846, 75,1028,89 and2962mgrespectively)weremixedandheated undermagnetic stirringto90^◦C.Then, three cyclesofprogres- siveheatingand cooling between90^◦C and 60^◦C were carried out,followedby anirreversibleshock inducedbydilutionwith 5mLofcooldeionizedwateraddedtothemixtureat80^◦C.For thesynthesisoffluorescentLNCs,thefluorescentcompoundNile Red (NR; Sigma–Aldrich, Saint-Louis, USA) was used as previ- ouslydescribed (Garcionetal.,2006).Briefly, NRwasdissolved in acetoneat 0.1% (w/w),and the 0.1%NR solutionin acetone wasincorporatedin Labrafac^® at1:10 (w/w).Then, LNCs were preparedas described above withcomplete evaporationof the acetone during the process. Size exclusion and high-pressure liquidchromatography (HPLC) assays demonstrated a complete

encapsulationandretentionofNRwithintheLNCs(Garcionetal., 2006).

2.2. Method1:IncorporationofDSPE-PEG2000-maleimideinto theLNCshellandpeptideconjugation

Firstly, 20mg DSPE-PEG2000-maleimide (2941.605g/mol, Avanti Polar Lipids, Alabaster, USA) powder diluted in HEPES buffer(0.1M,pH7.4),wasaddedto60mgofLNCssuspensionin ordertoobtainaﬁnalconcentrationof20mM.LNCsand DSPE- PEG2000-maleimidewereincubatedfor2hat60^◦C.ASepharose^® CL4-B column (Sigma–Aldrich),equilibrated withHEPES buffer, wasused to separate thefunctionalized LNCs fromfree DSPE- PEG2000-maleimide.TheLNCconcentrationofthemixturewas estimatedbyturbidityat580nmusingaMultiskan^® microplate spectrophotometer (Thermo Electron, Saint-Herblain, France).

Secondly,1.2mgfunctionalizedLNCscontainingDSPE-PEG2000- maleimide were incubated with different amounts (between 0.6␮gand6␮gofpeptides)ofL1papillomavirus-derivedpeptide (L1;sequence:CTSTTAKRKKRKLK,1313g/mol;Millegen,Labège, France) or control peptide (CP; sequence: CLVEETSFIDAGAPS, 1280g/mol;Millegen)overnightinHEPESbufferatroomtemper- atureundermagneticstirring.ASephadex^TMG25Mediumcolumn (GEHealthcareBio-SciencesAB,Sweden)equilibratedwithHEPES buffer was used to remove unbound peptides. Turbidimetry measurement was performed for each fraction to detect the nanocapsules.Then,theywerepooledanditisconcentrationwas assessedbyturbidimetricmeasurement at580nm.The amount of peptide graftedin theparticles was determined bya micro Bradfordcolorimetricproteinassaykit(microBCA,PerbioScience, Courtaboeuf,France).

2.3. Method2:Lipopeptidemonomerformationand post-insertioninLNCshell

A1mM DSPE-PEG2000-maleimidesolutionwaspreparedby dissolving4.3mgofDSPE-PEG2000-maleimidepowderin1.5mL of HEPES buffer. To synthesize lipopeptides, DSPE-PEG2000- maleimide solution wasincubated in the presence of different amounts of peptide(L1 orCP) overnight,at roomtemperature under magnetic stirring. The molar ratio of DSPE-PEG2000- maleimide/peptidewasvariedfrom1/0.9to1/2.

100␮LofcontrolLNCsat200mg/mLwereincubatedwith1mL oflipopeptidesolutionfor4hat37^◦Cundermagneticstirring.A Sephadex^TMG25mediumcolumnequilibratedwithHEPESbuffer was used to separate the peptide-LNCs (L1- or CP-LNCs)from freeDSPE-PEG2000-maleimide.Todeterminethepeptideconcen- trationsgrafted onto theLNCs, a microBCAassay kit wasused accordingtothemanufacturer’sinstructions. Theconcentration ofLNCswasevaluatedbyturbidimetricmeasurementsat580nm usingaMultiskan^®microplatespectrophotometer.

2.4. CalculationofthenumberofpeptidesatthesurfaceofLNCs followingcouplingbymethods1and2

Considering that all the excipients used in the formulation contribute to the formation of LNCs and the volume of these nanocapsulesismainlyduetotheLabrafac^®(whoserelativeden- sityis0.94),itwaspossibletoestimatethenumberofpeptides (NP)onthesurfaceofasinglenanoparticleineachsamplewith theformula:NP=([peptide]×N_A)/(V_Labrafac^®/V_LNC),where[peptide]is the amountof peptideas a molar concentration,N_A is theAvogadronumber,V_Labrafac^®isthevolumeofLabrafac^®inthe sampleand V_LNC is thevolumeofa singleLNCcalculatedfrom thesizemeasurementsemployingthevolumeofsphereformula, 4/3␲R³.

