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Development and evaluation of injectable nanosized drug delivery systems for
apigenin
Reatul, Karim; Palazzo, Claudio; Laloy, Julie; Delvigne, Anne-Sophie; Vanslambrouck,
Stéphanie; Jérôme, Christine; Lepeltier, Elise; Orange, Francois; Dogne, Jean-Michel;
Evrard, Brigitte; Passirani, Catherine; Piel, Géraldine
Published in:
International Journal of Pharmaceutics
DOI:
10.1016/j.ijpharm.2017.04.064
Publication date:
2017
Document Version
Publisher's PDF, also known as Version of record
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Citation for pulished version (HARVARD):
Reatul, K, Palazzo, C, Laloy, J, Delvigne, A-S, Vanslambrouck, S, Jérôme, C, Lepeltier, E, Orange, F, Dogne,
J-M, Evrard, B, Passirani, C & Piel, G 2017, 'Development and evaluation of injectable nanosized drug delivery
systems for apigenin', International Journal of Pharmaceutics, vol. 532, no. 2, pp. 757-768.
https://doi.org/10.1016/j.ijpharm.2017.04.064
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Development
and
evaluation
of
injectable
nanosized
drug
delivery
systems
for
apigenin
Reatul
Karim
a,d,*,
Claudio
Palazzo
a,
Julie
Laloy
b,
Anne-Sophie
Delvigne
b,
Stéphanie
Vanslambrouck
c,
Christine
Jerome
c,
Elise
Lepeltier
d,
Francois
Orange
e,
Jean-Michel
Dogne
b,
Brigitte
Evrard
a,
Catherine
Passirani
d,
Géraldine
Piel
aaLaboratoryofPharmaceuticalTechnologyandBiopharmacy,CIRM,UniversityofLiege,Liege,Belgium bNamurNanosafetyCentre,NARILIS,DepartmentofPharmacy,UniversityofNamur,Namur,Belgium c
CenterforEducationandResearchonMacromolecules(CERM),UniversityofLiege,UR-CESAM,Liege,Belgium
d
MINT,UNIVAngers,INSERM1066,CNRS6021,UniversitéBretagneLoire,Angers,France
e
UniversitéCôted’Azur,CentreCommundeMicroscopieAppliquée,Nice,France
ARTICLE INFO
Articlehistory:
Received3February2017
Receivedinrevisedform21April2017 Accepted25April2017
Availableonline27April2017
Keywords: Apigenin Liposome Lipidnanocapsule Polymericnanocapsule Injectablenanocarrier ABSTRACT
Thepurposeofthisstudywastodevelopdifferentinjectablenanosizeddrugdeliverysystems(NDDSs) i.e.liposome,lipidnanocapsule(LNC)andpolymericnanocapsule(PNC)encapsulatingapigenin(AG)and comparetheircharacteristicstoidentifythenanovector(s)thatcandeliverthelargestquantityofAG whilebeingbiocompatible.Twoliposomeswithdifferentsurfacecharacteristics(cationicandanionic),a LNCandaPNCwereprepared.Anovel tocopherolmodifiedpoly(ethyleneglycol)-b-polyphosphate block-copolymerwasusedforthefirsttimeforthePNCpreparation.TheNDDSswerecomparedbytheir physicochemical characteristics, AG release, storage stability, stability in serum, complement consumptionandtoxicityagainstahumanmacrovascularendothelialcellline(EAhy926).Thediameter andsurfacechargeoftheNDDSswerecomparablewithpreviouslyreportedinjectablenanocarriers.The NDDSsshowedgoodencapsulationefficiencyanddrugloading.Moreover,theNDDSswerestableduring storageandinfetalbovineserumforextendedperiods,showedlowcomplementconsumptionandwere non-toxictoEAhy926cellsuptohighconcentrations.Therefore,theycanbeconsideredaspotential injectablenanocarriersofAG.Duetolesspronouncedbursteffectandextendedreleasecharacteristics, thenanocapsulescouldbefavorableapproachesforachievingprolongedpharmacologicalactivityofAG usinginjectableNDDS.
©2017ElsevierB.V.Allrightsreserved.
1.Introduction
Apigenin(AG)isanaturalplantflavonoid(40,5, 7,-trihydroxy-flavone),widelyfoundinmanycommonfruitsandvegetablese.g. oranges,grapefruit, chamomile,tea, parsley, onions and wheat
sprouts(Pateletal.,2007;Zhengetal.,2005).Itshowedanumber ofsignificantbeneficialbioactivities i.e.anti-oxidant(Romanova etal.,2001),anti-inflammatory(Leeetal.,2007)andanti-cancer properties (Shukla and Gupta, 2010). Despite the numerous positiveeffects,AGischaracterizedbyverylowaqueoussolubility
Abbreviations:AG,apigenin;AL-AG, apigeninloadedanionicliposome;AL-blank,emptyanionicliposome;BCS,BiopharmaceuticalClassificationSystem;Chol, cholesterol; CL-AG, apigenin loaded cationic liposome; CL-blank, emptycationic liposome; DC-,dimethylaminoethane-carbamoyl chain; DC-Chol, 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterolhydrochloride;DLS,dynamiclightscattering;DMEM,Dulbecco’smodifiedEagle’smedium;DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine;DSPE-mPEG2000,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000]ammoniumsalt;EE,entrapmentefficiency;FBS,fetalbovineserum;HEPES,4-(2-hydroxyethyl)piperazine-1-ethanesulfonicacid;HPLC,high-performance liquidchromatography;Kolliphor1HS15,macrogol15hydroxystearate;LabrafacLipophileWL1349,caprylic/caprictriglycerides;LDH,lactatedehydrogenase;LipoidSPC-3, hydrogenatedphosphatidylcholinefrom soybean;LNC,lipidnanocapsule;LNC-AG,apigeninloadedlipidnanocapsule;LNC-blank, emptylipidnanocapsule;MPS, mononuclearphagocyticsystem;MWCO,molecularweightcut-off;NaCl,sodiumchloride;NDDS,nanosizeddrugdeliverysystem;NHS,normalhumanserum;NTA, nanoparticletrackinganalysis;PDI,polydispersityindex;PEG,poly(ethyleneglycol);PNC,polymericnanocapsule;PNC-AG,apigeninloadedpolymericnanocapsule; PNC-blank,emptypolymericnanocapsule;PS80,polysorbate80;TEM,transmissionelectronmicroscopy;UPW,ultra-purewater.
* Correspondingauthorat:INSERMU1066,MIcroetNanomédecinesTranslationnelles,IBS-CHUANGERS,4rueLarrey,49933AngersCedex9,France. E-mailaddresses:reatul.karim@ulg.ac.be,reatulkarim@gmail.com(R.Karim).
http://dx.doi.org/10.1016/j.ijpharm.2017.04.064
0378-5173/©2017ElsevierB.V.Allrightsreserved.