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M.Weylandetal./InternationalJournalofPharmaceuticsxxx (2013) xxx–xxx 3 2.5. Formationofnucleicacid-peptide-LNCcomplexes

Negativecontrol siRNA (controlsiRNA, sequence:5-GUCC- GGAAUUACCAUGAGU-3),asiRNAthatinhibitstheexpressionof CD133(CD133siRNA,sequence:5-CCCUUAAUGAUAUACCUGA-3) and a siRNA that inhibitstheexpressionof Bcl-2 (Bcl-2 siRNA, sequence:5-GUACGAUAACCGGGAGAUA-3)wereobtainedfrom Sigma–Aldrich.Fortheformationofcomplexes,90ngofDNAor 0.02nMofsiRNAswereincubatedfor90minatroomtemperature withdifferentamountsofcontrolLNCs,L1-LNCsorCP-LNCs(from 6to300␮g)inaﬁnalvolumeof20␮LinRNase-freewater.The concentrationofsiRNAswas1␮MandLNCsconcentrationvaried from3to9g/L.

2.6. CharacterizationofLNCs

Suspensions(controlLNCs,L1-LNCsandCP-LNCs)werechar- acterized using Zetasizer^® Nano Series DTS 1060 (Malvern InstrumentsS.A., Worcestershire,UK)fortheirsize,polydispersityindex(PdI)andzetapotential.Beforetheanalysis,theparticles werediluted1/60times(v/v)indeionizedwaterinordertoensure aappropriatesignalintensityatthedetector.

2.7. Gelretardationassays

Complexes, diluted in 1X TBE (Tris-Borate- Ethylenediaminetetraacetic acid (EDTA); Sigma–Aldrich) and gelloadingsolution(Sigma–Aldrich),wereloadedina1%agarose gelcontaining 0.5␮g/mLof ethidiumbromide(Sigma–Aldrich).

Electrophoresiswascarriedoutat100Vfor45minin1×TBEas runningbuffer.ThegelwasvisualizedunderUVlampsusingaVil- berLourmatChemiSmart3000transilluminator(Marne-la-Vallée, France).

2.8. Cellculture

Malignanthumangliomacells U87MGandCOS-7 cellswere obtainedfromtheAmericanTypeCultureCollection (ATCC-LGC Promochem,Molsheim,France). Theywerecultured at37^◦C/5%

CO₂inDulbecco’sModiﬁedEagleMedium(DMEM;Lonza,Verviers, Belgium) containing 4.5g/L glucose, 1% antibiotics from stock solution(10,000unitsofpenicillin,10mgofstreptomycin,25␮g amphotericin B/mL; Sigma–Aldrich) supplemented with 10% of foetalbovineserum(FBS;Lonza).Theywerepassagedwith0.5%

porcinetrypsinand0.2mg/mLEDTA(Lonza)whentheyreached 80% conﬂuence. The medium wasreplaced with every 3 days.

Undifferentiatedhumancolon carcinomaCaco-2 cells, obtained fromATCC-LGC Promochem, werecultivated in DMEM (Lonza) containing4.5g/Lglucose,1%antibiotics(Sigma–Aldrich)and1× non-essentialaminoacids(NEAA;Lonza)supplementedwith10%

ofFBS(Lonza).Thecellsweredissociatedinthesamewayasfor theglioblastomacellslinesandthemediumwasreplacedevery2 daysuntiltheyreached80%conﬂuence.

2.9. Luciferaseexpressionassaystoassesstransfectionefﬁcacy

Inordertotesttheefficacyofthetransfectionusingnanocar- riers,astandardizedluciferaseassaywasperformedonCOS-7cell linetransfectedwithfireflyluciferasegenecontainingpGL4luc2 plasmidtransfected withdifferenttransfectionagents as previ- ouslydescribed(Bousarghinetal.,2003a,2009;Combelasetal., 2010). Briefly, after washing with serum-free medium, COS-7 cellswereexposedtothepGL4luc2luciferasereporterplasmid (10␮g/mL;Promega,Madison,USA)inthepresenceoftheindi- catedtransfectionagent(controlLNCs,lipopeptidegrafted-LNCs orlipofectamine). Luciferase gene expressionwasmeasuredby

luminescenceassayusing theFireﬂy luciferaseassaykit (Inter- chimMontluc¸on,France).Theluminescencewasintegratedover 10susingaVictor2platereader(Wallac,PerkinElmer).Theresults wereexpressedascountspersecond(cps)perwell.