ContentslistsavailableatScienceDirect
International
Journal
of
Pharmaceutics
(1.35
m
g/mL) and highpermeability(log Pvalue2.87)(Lietal., 1997)and it is then a Biopharmaceutical Classification System (BCS)IImolecule(Zhangetal.,2012).Takingtheseintoaccount, thedevelopmentofitsinjectableformulations,usefultoovercome theconstraintofloworalbioavailability,ischallengingasAGis insoluble in most biocompatible solvents (Zhao et al., 2013). Therefore,useofAGforinvivostudiesislimited.Oneofthemostinterestingstrategiestoovercomethisissueis toencapsulatetheAGinnanosizeddrugdeliverysystems(NDDSs). NDDSs,alsoknownasnanocarriers,arepromisingandversatile approachfordelivery ofhydrophobic drugs(Karimetal., 2017; Lainé et al., 2014) with several advantageous properties i.e. adaptablecharacteristicswitheasilymodifiablesurface,capacity toentraplargequantitiesofhydrophobicdrugandprotectitfrom degradation,improvebioavailability,releasedruginacontrolled manneroverextendedperiod,prolongplasmacirculationhalf-life andincreasepharmacologicaleffects(Peeretal.,2007;Zhangetal., 2008). Moreover, they can be modified for site-specific drug deliverywhich reduceside-effectsandimprovethetherapeutic window (Karim et al., 2016). Among various nanocarriers, liposome(Eavaroneetal.,2000;FelgnerandRingold,1989),lipid nanocapsule(LNC)(Allardetal.,2008;Lamprechtetal.,2002)and polymer-basednanocapsule(PNC)(De Meloet al.,2012; Mora-Huertaset al.,2010)have beenwidelystudied.Althoughthese nanocarriers can be generally considered as vesicular systems, theircompositionandmorphologyaresignificantlydifferentfrom each other. Liposomeshave structuralsimilarities withcellular organellesandaremadeofphospholipidbilayer(s)surroundingan aqueous core. Due to their particular structure, liposomes are capabletoencapsulatebothlipophilicdrugs(inthelipidic-bilayer (s))andhydrophilicdrugs(inthecore).Incomparison,PNCshavea solid polymer-shell surrounding an oily core, where lipophilic drugsareencapsulated.StructureofLNCsisahybridamongPNCs andliposomes;characterizedbyanoily-liquidcoresurroundedby asolidlipidshell.AllthreeNDDSs,i.e.liposome(Sharmaetal., 1996), LNC (Zanotto-Filho et al.,2013) and PNC(Mora-Huertas etal.,2012)havebeenwidelystudiedtoimprovethedeliveryof poorlywatersolubledrugs.Additionally,thesenanocarrierscanbe designed for parenteral administration, in order to bypass absorptionprocess and maximize the drug bioavailability. AG-loaded injectable NDDSs can be particularly beneficial for treatmentofnumeroustypesofcancers(e.g.coloncancer,brain cancer,breastcancer,livercancer,prostatecancer,cervicalcancer, thyroid cancer, skin cancer, gastric cancer etc.) due to the promisingactivityofAG[reviewedindetailbyPateletal.,Shukla andGupta](Pateletal.,2007;ShuklaandGupta,2010)andthe capabilityofNDDSstoextravasateandpreferentiallyaccumulate intumorsbyenhancedpermeabilityandretentioneffect(Fuetal., 2009;Iyeretal.,2006).However,afteradministrationintoblood circulation, the NDDSs can be destabilized by plasma proteins leading to premature drug release. Moreover, they can form aggregatesoradsorbasignificantamountofplasmaproteinsand forma “protein-corona” (Palchetti et al.,2016).If opsonins are adsorbedonthesurface,theNDDSsaresubsequentlycapturedand rapidlyeliminatedfromthesystemiccirculationbymononuclear phagocyticsystem(MPS) (apartoftheimmune system)which restrictstheirbloodcirculationtime.Formationofaggregatescan alsoresultrapidNDDSuptakebyMPS(Heetal.,2010).Therefore, stability of NDDSs in serum and low complement protein consumption are necessary for developing of safe and long-circulatingnano-therapeuticsforfutureclinicaluse(Lietal.,2015; Mooreetal.,2015).
Different NDDSs are prepared from diverse ingredients and have variations in morphology, surface characteristics, drug loadingcapacity,drugreleaserates,toxicityetc.Thepurposeof this study was to develop and compare the characteristics of
differentinjectableAG-NDDSsinordertoidentifythenanocarrier (s) that can deliver the largest quantity of AG while being biocompatible. Twoliposomes withdifferentsurface character-istics(anionicandcationic),aLNCanda PNCwereprepared.A novel block-copolymer i.e. tocopherol modified poly(ethylene glycol)(PEG)-b-polyphosphate(Fig.S1inSupplementarymaterial) (Vanslambrouck, 2015) was used for the first time for PNC preparation.Differenttechniqueswereusedforpreparationofthe nanocarriers,andtheso-obtainedNDDSswerephysicochemically characterized. Moreover, stability of theNDDSs in fetal bovine serum(FBS)andtheircomplementproteinconsumptioninnormal humanserum(NHS)wereevaluated.Toxicityofthenanocarriers wasassessedagainstahumanmacrovascularendothelialcellline toevaluatetheirbiocompatibility.
2.Materialsandmethods 2.1.Materials
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine(DPPC), 1,2-dio-leoyl-sn-glycero-3-phosphoethanolamine (DOPE), 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (DSPE-mPEG2000)werepurchasedfromAvantiPolar Lipids,Inc.
(USA). Cholesterol (Chol), 4-(2-hydroxyethyl)piperazine-1-etha-nesulfonicacid(HEPES),sodiumchloride(NaCl)andmacrogol15 hydroxystearate(Kolliphor1HS15)werepurchasedfrom Sigma-Aldrich (Germany). Hydrogenated phosphatidylcholine from soybean (Lipoid S PC-3) was provided from Lipoid GmbH (Germany), caprylic/capric triglycerides (Labrafac Lipophile WL1349) was supplied by Gattefosse (France). Polysorbate 80 (PS80)waspurchasedfromMerck(Germany).AGwaspurchased fromIndis NV (Belgium). The tocopherolmodified PEG-b-poly-phosphatecopolymer(PEG120-b-(PBP-co-Ptoco)9)wassynthesized
byorganocatalyzedring-openingpolymerization(Clementetal., 2012)ofabutenylphosphateringfroma monomethoxy(polyeth-ylene glycol) macroinitiator (MeO-PEG-OH, MW 5000g/mol, Aldrich) (Yilmaz et al., 2016) followed by the grafting of a tocopherolderivativeonthepolyphosphatebackbonebythiol-ene reaction(Baetenetal.,2016)(Fig.S1inSupplementarymaterial). Ultra-purewater(UPW)wasobtainedfromaMilliporefiltration system.Alltheotherreagentsandchemicalswereofanalytical grade. Normal human serum (NHS) was provided by the “EtablissementFrançaisduSang” (Angers,France).Sheep eryth-rocytes and hemolysinwere purchased from Eurobio (France). EAhy926cells(humanumbilicalendothelialcellline), Penicillin-Streptomycin,andDulbecco’smodifiedEagle’smedium(DMEM) wereprovidedbyLonza(Belgium).Fetalbovineserum(FBS)was providedbyBiologicalsIndustries(USA).
2.2.PreparationofNDDSs
2.2.1.Preparationofcationicliposomesandanionicliposomes Thecationicandanionicliposomeformulations(CL-AGand AL-AGrespectively,compositionshowninTable1,CLcompositionis modifiedfrom(Bellavanceet al.,2010)) wereprepared bythin lipid-film hydration,extrusion(Weiet al.,2015)and PEG post-insertionmethod.Briefly,AGandtheexcipientsweredissolvedin absoluteethanolandthendriedinarotaryevaporatorat30Cfor 1h to form a dry lipid film. Subsequently, the dried film was hydratedwithHEPESbuffer(pH7.4)andhardlyagitatedfor15min. Afterwards, the lipid dispersion was extruded consecutively through0.4
m
m(3),0.2m
m(3)and0.1m
m(3)polycarbonate membranes (Nucleopore1, Whatman) at 50C (above phase transition temperature of DPPC) to obtain primary liposomes.DSPE-mPEG2000(inHEPESbuffer)wasaddedtothesurfaceofthe
primaryliposomesbypost-insertiontechnique,byincubationat 50Cfor30min.Theliposomeformulationswerethenpurifiedby dialysis(MWCO20kD,Spectra/Por1biotechgradecelluloseester membrane,SpectrumLabs,Netherlands)againstHEPESbuffer(pH 7.4)at4Cfor21hcycles.
Blank liposomes (CL-blank and AL-blank) were prepared followingthesameprocedurebutwithouttheadditionofAG. 2.2.2.Preparationoflipidnanocapsules
Theapigenin-loadedLNCs(LNC-AG)werepreparedusingphase inversiontemperaturetechnique(Laineetal.,2014).Inbrief,AG (0.2%w/w),Kolliphor1HS15(16.5%w/w),Lipoid1SPC-3(1.5%w/ w),LabrafacLipophileWL1349(20.1%w/w),DSPE-mPEG2000(1.9%
w/w),NaCl(1.7%w/w)and UPW(58%w/w)weremixedunder magnetic stirring at 60C. Three heating-cooling cycles were performedbetween90Cand60C.Duringthelastcoolingstep, whenthetemperaturewasinthephaseinversionzone(78–83C),
ice-coldUPWwasadded(finalconcentration69.8%w/w)toinduce irreversibleshockandformtheLNC-AG. Thenanocapsuleswere then diluted with HEPES buffer and passed through 0.2
m
m celluloseacetatefiltertoremoveanyaggregates.Purificationwas donebydialysismethodasdescribedin2.2.1.BlankLNCs(LNC-blank)werepreparedbythesameprocedure asLNC-AG,butwithouttheadditionofAG.
2.2.3.Preparationofpolymer-basednanocapsules
The apigenin-loaded PNCs (PNC-AG) were prepared using nanoprecipitation technique followed by solvent evaporation undervacuum(Mora-Huertasetal.,2012).Inshort,AG(1.2%w/ w),PEG120-b-(PBP-co-Ptoco)9(20.1%w/w),Lipoid1SPC-3(10.8%
w/w),LabrafacLipophileWL1349(67.9%w/w)weredissolvedin ethanol:acetone(1:3v/v).Subsequently,thesolutionwasslowly injected(0.8mmneedle)intoanaqueoussolutionofPS80(0.25% w/v)undermagneticstirringat400rpm.After10minstirring,the organicsolventwas completely removedbyevaporation under reducedpressure at40C. The PNC-AGwas purifiedbydialysis methodasdescribedin2.2.1.
BlankPNCs(PNC-blank)werepreparedinthesameprocedure asPNC-AG,butwithouttheadditionofAG.
2.3.Sizedistribution,zeta-potentialandmorphology
Themeandiameterandpolydispersityindex(PDI)oftheNDDSs weredeterminedbydynamiclightscattering(DLS)techniqueusing Zetasizer Nano ZS (MalvernInstruments Ltd, UK). NDDSs were diluted100-foldsinUPWbeforetheanalysis.Themeasurements wereperformedatbackscatterangleof173.Themeasuredaverage valueswerecalculatedfrom3runs,with10measurementswithin eachrun.
Additionally,thesizedistributionoftheNDDSswasdetermined usingnanoparticletracking analysis(NTA), whichcomplements
the DLS measurements. The NTA was carried out using the NanoSightNS300(MalvernInstrumentsLtd,UK).Briefly,theNDDS samplesweredilutedtooptimumconcentrationswithUPWand were infused in the sample chamber using a syringe pump at 30
m
L/min rate. A 405nm laser was used to illuminate the particles, and their Brownian motion was recorded into three 60s videos (25fps) using the sCMOS type camera of the instrument.Subsequently, theNTAsoftware(NTA3.2DevBuild 3.2.16)analyzedtherecordings,trackedthemotionoftheparticles andcalculatedthediameteroftheparticles.Theexperimentwas performedintriplicate.Zetapotential of thenanocarrierswas measuredusing laser Dopplermicro-electrophoresistechniqueusingZetasizerNanoZS (MalvernInstrumentsLtd,UK).
Morphology and size of the NDDSs were visualized by transmissionelectronmicroscopy(TEM)usingnegativestaining technique.Briefly,adropoftheNDDSdispersionwasplacedfor 30sonaTEMcoppergrid(300mesh)withacarbonsupportfilm. The excess dispersionwas removedwith a filterpaper. Subse-quently,stainingwasdonebyaddingadropof1%(w/v)aqueous solution of uranyl acetate on thegrid for 1.5min, followed by removalofexcesssolution.TheTEMobservationswerecarriedout withaJEOLJEM-1400transmissionelectronmicroscopeequipped withaMoradacameraat100kV.