2.10. CellularuptakeofNR-LNCsbyﬂowcytometry

U87MGcellswereplatedat6×10⁴perwellsin6-wellplatesin 2mLofDMEMcontaining10%FBSand1%antibiotics.After3days incubationat37^◦C/5%CO₂,serumdepletionwascarriedoutfor 24hbyreplacingtheoriginalmediumbyserum-freeDMEMwith 1%N1supplement(Sigma–Aldrich)and1%antibiotics.U87MGcells werethenincubatedfor2hinserum-freemediumwithNR-LNCs, L1-NR-LNCsor CP-NR-LNCs,derived frommethod1 or method 2,ataconcentrationof100␮g/mL.Cellswerethendetachedby trypsinization. Aftercentrifugation,theywere re-suspendedfor 1minin0.4%(w/v)trypanbluesolutioninHBSS(Lonza)toquench theextracellularfluorescence,thusenablingthedeterminationof thefractionthatwasactually internalized.Thetreated samples weresubsequentlywashedtwiceinPBSandre-suspendedinPBS containing2%formaldehyde(Prolabo,FontenaysousBois,France) and0.02%azide(Sigma–Aldrich)beforeanalysisbyflowcytometry inatleasttriplicateexperiments,with3000to10,000cellsmea- suredineachsample.ABDFACSCalibur^TM fluorescent-activated flowcytometerandtheBDCellQuest^TMsoftware(BDBiosciences, SanJose,California)wereusedtoperformflowcytometry.Analy- siswascarriedoutusingWinMDI2.9software(ScrippsInstitute, LaJolla,CA,USA).Thesameexperimentationwasrealizedwith fluorescentsiRNAscoupledwithAlexa488(Sigma–Aldrich).

2.11. siRNAinterferencewithBcl-2expressioninU87MGand CD133expressioninCaco-2cells

U87MG or Caco-2 cells were plated in 6-well plates with 2.5×10⁵ cellsperwellin2mLofDMEMcontaining10%FBS,1%

antibioticsand1×NEAAfor24hat37^◦C/5%CO₂.Serumdeple- tionwascarried out for24hbyreplacing theoriginalmedium by serum-free DMEM added with 1× N1 supplement and 1%

antibiotics. U87MG or Caco-2 cells were transfected for 2hat 37^◦C/5%CO₂ in serum-freemediumwith25nMof siRNAsand 100␮g/mL LNCs (50␮L suspension in the 2mL wells). N-TER reagent(Sigma–Aldrich)wasusedasthecontrolagentoftrans- fection and the transfection was performed according to the manufacturer’sinstructions.Thecellswereincubatedwith5␮g/mL AC133antibody(MiltenyiBiotech,Paris,France)orwith4␮g/mL Bcl-2antibody(SantaCruzBiotechnology,Heidelberg,Germany) or withIgG1isotype control (BDBiosciences,Le Pont-de-Claix, France)for1hat4^◦CinPBScontaining5%FBSand0.02%sodium azide. Cellswerewashed threetimes inPBS containing5%FBS and 0.02% sodiumazide,and incubated for30minat4^◦C with FITC-conjugatedgoatanti-mouseIgGF(ab’)2fragmentpolyclonal antibody(Dakocytomation,Trappes, France)at20␮g/mLinPBS containing5%FBSand0.02%sodiumazide.Followingthreemore washes inPBScontaining5%FBS and0.02% sodiumazide,cells werere-suspendedinPBScontaining2%formaldehydeand0.02%

sodiumazide.ABDFACSCalibur^TMandtheBDCellQuest^TMsoftware wereusedtoacquiretheﬂowcytometrydataandtheanalysiswas carriedoutusingWinMDI2.9software.

2.12. ReversetranscriptionandquantitativePCR(RTqPCR)

ThecellsarerecoveredandRNAwasextractedusingtheRNeasy Minikit(QiagenS.A.,Courtaboeuf,France),includedaQIAshredder step(Qiagen),andaﬁrstDNasetreatmentwithaRNase-FreeDNase Set(Qiagen).TheDNasetreatmentwascarriedoutwithaTurbo DNaseFreekit(Ambion,LifeTechnologies,Paisley,UK)inaﬁnal

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volumeof25␮L.RNAwasprecipitatedinethanol,andthequali- tativeanalysisofRNAfromtheextractwasrealizedonanAgilent NanoRNAchip.Reversetranscription(RT)reactionfrom0.75␮gof RNAwasmadewithanAccuScriptHighFidelity1ststrandkit(Agi- lentTechnologies,lesUlis,France).QuantitativePCRwasrealized with1␮LoftheRTsamplebyaBrilliantIIFASTSYBR^®GreenQPCR MasterMixes(Agilent).qPCRwasmadeinMx3005P(Agilent),with theprimerconcentrationof400nM.Theexpressionofthreegenes wasanalyzed:␤-actinandhypoxanthine-guaninephosphoribosyl transferase,HGPRT,ascontrolsandCD133.TheprimersforCD133 werethoseusedinthepublicationofPlatetetal.,2007).Thedata wereanalyzedbyREST2009software.