2.4.ApigenindosageviaHPLCmethod
Toquantifytotal(encapsulatedandunencapsulated)AG concen-tration, CL-AG, AL-AG, and LNC-AG were broken by mixing vigorouslywithanappropriatevolume(7-foldsdilutionforCL-AG and AL-AG,40-foldsdilutionfor LNC-AG)for ofethanolto keep dissolvedAGconcentrationbetween5and50
m
g/mL.PNC-AGwas processedinthesimilar way,except ethanol:acetone(1:3v/v,7-folds dilution)wasusedasthesolvent.ToquantifyunencapsulatedAG concentration,formulationswereplacedoncentrifugal concentra-tor devices with polyethersulfone membrane (MWCO 30kD, Vivaspin500,SartoriusAG)andcentrifugedat14500gfor20min toseparatethefreeAGfromtherestoftheformulation.Thefiltrates containingunencapsulatedAGwerecollectedandethanol(2-folds) wasaddedtosolubilizeany undissolveddrug.AGdosageinthe abovementionedsampleswasperformedbyavalidatedmethodin a HPLC system (LC Agilent 1100 series, Agilent Technologies, Belgium).AnAlltimaTMHPC18analyticalcolumn(2504.6mm,5
m
m,GraceDivisonDiscoverySciences,Belgium)wasusedat30C. UPWandacetonitrile(55:45,v/v)wasusedasmobilephase.Flow rate was 1mL/min, injection volume was 10m
L and AG was quantifiedby anUVdetectoratl
of340nm.Analysisofthedatawas performedbyOpenLabHPLCAgilentsoftware.Retentiontimefor AGwas4.9min.2.5.Entrapmentefficiency(EE)
EEð%Þ¼ðTotalAGconc:inNDDSunencapsulatedAGconc:inNDDSÞ100 TheoriticalAGconc:inNDDS
*DeterminedbyHPLC(2.4)
2.6.Massyieldanddrugloadingcapacity
MassyieldofNDDSswascalculatedbygravimetricanalysisof thedriedNDDSsdispersions.Briefly,200
m
LofNDDSswere freeze-dried(Drywinner8,Heto-HoltenA/S,Denmark)overa24hcycle. Weight of the dried nanocarriers were measured (weights of HEPESbufferandNaClweretakenintoaccount)andmassyield(%)Table1
MolarratioofingredientsofCL-AGandAL-AG.
Ingredient Molarratio
CL-AG AL-AG
DPPC 1 1
DC-Chol 0.77 –
Chol – 0.77
DOPE 0.77 0.77
DSPE-mPEG2000(duringdrylipidfilmformation) 0.01 0.01
DSPE-mPEG2000(postinsertion) 0.04 0.04
wascalculatedusingthefollowingequation: Massyieldð%Þ¼TheoreticalWeightof200weight
m
Lof200NDDSm
L100NDDSDrug loading capacity was calculated using the following equation:
Drugloadingcapacity mgAG mgNDDS
¼AmountWeightofAGof200in200mLmNDDSLNDDSðmgÞðmgÞ
*DeterminedbyHPLC(2.4)
2.7.InvitrodrugreleaseprofileoftheNDDSs
Invitrodrugreleaseprofilesofthenanocarrierswerestudied withthedialysismethod.Inbrief,1mLofAGloadedNDDSswere takeninadialysisbag(MWCO20kD,Spectra/Por1biotechgrade cellulose ester membrane, SpectrumLabs, Netherlands) and dialyzedagainstHEPES buffer(pH 7.4)(200/1 acceptor/donator volumeratiotoobtainsinkcondition)at37C,stirredat75rpm
(SW22, Julabo GmbH, Germany). The concentration of AG was determinedbyHPLCmethoddescribedin2.4.
2.8.Storagestability
TheNDDSswerekeptat4Candsampleswerewithdrawnat day0,1,3,7and14.Stabilityduringstoragewasevaluatedbysize, PDI (using method described in 2.3), AG content (using HPLC methoddescribedin2.4)andAGleakage.TheleakageofAGfrom NDDSswasassessedusingthemethodtoquantifyunencapsulated AGmentionedinSection2.4.
2.9.StabilityoftheNDDSsinserum
StabilityoftheNDDSsinFBSwasevaluatedbyfollowingtheir sizedistributionagainsttime(Lietal.,2015;Palchettietal.,2016). ThenanocarrierformulationsweredilutedusingHEPESbuffer(pH 7.4)tooptimumconcentrations(200
m
glipid/mLforCL-AG,AL-AG andPNC-AG;500m
glipid/mLforLNC-AG)andmixedwithFBSat 1:1 ratio (v/v) at 37C. The mixture, along with nanocarrier dispersionandFBS(controls),wereincubatedat37Cat75rpmin a shaking water bath (SW22, Julabo GmbH, Germany). At predetermined time intervals (1min, 30min, 1h, 2h, 4h, 6h and24h),20m
Lofsampleswerewithdrawn,diluted50-foldswith UPW and size distribution was measured via DLS method describedin2.3.2.10.ComplementconsumptionbytheNDDSs
Complement activation was evaluated by measuring the residualhemolyticcapacityof NHStowardsantibody-sensitized sheeperythrocytesafterexposuretothedifferentNDDSs(CH50 assay)(Cajotetal.,2011).Inbrief,aliquotsofNHSwereincubated with increasing concentrations of the NDDSs at 37C for 1h. Subsequently,thedifferentvolumesoftheNHSwereincubated
withafixedvolumeofhemolysin-sensitizedsheeperythrocytesat 37C for45min.Thevolumeof serumthatcanlyse50% ofthe erythrocyteswascalculated(“CH50units”)foreachsampleand percentageofCH50unitconsumptionrelativetonegativecontrol wasdeterminedasdescribedpreviously(Vonarbourgetal.,2006). ParticlenumberintheNDDSdispersionswasdeterminedbyNTA describedinSection2.3andparticleconcentrationpermLofNHS wascalculatedaccordingtofollowing
equation-ParticlenumberpermLofNHS
¼Particleconc:inNDDSdispersionvol:ofNDDSadded vol:ofNHS
Subsequently,surfaceareaoftheNDDSspermLofNHSwas calculatedaccordingtothefollowing
equation-Surfacearea¼ParticlenumberpermLof NHS
p
ðaverageparticlediameterÞ2
The CH50 unit consumption by the different NDDSs were comparedbyplottingthepercentageofCH50unitconsumptionas afunctionoftheirsurfacearea.
2.11.InvitrocytotoxicityofNDDSsonendothelialcellline
Theendothelialcells(EAhy926)wereseededina96-wellplate at a density of 12.5103 cells/well and incubatedfor 24h. AG
solution(inDMSO)andNDDSs,ata concentrationof0.6
m
Mto 40m
M, were added to the cells in 200m
L of cell media and incubatedfor24h.Cytotoxicityofformulationswasdeterminedby evaluating cell viability using methyl tetrazolium (MTS) assay (CellTiter 961 Aqueous One Solution Cell Proliferation Assay, Promega, WI, USA) and cell necrosis by lactate dehydrogenase (LDH) assay (Cytotoxicity Detection KitPLUS, Roche, Basel,Switzerland),accordingtomanufacturer’sinstructions. 2.12.Statisticalanalysis
Resultsobtainedfromtheexperimentswereanalyzed statisti-callyusingGraphPadPrism1software.Meanandstandarddeviation (SD)weredeterminedandvaluesarerepresentedasMeanSD.One wayanalysisofvariance(ANOVA)wasperformedintherespective fields with Bonferroni post-test to compare among individual groups, andDunnett’spost-testtocompare withcontrol. P-valueless than0.05(p<0.05)wasconsideredtobestatisticallysignificant. 3.Results
3.1.CharacteristicsoftheNDDSs
Particle size and zeta potential of the developed NDDSs (determinedbyDLS)areshowninTable2.Themeanhydrodynamic diameteroftheAGloadedliposomes(CL-AGandAL-AG)andthe PNC-AGwascomparable(p>0.5).Thesizesofthesenanocarriers were around 143nm. The mean diameter of the LNC-AG was significantly (p<0.001) smaller (59nm) compared to theother
Table2
PhysicochemicalcharacteristicsoftheNDDSs.
Characteristics CL-AG AL-AG LNC-AG PNC-AG
Meandiameter(nm)a 1441 1426 592 1457
PDI 0.040.01 0.120.02 0.110.03 0.110.02
Zetapotential(mV) 43.21.2 27.42.3 24.96.0 16.24.4
EE(%) 712 341 825 844
Massyield(%) 803 865 722 814
Drugloadingcapacity(mgAG/mgNDDS) 16.50.2 6.50.3 6.20.5 14.30.6
a
NDDSs.All fourNDDSswere monodispersedwithPDI<0.2.The meandiameteroftheCL-AG,AL-AG,LNC-AGandPNC-AG deter-minedbyNTAanalysiswere133nm,136nm,54nmand141nm respectively.Thiswasinagreementwiththeresultsobtainedfrom DLS.Morphology oftheNDDSs wasvisualizedbyTEMimages (Fig.1). The NDDSs were nearly spherical and the liposomes were unilamellar. Additionally, mean sizes of CL-AG, AL-AG, LNC-AG andPNC-AGdeterminedbyTEMwere111nm,214nm,55nmand 87nmrespectively.Meansize(determinedbyTEM)ofCL-AGand LNC-AGwerecomparablewiththeresultsobtainedbyDLSandNTA, whereasaveragesizeofAL-AGandPNC-AGwerequitedissimilar. Thiscanpossiblyoccurduetothedifferencesofthephysicalstateof thesamples(dry vshydrated state) andsize calculation techniquesof TEMcomparedtoDLS(numbervsintensityweighted).
The zeta potential of the CL-AG was 43.2mV and was significantlydifferent(p<0.001)compared totheotherNDDSs, whichwerenegativelycharged.SurfacechargeofAL-AG,LNC-AG andPNC-AGwere27.4mV,24.9mVand16.2mVrespectively. However, only the zeta potentials of AL-AG and PNC-AG were significantlydifferent(p<0.05)amongthesethreeNDDSs.
EE of the CL-AG was 71% which was significantly higher (p<0.001) compared tothe AL-AG. Moreover,EE of the nano-capsules were significantly higher compared to the CL-AG (p<0.05)andAL-AG(p<0.001)butwerecomparable(p>0.05) witheachother(82%forLNC-AGand84%forPNC-AG).
Mass yields of the NDDSs were between 72 and 85%. The highestyieldmasswasobservedforAL-AG,followedbyLNC-AG, PNC-AG and CL-AG. No significant difference (p>0.05) was observed for yield mass of CL-AG and PNC-AG. Drug loading capacityinCL-AGandPNC-AGweremorethan2-foldhigher(16.5 and 14.3
m
gAG/mgNDDS respectively) compared to AL-AG and LNC-AG(6.5and 6.2m
gAG/mgNDDSrespectively). Drug loading capacityoftheNDDSsweresignificantlydifferent(p<0.01)from eachother,exceptforAL-AGandLNC-AG.3.2.InvitrodrugreleaseprofileoftheNDDSs
Thedrugrelease(%)fromtheNDDSswasplottedagainsttimeto obtaintheirdrugreleaseprofiles(Fig.2).Fasterreleaseprofiles
were observed for the liposomes in comparison to the nano-capsules. Although initial release from CL-AGs was slower compared totheAL-AGs,theliposomes releasedabout85–91% drugafter6h.
Incomparison,thenanocapsulesshowedabiphasicandmore sustained release profile, with a faster release rate up to 8h, followedbyamuchslowerrateupto72h.Moreover,therelease ratesofLNC-AGandPNC-AGwereverycomparableupto24h,with a release of 54% and 58%drug respectively.However, thedrug releaserateofPNC-AGwasrelativelyquickerafter24hcompared toLNC-AG.After72h,theLNC-AGreleased63%drugwhereas PNC-AGreleased85%drug.
3.3.StoragestabilityoftheNDDSs
Stability of the NDDSs during storage was evaluated using severalparametersi.e.sizedistribution(meandiameterandPDI), AG concentration and drug leakage. Mean size and PDI were determinedtoevaluatethephysicalstabilityofthenanocarriers, AGconcentrationwillprovideinformationaboutchemicalstability of the drugwithin the NDDSs,whereas drug leakage willgive evidenceoftherobustnessoftheNDDSsduringstorage.Sizeofall four NDDSs werestable throughout thestudy period (Fig.3a).
Fig.1.RepresentativetransmissionelectronmicroscopyimagesofCL-AG,AL-AG,LNC-AGandPNC-AG(whitebar:200nm).
Fig.2.InvitrodrugreleasefromCL-AG(),AL-AG(&),LNC-AG(&)andPNC-AG (!).
Moreover,PDIofthenanocarrierswerebelow0.2 upto14days showingthattheformulationsremainedmonodispersed.
AGconcentrations(%ofinitial)inthenanocarriersareshowed in Fig. 3b. The AG% in AL-AG, LNC-AG and PNC-AG remained unaffected, signifying the stability of the drug in these nano-carriers. However, AG% in CL-AGs gradually reduced to 90% of initialconcentrationafter 14days, demonstrating possibledrug degradationinthisNDDS.NodrugleakagefromanyoftheNDDSs wasobservedupto14days.
3.4.StabilityoftheNDDSsinserum
Stability of theNDDSs inserum was evaluatedbyfollowing theirsizedistributioninFBS(at37C,75rpm)againsttimeusing DLSin order todetect any alteration in their diameterand to determinepossibleparticledestabilization,aggregationorprotein corona formation. Additionally, the NDDS dispersions and FBS werealsoincubatedundersameconditionsascontrols.
AsDLSshowssizedistributiongraphsinrelativeintensity(%), theheightofanunimodalpeak willbehighercomparedtothe samepeakinamixedmultimodalsample.Moreover,focusinga particularsizerange (e.g.0–50nmor 100–300nm)and getting informationaboutaspecificpeakfromamixtureisnotpossiblein DLS.Therefore,peakheightsofNDDS-FBSmixtureswere normal-izedintheoverlaidsizegraphs(FBS,NDDS,NDDS+FBS)(Fig.4a) foreasierqualitativecomparisonwiththecontrols,whileposition ofthenormalizedpeaksstillprovidedinformationaboutpossible coronaformation.