2.13. Statisticalanalysis

XLSTAT2012Version2012.6.09(AddinsoftParis,France)was usedfordataanalysis.Statisticalsigniﬁcanceforeachexperiment wasdeterminedbyDunnett’stest.Thetestswereconsideredas signiﬁcantifthepvalueswerelessthan0.05.

3. Resultsanddiscussion

3.1. FunctionalizationofthesurfaceofLNCsfornucleicacid delivery:evaluationoftwopost-insertionmethodsforthe L1-papillomavirus-derivedpeptide

Toinvestigate thebeneﬁt of attachingan L1 papillomavirus derived-peptidetoLNCstoimprovethedeliveryofnucleicacids tocancercells, theability ofcontrol LNCs tobindto DNAwas ﬁrstinvestigated.AsshownonFig.1A,afterincubationof90ng ofDNAwithincreasingamountsofLNCs(between6to300␮g), controlLNCswerenotcapableofretainingDNAwithinthewells of agarose gel, indicating their low ability to form complexes withDNA.

In orderto complexLNCs withnucleic acids, thepossibility offunctionalizingthembycouplingtheL1-peptidederivedfrom humanpapillomavirustype-16capsidprotein(Fig.1B)ontotheir surfacewasinvestigated.SubmicronsizesLNCsweresynthesized asdescribedinthematerialsandmethodssection.Twomethods ofattachmentoftheL1-peptideontothesurfaceofLNCs,bothper- formedonpre-formedLNCs,wereinvestigated(Fig.1C).Method1 usedabi-functionalDSPE-PEG2000-maleimidepolymercontain- ingasulfhydryl-reactivemaleimidegroupthatwasﬁrstinserted intotheLNCsand thenallowedtoreactwiththethiol-function presentsontheN-terminalcysteineoftheL1-peptideorCP-peptide (ascontrol)(Fig.1C andD). In contrast,in method2 thesame bi-functionalpolymerwasﬁrstcoupledwiththethiolatedpep- tidetoformalipopeptide,whichwastheninsertedintotheLNCs (Fig.1C).In ordertoreducepotentialalterationsinthepeptide whenitwascombinedwiththeDSPE-PEG,thetemperatureofthe insertionstep wasdecreased from60^◦Cto37^◦C,and thereac- tiontimewasdoubled,from2hto4h.Astericexclusioncolumn wasusedtoseparatepeptideboundtoLNCs fromfreepeptide (Fig.1E).Thus,differenttypesofLNCswereobtainedallpresenting amonomodalsizedistribution(Table1):controlLNCs(composed exclusivelyofatriglyceridecoreandashellcontaininglecithinand anamphiphilicsurfactant,thehydroxystearateofpolyethylenegly- col)andfunctionalizedLNCscarrying DSPE-PEG2000-maleimide (M-LNCs,method1),L1-LNCsorCP-LNCsfrommethod1(10␮g ofpeptidefor20mgLNCs)andL1-LNCsorCP-LNCsfrommethod 2(1mgof peptidefor20mg LNCswasused)(Fig.1C,Table1).

ThemicroBCAassaywasusedtodeterminetheyieldofpeptide incorporationintoLNCsandindicatedthatabout85%ofthepep- tidesusedwereincorporatedontotheLNCsurfacewhateverthe method(Fig.1E).Withmethod1,weobtainedbetween16 and

160peptidesperLNC,whereaswithmethod2,thisnumbervaried between2066and2327peptidesperLNC(Table2).Theinsertion ofDSPE-PEG2000-maleimide withintheLNCshellbymethod1 resultedinamarkedreductionofthezetapotential(from−6to

−37mV)andaclearincreaseinsize(from50to70nm).Whatever thepeptideused,controlorL1,forfunctionalizationofLNCsusing method1,thezetapotentialoftheparticleremainedclearlynega- tive(−26/−38mV)(Table1).Incontrast,duringmethod2,thezeta potentialofnewlyformednanocarriersappeareddirectlydepen- dentonthepeptideusedandreﬂectedeitherthepositivechargeof theL1-peptideorthenegativechargeoftheCP-peptide(Table1).

Hence,thezetapotentialofL1-LNCswas+17mVwhilethatofCP- LNCswas−27mV.Interestingly,thepost-insertionprocedureused inmethod2afterthesynthesisofthelipopeptideresultedinamod- eratechangeoftheLNCsize(from50to60nmforallLNCs)(Table1).