Throughoutthestudyperiod,sizedistributionsofthecontrol NDDSswereunimodalandtheirdiameterdidnotchange(Fig.4). Therefore,nosigns ofparticleaggregationor degradationwere observed.SizedistributionofthecontrolFBSremainedbimodal (more frequent, peaks around 10–15nm and 30–50nm) or trimodal (less frequent additional small peak around 200nm) up to6hof the study. However, larger aggregates were often observedincontrolFBSafter24hwithpeaksaround300–500nm. Upto6h,sizedistributionsoftheNDDS-FBSmixturesfor CL-AG,AL-AGandPNC-AGweretrimodal,showingthepeaksofthe freeproteins (around 10–15nm and 30–50nm)and theNDDSs (peaksaround120–150nm).Positionof thepeaksofNDDS-FBS mixtureswerecomparablewiththecorrespondingcontrolpeaks. However, size distribution graph for LNC-AG-FBS mixture was bimodalasoneofthepeaks(around30–50nm)ofFBSoverlapped withthepeakofLNC-AG,resultingawidercombinedpeakinstead oftwoseparatepeaks.Moreover,higherconcentrationofLNC-AG wasnecessary,comparedtoCL-AG,AL-AGandPNC-AG,toobserve itspeaksinFBSduetotheoverlappingpeaks.However,thepeakof the NDDS was identifiable due to the increased height of the
secondpeakintheLNC-AG-FBSmixture.Thepositionof LNC-AG-FBSpeakswerecomparablewiththecontrols(LNC-AGandFBS), like theotherNDDSs. Therefore, upto 6h, noneof the NDDSs showed any signs of particle aggregation or adsorbing large amountofserumproteins,demonstratingtheircolloidalstability inserum.
TherespectivepeaksofCL-AG,AL-AGandLNC-AGinFBSshifted towardlargerdiametersafter24h.However,itisdifficulttocome intoconclusionthattheaugmentationofdiameterisduetoprotein adsorptionandcoronaformationaroundtheNDDSsurface,ordue to particle aggregation as the control FBS showed aggregated particle peaks around 300–500nm after 24h. However, in the experimentwithPNC-AG,theNDDSpeakinFBSmixturedidnot shifttowardhighervalueafter24h,andthecontrolFBSalsodid notshowanypeaksoflargeaggregatedparticles.
3.5.ComplementconsumptionbytheNDDSs
The complement consumption by thedifferentNDDSs were measured by CH50 assay. Their percentage of CH50 unit consumption was plotted as a function of the particlesurface areapermLofNHS(Fig.5).Asusuallyobserved,thecomplement consumption for thefournanocarriers was increasingwiththe amountofNDDSaddedinNHS.TheLNC-AGshowedthelowest CH50consumptionandreachedonly2.1%at800cm2/mLofNHS.
The complement consumption of AL-AG and CL-AG increased graduallyandreached17.5%at852cm2/mLofNHSand26.8%at
752cm2/mLofNHSrespectively.AlthoughthePNC-AGconsumed higher CH50 unitsat smallersurface areas thanthe others, its consumption increased slowly when more nanoparticles were addedandreached23.7%at838cm2/mLofNHS.
3.6.InvitrocytotoxicityoftheNDDSsonendothelialcells
Cytotoxicity of AG solution and the NDDSs on EAhy926, a humanendothelialcellline,wasevaluatedinvitrobytwodifferent assaysi.e.MTSandLDHassay(Fig.6).Thedrugsolutiondidnot showany significanttoxicityin bothassays.TheCL-AGand CL-blank showed no signs of toxicity up to 2.5
m
M. However, significant reduction of cell viability was revealed at concen-trations10m
M,correspondingto171m
g/mLofCL-AG(Table3). Correspondingly,significantnecrosiswasobservedinLDHassayat similarconcentrationsofCL-blankandPNC-blank.However,the AL-blank, AL-AG, LNC-blank, LNC-AG and PNC-AG showed no significantreductionincellviabilityoranysubstantialcellnecrosis atthetestconcentrations.Overall,theresultsobservedinMTTand LDHassayswerecomparableandthenanocarrierswerenontoxicFig.3.StabilityprofilesoftheNDDSsat4Cupto14days:a)MeandiameterofCL-AG(),AL-AG(&),LNC-AG(&)andPNC-AG(!)at4Cupto14days;b)Apigenin
uptohighconcentrationsoftheNDDSs(AGconc.and correspond-ingNDDSconc.areshowninTable3).
4.Discussion
Theaimofthepresentstudywastodevelopinjectabledosage formsofAG,toallowtheiruseforinvivostudies.AGshowedmany promising pharmacological activities, but its in vivo use is restricted due tovery low aqueoussolubility. As a result, very slowdissolutionwouldoccurafteroraladministration,which is theratelimitingstepcausingslowabsorptionandlow bioavail-ability(Zhangetal.,2013).Infact,astudyonpharmacokineticsand metabolism ofAGreportedthat thedrugreachedthesystemic circulation24hafteroraladministration(Gradolattoetal.,2005). Parenteral administration of AG solution formulations can overcome theproblem ofbioavailability, but face challenges of shortplasmahalf-life(90–105min)(Wanetal.,2007;Zhangetal., 2013)andnonspecifichightissuedistribution(Wanetal.,2007). Moreover,rapid crystallizationmayoccurwhen these formula-tions are injected into blood which reduces its availability at
Fig.4.a)SizedistributionprofilesofCL-AG,AL-AG,LNC-AGandPNC-AGinFBSat37Cand75rpmafter6h.b)DiameterofCL-AG,AL-AG,LNC-AGandPNC-AGinFBSat37C
and75rpmupto24h.
Fig.5. Complementconsumptionat37CbyCL-AG(),AL-AG(&),LNC-AG(&)and PNC-AG(!).
diseasedtissue(Engelmannetal.,2002).Infact,Engelmannetal. observedenlargedabdominallymphnodesinmicecausedbyAG deposition after treatment with such formulation of the drug (Engelmannetal.,2002).Hence,itisnecessarytodevelopsuitable drugcarriersystemsforAGwithsufficientstabilityduringstorage and in serum. Therefore, three types of NDDSs of AG were developed inthis studyi.e. liposomes,LNCand PNC;and were evaluated as potential injectable formulations of AG. For PNC
Fig.6.CytotoxicityofAG,CL-AG,CL-blank,AL-AG,AL-blank,LNC-AG,LNC-blank,PNC-AGandPNC-blankonEAhy926cells.Thecellsweretreatedfor24h.Attheendofthe incubationperiod,cellviabilitywasdeterminedbytheMTSreductionassayandcellnecrosiswasquantifiedbyLDHassay,asdescribedinSection2.11.(OnewayANOVAwith Dunnett’spost-test.p<0.1isdenotedby(*),p<0.01by(**)andp<0.001by(***)).
Table3
AGconcentrationsandcorrespondingNDDSconcentrations.