Althoughmethod1waspreviouslyreportedtoallowthegrafting ofimmunoglobulinontothesurfaceofLNCswithagoodefﬁciency (Beduneauetal.,2008),itisimportanttonotethenecessityofusing alargeexcessofDSPE-PEG2000-maleimidetoobtainasigniﬁcant amountofantibodyorpeptideincorporation.Sincethemolarratio betweenpeptideandDSPE-PEG2000-maleimidewasabout1/23, manysulfhydryl-reactivemaleimidegroupswerenotfunctional- izedafterthechemicalreactionwithmethod1.Inlinewiththis,it isimportanttonotethatthequantityofpeptideusedforincorpo- rationwithmethod1(10␮gfor20mgLNCs)wasmuchlowerthan theamountusedwithmethod2(1mgfor20mgLNCs).However, increasingtheamountofpeptideduringthethiol-maleimidereac- tiondidnotresultinanyimprovementofitsincorporation,indicat- ingalimitedaccessofthethiolatedpeptidetosulfhydryl-reactive maleimidegrouppresentatthesurfaceofLNCs.Thus,afterusing method1,thezetapotentialofLNCsremainedessentiallythatofthe freenegativesulfhydryl-reactivemaleimidefunctions.Incontrast, method2permitteda3-foldreductionoftheamountoftheDSPE- PEG2000-maleimideperLNC,andanincreaseinthenumberofpep- tidespresentatthesurfaceoftheLNCbyafactorupto150(Table2).

SincethemolarratiobetweenDSPE-PEG2000-maleimide/peptide was about 1/1, this very efﬁcient incorporation indicated that notonlythethiol/maleimidereactionwascompletebutalsothe post-insertionofthelipopeptidemonomer.Asa result,thesur- faceofLNCsfunctionalizedviamethod2appearedtobetotally biomimeticanddisplayedthepropertiesoftheL1-orCP-peptides characteristics.

3.2. EvaluationoftheabilityofLNCstobeinternalizedbyU87MG gliomacellsdependingonthemethodofL1-peptideincorporation

Inordertofurtherinvestigatethedifferencesbetweenthetwo methodsofpeptideincorporation,LNCinternalizationbyU87MG glioma cells wasstudied. Thus, peptide-LNCs incorporatingthe fluorescenttracerNileredwereincubatedwithU87MGcellsfor 2hbeforedeterminingLNCuptakebyfluorescence-activatedflow cytometry.Todiscriminatebetweenassociationwiththecellsur- faceandtrueinternalization, Nileredextracellularfluorescence wasquenchedbytheadditionoftrypanblue,thenonquenched fractionthusrepresentinginternalizedLNCs(Paillardetal.,2010).

AsshowninFig.2,Nilereduptakewasconsiderablylowerwhen method1 was usedto functionalizethe LNCs (about −40% for M-LNCs and L1-LNCs, and −20% for CP-LNCs) than when LNCs functionalizedbymethod2wereused(80%forL1-LNCsand70%

forCP-LNCs).Althoughnegatively-chargednanocarriershavebeen describedtobeinternalizedbyU87MGcells,thelowzetapotential ofthenanoparticlespreparedbymethod1mayhavereducedtheir interactionwiththecell-surface negativecharges(Changetal., 2012).AnotherimportantconsiderationisthefreePEG-maleimide functionalgrouppresentatthesurfaceofthelipidnanocarrierspre- paredbymethod1mayhavegreatlyreducedtheiruptakethough

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M.Weylandetal./InternationalJournalofPharmaceuticsxxx (2013) xxx–xxx 5

Fig.1. InteractionofcontrolLNCswithnucleicacidsandfunctionalizationusingtheL1-peptide.A)GelretardationassayofcontrolLNCsincubatedwithplasmidDNA.The amountofDNAamountineachwellwas90ngwhiletheamountofLNCwasincreasedfrom120to300␮g.B)SchematicillustrationoftheL1-peptideoriginfromhuman papillomavirustype16capsidrepresentinghumanpapillomavirustype16model(HPV-16),isolatedcapsomer,andL1-peptide.Notethat,althoughtheC-terminalpartof theL1proteinissituatedinsidetheHPV-16capsid,itsexactlocalizationremainsunknown.C)SchematicrepresentationofthecouplingpeptidetoLNCsusingtwomethods:

i)method1:theinsertionofDSPE-PEG2000-maleimideintheLNCshellismadebeforeasulfhydryl-malemidereactionthatallowsthegraftingofthepeptide,ii)method2:

thesulfhydryl-malemidereactionisﬁrstcarriedouttoformaDSPE-PEG2000-peptidemonomerthatistheninsertedintheLNCshell.D)Reactionofthesulfhydrylgroup ofthepeptidecysteineandthemaleimideextremityoftheDSPE-PEG2000-maleimideleadstoastablethioetherbonds.E)MicroBCAdeterminationofpeptideperfraction recoveredintheSephadex^TMG25mediumcolumn.ThelargerpeakcorrespondstopeptidesinsertedwithinLNCs(85%)whilethelowerrepresentsfreepeptide(9%).

repulsion,sequestrationwithintheextracellularmatrixoradsorp- tion ofproteins containingcysteine thiol groups (Salvati et al., 2013).TakingintotherelativelylowuptakeofLNCspreparedwith method1thatwaspossiblyduetolowpeptideincorporationand thepresenceofexcessfreemaleimidefunctionalgroupatthesur- face,allfurtherexperimentswerecarriedoutusingLNCsprepared bymethod2.

3.3. DevelopmentandbiologicalevaluationofDNA-L1-LNC complexes

Inorder toanalyzetheformationofcomplexesbetweenL1- LNCs and nucleic acids, theinteraction of LNCs with DNA was investigatedbyagelretardationassay.Forthispurpose,increas- ingamountsofLNCs(6–300␮g)wereincubatedwithaconstant

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Table1

Physicochemicalcharacteristicsofcontrolandpeptide-LNCsobtainedwithmethod1and2,includingmeandiameter,PdIandzetapotential.Datawereobtainedfromthree independentexperiments(n=3).PdI:polydispersityindex.Dunnett’stest:*p<0.001,comparisonwithcontrolLNCs;^◦p<0.001,comparisonbetweenmethod1andmethod 2.

Formulations Meandiameter(nm) PdI Zetapotential(mV)

ControlLNCs LNC 49.4±1.4 0.04±0.01 −6.2±0.4

NR-LNC 49.1±1.0 0.03±0.01 −5.6±1.2

PeptideLNCs(method1) M-LNC 71.8±1.6* 0.03±0.01 −37.4±2.6*

L1-LNC 71.2±1.2* 0.06±0.01 −38.8±1.5*

CP-LNC 72.5±1.5* 0.07±0.02 −36.2±0.7*

M-NR-LNC 70.8±2.0* 0.18±0.03 −26.9±1.3*

L1-NR-LNC 71.8±1.4* 0.12±0.02 −34.2±1.6*

CP-NR-LNC 71.4±1.2* 0.09±0.02 −33.6±1.2*

PeptideLNCs(method2) L1-LNC 61.5±2.5*^,◦ 0.106±0.02 +17.9±0.5*^,◦

CP-LNC 58.5±1.6*^,◦ 0.087±0.01 −28.4±1.5*^,◦

L1-NR-LNC 57.3±2.1*^,◦ 0.049±0.02 +17.1±1.3*^,◦

CP-NR-LNC 59.9±1.8*^,◦ 0.201±0.02 −26.3±1.8*^,◦

Table2

QuantiﬁcationsoftheamountofpeptideperLNCassociatedbymethod1and method2expressedasthemassofDSPE-PEG2000-maleimidepermassofLNC, asmassofpeptidepermassofLNCandbynumberofpeptidesperLNC.

␮gof DSPE-PEG2000- maleimide/mgof LNCs

␮gof peptides/mg ofLNCs

Peptides/LNC

Method1 333␮g 2␮g between16and160

Method2 99␮g 44␮g between2066and2327

amountofplasmidDNA(90ng)in20␮Lfor90minatroomtem- perature.WhileL1-LNCswereabletodelayorblockthemigration ofDNAintheagarosegel(Fig.3A),CP-LNCsdidnotchangethis DNAmigrationwhatevertheconcentrationtested(Fig.3B).Deco- rationoftheLNCshellwithL1-peptidewas,therefore,responsible fortheacquisitionofanewfunctionality:theformationofcom- plexeswith DNA. Full complexation of the 90ng of DNA used occurredwhen240␮gofL1-LNCswereused(Fig.3A).Inorderto definethebiologicalsignificanceofthisnewpropertyofinterac- tionofL1-LNCswithDNA,transfectionofCOS-7cellswithaplasmid DNA(pGL4luc2)expressingthefireflyluciferasewasinvestigated usingdifferentLNCformulationsandlipofectamineasatransfec- tioncontrol.Luciferaseexpressionwasfollowedbyluminescence asdescribedabove(Section2.9).Althoughluciferaseactivitywas detectedinthepresenceoflipofectamine,noluciferaseexpression wasdetectedwhentransfectedwithDNAcomplexedwithcontrol, non-functionalizedLNCs(Fig.3C).FunctionalizationofLNCswith

0 20 40 60 80 100 120

NR-LNC L1-NR-LNC CP-NR-LNC M-NR-LNC

Nile red uptake (au)

Method 1 Method 2

**

*, ^##

*,^##

Fig.2. ComparisonoftherelativeNilereduptakebyU87MGcellsbetweenthe LNCs(NR-LNC,L1-NR-LNCandCP-NR-LNC)synthesizedbymethod1andmethod2.

U87MGcellsweretransfectedduring2hat37^◦Candcellularuptakewasdeter- minatebyﬂowcytometry.Dunnett’stest:*p<0.05;**p<0.01,comparisonwith NR-LNCs;^##p<0.01,comparisonbetweenmethod1andmethod2.