AGconc.(mM) CorrespondingNDDSconcentration(mg/ml) CL-AG AL-AG LNC-AG PNC-AG
40.0 683 1678 1744 756
10.0 171 420 436 189
2.5 43 105 109 47
preparation, a novel tocopherol modifiedPEG-b-polyphosphate block-copolymer was used for the first time. The amphiphilic surface active properties of the polymer can aid to improve nanocarrier stability which has been already described in the literature(Lopalcoetal.,2015).Theuseofpolyphosphatebackbone insteadofcommonlyutilizedpolylactideorpolyglycolidechainsis more biocompatible as the degradation products of polyphos-phates do not create extreme acidic environment (Yilmaz and Jerome,2016).Thepresenceoftocopherolonthepolyphosphate chainhelpstoimproveentrapmentofhydrophobicdrugslikeAG (Tripodoetal.,2015).Additionally,itmayimprovethestabilityof AGbyactingasanantioxidantandprotectingitsphenolgroups fromoxidation.
Three key physicochemical properties of the nanocarriers influencetheirinvivobehavior:particlesize,surfacechargeand surfacecoating(Straubingeretal.,1993).Thesepropertiesmustbe optimizedinordertoachievefavorabledrugdelivery.Particlesize is an important parameter which hasprofound impact on the uptakeofNDDSsbyMPS.TherateofMPSuptakeincreasesassize ofNDDSsincreases(Senioretal.,1985).SizeoftheCL-AG,AL-AG and PNC-AG were comparable, but the LNC-AG had smaller diameter.ComparedtoAL-AG,thelipidbilayerofCL-AGhadan additionaldimethylaminoethane-carbamoyl(DC-)chainon cho-lesterolmolecules whichimpartedasignificantpositivesurface charge (as ionically bonded chloride ion dissociates from the hydrochloridesalt of tertiary aminegroup of the DC-chain in aqueous environment) without affecting vesicle size. Although mostcomponentsofthenanocapsulestructuresoftheLNC-AGand PNC-AGweresimilar,theratiooftheexcipientsandmanufacturing techniquesweredifferent,resultinginnanocapsuleswith dissimi-larsizes.However,themajorfactorgoverningthehighersizeofthe PNC-AG is possibly its higher weight fractionof core-oil (69%) comparedtoLNC-AG(50%),whichisinaccordancewithprevious reports (Heurtault et al., 2003; Lertsutthiwong et al., 2008). Though,sizeofalltheNDDSswerewithinacceptablelimitsand comparable with previously studied systemically administered nanocarriers(Fuetal.,2009;Laineetal.,2014;Limetal.,2015; Mosqueiraetal.,2001).
Thesurfacechargeofnanocarriersisanothercriticalparameter thatisimportantfromtwoperspectives-storagestabilityandin vivo distribution. Charged NDDSs are less prone to particle aggregationandmorestableasdispersions,comparedtoneutral nanocolloids. Moreover, their cellular-interaction capacity and possibility of intracellular drug delivery are generally higher, comparedtoneutralNDDSs.Macrophageengulfmentofcharged nanocarriers increases as intensity of surface charge amplifies, whereas non-phagocytic cellular uptake increases the charge movestowardscomparativelymorepositivevalue(Heetal.,2010). ThesurfacechargesoftheAG-loadedNDDSs canbeorderedas follows-CL-AG>PNC-AG>LNC-AGAL-AG,wherezetapotential ofonlyCL-AGwaspositive.
ThepresenceofadditionalDC-chainonthecholesterolofthe lipid bilayer of CL-AG not only altered the zeta potential, but improvedtheEEby2folds,comparedtoAL-AG.Thisispossiblydue tochargedinteraction betweenAGand the DC-chain. AGis a weaklyacidicmoleculehavingtwopKavalues(6.6and9.3)(Favaro etal.,2007),andthereforewillbepartiallydeprotonatedatthepH ofthebuffer(7.4)withanequilibriumbetweenmono-anionicand neutralspecies(Papayetal.,2016;Tungjaietal.,2008).Therefore, theneutralspeciescanbeentrappedinthelipidbilayerandthe mono-anionicspeciescaninteractwiththepositivelycharged DC-chainontheCL-AGsurfaces,resultingsignificantlyimproveddrug entrapment. Yuan et al. observed similar electrostatic and/or hydrogen bond formation among AG and positively charged humanserumalbumin(Yuanetal.,2007).Additionally,Papayetal. reported probable electrostatic repulsion among AG and
sulfobutylether-
b
-cyclodextrin, due to presence of negatively charged sulfo groupin the cyclodextrin which weakened their complexationaspHwasincreased(Papayetal.,2016).Moreover, the ionized DC- chain is present both at the inner and outer surfacesofthelipidbilayerfacingtowardstheaqueouscoreand the surrounding aqueous environment respectively. As AGwas addedduringtheformationofdrylipidfilm,wehypothesizethat AGmightbeelectrostaticallyattachedwithbothinnerandouter surfacesduring formationof theliposome.Thephenomenon is more evident fromthedrugrelease characteristicsand storage stability(chemical)ofCL-AGwhichisexplainedintherespective partsofthediscussion.ThenanocapsuleshadhighEEwhichcanbe possiblyattributedtothepresenceofanoilycore.Drug loading capacity of the CL-AG was 2.5 folds higher compared to AL-AG, due to the presence of the additional positivelychargedDC-chain.Similarly,drugloadingcapacity of PNC-AGwas2.3foldsmorecomparedtoLNC-AG,possiblydueto presenceofhigher%ofcoreoilinitsformulation(Lertsutthiwong etal.,2008).
Amajorparameterevaluatedinthisstudywasthedrugrelease characteristics of the different NDDSs, to determine their feasibilityasextendedreleasecarriersofAG.Inprevious experi-ments, plasma concentrationof AG was highafter intravenous administration, but it rapidly fellwith a half-lifearound 1.75h (Wanetal.,2007), whichcanbedue toeithercrystallizationof apigenininphysiologicalpH(Engelmannetal.,2002),orformation of its metabolites (Gradolatto et al., 2004). To prolong the pharmacologicalactivity,dosageformshavingprolongedplasma circulation time and extended release profile canbe beneficial. Drugreleaseat37CinHEPESbuffer(pH7.4,285–295mOsm/kg) fromtheliposomeswasmorerapidcomparedtothenanocapsules, which can be attributed to the different composition and morphologyofthetwotypesofnanocarriers.Thenanocapsules have an oily coresurrounded by hydrophobic and amphiphilic polymers.Therefore,majorityofhydrophobicdrugslikeAGwillbe encapsulatedinthecoreofsuchnanocarriers.However,liposomes entrapmajorityofAGwithintheirlipidbilayerandthusreleased thedrugmoreeasilycomparedtonanocapsules.Thereleaserate fromCL-AGwascomparativelyslowerthanAL-AG,whichcanbe attributedtotwo possiblereasons.Firstly, thepossible electro-staticattractionofAGwiththepositivelychargedDC-chaincan hinderthemovementofthedrugfromthelipidbilayer.Secondly, thelikelihoodofpresenceofaportionoftheentrappedAGatthe innersurfaceofthelipidbilayerofCL-AG(assomeDC-chainswill bepresentatthatsurface)whichprovidesmoreobstaclesinthe movementpathwayof thedrug.However,thenanocapsulesi.e. LNC-AG andPNC-AG showedmuch sustainedrelease character-isticsthantheliposomes.Thereleaserateofthenanocapsuleswas comparable upto 24h, but the PNC-AG showedslightly faster release rate afterwards, compared to LNC-AG. The higher% of excipientspresentontheLNC-AGshell(50%comparedto30%for PNC-AG)mayhaveproducedathickerwallwhichcontributedto the slower release rates at the later stages of the experiment (Watnasirichaikuletal.,2002).