L1-orCP-peptidealmosthadnoimpactontheexpressionofpDNA transfected(Fig.3C).ConsideringthefactthatLNCentryintocells wasdemonstratedforbothtypesofLNCscombinedwithDNA:L1- LNCs+DNA(formingcomplexes)andCP-LNCs+DNA(notforming complexes)(Fig.3D),thisobservationsuggeststhattheDNAtrans- portedbyLNCscannotreachthenucleusoftransfectedcells.While theycanformcomplexeswithDNA,L1-LNCsdonotappeartobean effectivetransfectionagent.Therefore,theirutilityforthedelivery ofsiRNAstocancercellswasinvestigated.

3.4. Development,biologicalevaluationandphysicochemical characterizationofsiRNA-L1-LNCcomplexes

InordertodeterminetheabilityoftheLNCsdevelopeddur- ingthis work tocomplex withsiRNAs,a siRNA gel retardation assay in the presence of differentLNCs was performed. About 133ngofsiRNAwasincubatedfor90minatroomtemperaturewith 60–180␮gofLNCsin20␮LofTBEbuffer(correspondingﬁnalcon- centrations:1␮MsiRNAsand3–9g/LLNCs)beforeelectrophoretic migration.AsshowninFig.4A,gelretardationdemonstratedthe complexationofsiRNAstotheL1-LNCsbutnottothecontrolorCP- LNCs.About180␮gofL1-LNCsfor133ngofsiRNAswasfoundtobe thebestconditionforcompletecomplexationofsiRNAs(Fig.4A).

To further investigate the complexation of siRNA with functionalized-LNCs,thephysicochemical propertiesofpeptide- LNCscombinedwithsiRNAwereanalyzed.Asshown inTable3 (incomparisonwithTable1), a 12-nmincrease insizeof LNCs wasobservedwhen133ngofsiRNAwasincubatedwith180␮g ofL1-LNCsin20␮L(L1-LNC+siRNA).Incontrast,nochangeinsize wasseenwhensiRNAwasincubatedwithCP-LNCs(Table3).In termsofzetapotential,aslightdecreaseinpositivecharge was observedforL1-LNCsincubatedwithsiRNAwhileaslightincrease innegativechargewasobservedforCP-LNCsincubatedwithsiRNA (Table3).TheseobservationsindicatethatalthoughsiRNAsmodify thechargeatthesurfaceofL1-LNCsandCP-LNCs,signiﬁcantcom- plexformationonlyoccurswithL1-LNCs,asshownbytheincrease insizewhencombinedwithsiRNAsandthedelayintheirmigration inagarosegel.

ThespeciﬁcsilencingcapacityofthesesiRNA-bearingLNCswas thenstudiedinlivingcells.Twomoleculartargetswerechosen:

Table3

Physicochemicalcharacteristicsofpeptide-LNCscomplexedwithsiRNAsobtained withmethod2,includingmeandiameter,PdIandzetapotential.Datawereobtained fromthreeindependentexperiments(n=3).PdI:polydispersityindex.

Meandiameter(nm) PdI Zetapotential(mV) L1-LNCs+siRNA 73.4±2.2 0.191±0.05 +11.3±1.9 CP-LNCs+siRNA 60.5±0.7 0.095±0.04 −22.9±0.5

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M.Weylandetal./InternationalJournalofPharmaceuticsxxx (2013) xxx–xxx 7

Fig.3.GelretardationassayofDNAandfunctionalstudy.A)GelretardationassayofDNAcomplexedwithL1-LNCsB)andwithCP-LNCs.TheamountofDNAwasthesamein eachwell:90ng.TheamountofLNCswasincreasedfrom6to300␮g.Lane1:DNA.Lanes2to10:L1-orCP-LNCs+DNA.C)EfﬁciencyoftransfectionofCOS-7cellsbydistinct LNCsformulationsinthepresenceofthepGL4luc2plasmidassessedbyluminescencemeasurements.Datawereobtainedfromthreeindependentexperiments(n=3).cps:

countpersecond.D)FlowcytometryanalysisoftherelativeNilereduptakewithinU87MGcellsusingdifferentLNCsformulationscombinedwithDNA(NR-LNC+DNA, L1-NR-LNC+DNA,CP-NR-LNC+DNA).Datawereobtainedfromthreeindependentexperiments(n=3).Dunnett’stest:*p<0.05,comparisonwithNR-LNC+DNA.

CD133,themRNAexpressionofwhichwasinvestigatedinCaco- 2 cells, and Bcl-2, theexpression of which wasinvestigated in U87MGcells.AsshowninFig.4B,RTqPCRassaysdemonstrateda potentactivityofCD133siRNAscombinedwithbothN-TERpeptide andL1-LNCsonCD133mRNAexpressioninCaco-2cells.Incon- trast,CD133siRNAscombinedwithCP-LNCsandcontrolsiRNAsin allformulationstestedhadnoeffectonCD133mRNAexpression (Fig.4B).AsfarastheBcl-2targetwasconcerned,ﬂowcytometry analysisdemonstratedthattreatmentofU87MGcellswithBcl-2 siRNAscombinedwithL1-LNCsresultedinastronginhibitionof theexpressionoftheproteinwithaﬂuorescenceintensityaslow asthatobtainedwithanIgG1controlimmunoglobulin(Fig.4C).

AllotherformulationscontainingthespeciﬁcsiRNA,includingthe N-TERpeptide,didnotproduceanysigniﬁcanteffectontheBcl-2 proteinexpression(Fig.4C).Althoughthegenesuppressionstud- iesweremadeattheproteinlevel,thisobservationcontrastedwith thatmadeonCD133mRNAexpressioninCaco-2cells,inwhichthe N-TERreagenthadasimilareffecttoL1-LNCs.Thissuggeststhatthe modalitiesofsiRNAsilencingtriggeredbyN-TERandL1-LNCsare distinct.

Among the mechanisms that may explain these differences in behaviour between the different nanoparticle systems are differences in endocytosis pathways that have been described for the uptake of non-viral gene delivery systems, including clathrin-mediated endocytosis, caveolae-mediated endocytosis andmacropinocytosis(Khaliletal.,2006;Torchilin,2009).Tocon- siderthis possibility,theuptakeoffluorescentsiRNAsintocells wasinvestigatedwhencombinedwithN-TERpeptide,L1-LNCsor CP-LNCs.AsshowninFig.4D,N-TERpeptidewasmoreefficient thanL1-LNCsinpromotingsiRNAsinternalizationbythecells.Con- firmingthehypothesisthatsiRNAscannotbeinternalizedalone, siRNAscombinedwithCP-LNCswerenotfoundinthecellsafter twohoursofincubationat37^◦C(Fig.4D).Inparallel,theuptakeof Nilered-labelledLNCscomplexedwithsiRNAswasfollowedinthe samecells.Interestingly,althoughbothL1-andCP-LNCscombined withsiRNAswereinternalized,a more pronounceduptake was observedwithL1-LNCsthatwerecomplexedwithsiRNAs(Fig.4E).

Thispointdemonstrated,therefore,thatsiRNA-L1-LNCcomplexes areeffectivelycapturedbycells,probablyduetotheirsizeandtheir positive surfacechargeallowinginteractionwiththenegatively

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Fig.4.GelretardationassayofsiRNAsandfunctionalstudy.A)GelretardationassayofsiRNAscomplexedwithcontrolLNCs,L1-LNCsandCP-LNCs.siRNAisretainedwhen itiscomplexedwithL1-LNCs.siRNAconcentrationwasthesameforeachwell:1␮M.TheamountofLNCsineachwellwasincreasedfrom60to180␮g.B)RTqPCRanalysis ofCD133expressioninCaco-2cellsaftertransfectionwithcontrolsiRNAandCD133siRNAindifferentformulations(N-TER,L1-LNCandCP-LNC).Dataarenormalizedwith respectto␤-actinandwereobtainedfromthreeindependentexperiments(n=3).Dunnett’stest:**p<0.01,comparisonwithuntreatedcontrolcells.C)Flowcytometry analysisofBcl-2expressioninU87MGcellsaftertransfectionwithcontrolsiRNAandBcl-2siRNAindifferentformulations(L1-LNC,CP-LNCandN-TER).Datawereobtained fromthreeindependentexperiments(n=3).Dunnett’stest:**p<0.01,comparisonwithcellsweretreatedwithBcl-2.D)Flowcytometryanalysisoftherelativeﬂuorescent siRNAuptakefromdifferentformulations(N-TER,L1-LNCandCP-LNC).Datawereobtainedfromthreeindependentexperiments(n=3).Dunnett’stest:**p<0.01and*p<0.05, comparisonwithuntreatedcontrolcells;^#p<0.05,comparisonbetweensiRNA-N-TERpeptideandsiRNA-L1-LNC.E)FlowcytometryanalysisoftherelativeNilereduptake withinL1-LNCandinCP-LNC.Datawereobtainedfromthreeindependentexperiments(n=3).Dunnett’stest:**p<0.01,comparisonwithuntreatedcontrolcells.

chargedlipidsin theouterplasma membrane leaﬂet(Table3).

Althoughboth N-TERand L1-LNCspromoted siRNAsuptake by cells,therouteofendocytosis,thecontributionofeachpathway andthecapabilitytoescapefromtheendolysosomalcompartment maybedifferent(Paillardetal.,2010),thusexplainingthedifferent efﬁciencyofsilencing.TheintracellulardistributionofthesiRNAs,

whichmaydependonthecarriersystem,alsorepresentsanimpor- tantparametertogetherwithpotentialvariationsinthelocaliza- tionoftheselectedmRNAtargetswithinthecell.Finally,therelease kineticsandthestabilityofthesiRNAswhencomplexedtotheir carrierisalsoapivotalissueandmaydifferbetweenN-TERand L1-LNCs.