NodrugleakagewasobservedfromanyoftheNDDSsduring thestoragestabilitystudy(14daysat4C)showingtherobustness ofthenanocarriers.Sizesofallthenanocarrierswerealsostable under the same conditions, and all of them remained mono-dispersed,demonstratingphysicalstabilityoftheNDDSs. Howev-er,theAGconcentrationgraduallydecreasedto90%after14daysat 4CincaseofCL-AGs.Basedonthedifferenceintheformulation, EEanddrugloadingcapacityoftheliposomes,alargeportionof theentrapped AGmaybepresentat thesurfacesoftheCL-AG whichputsthedrugincontactoftheaqueousenvironmentduring storage.Thismayleadtochemicalmodificationordegradation,as pureapigeninisknowntobeanunstablemolecule(Pateletal.,
2007)in aqueousenvironmentswithpHbelow8.25(Xuetal., 2006).Furtherstudywouldbenecessarytoconfirmthe mecha-nismofthepossibleapigenindegradation.However,AG concen-trationdidnotalterthroughouttheexperimentperiodforAL-AG, LNC-AG and PNC-AG. Therefore, these NDDSs can be used to improveaqueoussolubilityandstabilityofAG.NoneoftheNDDSs showedanydrugleakageat4Cupto14days.
Thebehaviorofnanocarriersatthe“bio-nanointerface”must beevaluatedtopredicttheirinvivofate(Neletal.,2009;Palchetti etal.,2016).Formationofproteincoronaaroundnanocarriersis dependent on their composition, diameter, shape and surface propertiesalongwithseveralexperimentalparameters,e.gprotein concentration, temperature, incubation time and incubation condition (static vs dynamic) etc. (Caracciolo, 2015; Palchetti etal.,2016).Fromrelativelysmalltohugeamountsofproteinsmay getadsorbedonthenanovectorsurface,andifplasmaopsoninsare adsorbed, the NDDSs will be removed from the systemic circulationbytheMPS(Palchettietal.,2016).Additionally,NDDSs can be destabilized or form aggregates in presence of serum proteins which will alter theirinvivo fate. Afterinjectioninto systemic circulation, NDDSs will be dispersed, diluted and surroundedbyhighamountsofproteinsveryquickly.Therefore, itisnecessarytokeepnanocarrierconcentrationaslowaspossible, comparedtoserum proteins, while evaluatingtheirstability in serum. The total concentration of serum proteins (average concentration calculated based on manufacture specifications) was188foldshigherthanconcentrationoftheCL-AG,AL-AGand PNC-AG,and75foldshigherthantheLNC-AG,inthe nanocarrier-FBSmixturesusedinthisstudy.NoneoftheNDDSsshowedany signsofparticleaggregationorproteincoronaformationupto6h, whichindicatesthatthePEGchainsontheirsurfacewereadequate toefficientlyrepeltheserumproteins.Althoughsomepeaksof largeraggregateswereobservedinDLSafter24hincaseofCL-AG, AL-AGandLNC-AG,itisnotconclusivethattheyadsorbedproteins ontheirsurfaceasthecontrolFBSalsoshowedpeaksaround200– 500nmatthis time point.The nanocarriersmaygraduallylose theirabilitytorepelproteinsduetodesorptionofPEGchainsfrom theirsurface,which occurs in a time-dependentway(Nag and Awasthi,2013;Nagetal.,2013).Conversely,thelargerpeaksonthe NDDS-FBSmixturealsomayappearduetotheaggregatedparticles fromFBS, which overlapped thepeaks of thenanocarriers and shiftedthepeakstowardahighervalue.ThePNC-AGdidnotshow anysignsofparticleaggregationorproteinadsorptionupto24h, butitspeakshiftedslightlytowardssmallerdiameter (approxi-mately35nm).AsthePNC-AGwaspreparedusing nanoprecipi-tationtechnique,tinyaqueouscavitiesmaygetentrappedwithin itsoilycore(Rabaneletal.,2014).WaterfromNDDScoremaynot escapetotheexteriorduringstorageastheparticlecoreandshell membranearelessfluidatcolderstoragetemperature(therefore nochangein particlesize is observed duringstorage), but can graduallycomeoutwhentheNDDSisatphysiologicaltemperature inpresenceofserumduetoalteredosmoticpressure(Wolfram et al., 2014). Finally, the experiment showed that all of the developed NDDSs (CL-AG, AL-AG, LNC-AG and PNC-AG) were stableafterlargedilutioninserumand didnotformsignificant aggregatesorproteincoronaforextendedperiodwhich demon-stratestheirprolongedstabilityinserum.
Additionally,complementconsumptionoftheNDDSsinhuman serumwasevaluatedbyCH50assay,ashighconsumptioncanlead to a rapid activation of the complement system and can be followedbyclearance frombloodstream. TheCH50 assayis an efficient technique for measuring the activation of the total complement system. It correlates well withother complement activationevaluationmethodsi.e.crossedimmunoelectrophoresis andenzyme-linkedimmunosorbent assay,andrepresentsalsoa goodpreliminaryexperimenttopredictthestealthpropertiesof
nanocarriersintendedforsystemicadministration(Meerasaetal., 2011). However, previous studies reporting complement con-sumption ofnanocarriers (Cajotet al., 2011; Vonarbourg et al., 2006) plotted percentage of CH50 unit consumption against theoreticalsurfaceareaoftheparticleswhichwascalculatedusing anarbitrarydensityvalue.Incontrast,NTAwasusedinthisstudy todeterminethenumberofnanocarrierspermLofNHS.Surface areawascalculatedusingthisnumberandthemeandiameterof the NDDS. The previous method of theoretical surface area calculationproducedvaluesmuch higher(1474–1616cm2/mLof
NHSfortheNDDSsamplesinthisstudy)thantheactualsurface areaobtainedbyNTA.Therefore,carefulconsiderationisnecessary when comparingthe resultswith previousreports. The lowest CH50 unit consumption was observed for LNC-AG, which is in agreementwiththeresultsdescribedbyVonarbourgetal.andcan bepossiblyattributedtoitssmallersize(Vonarbourgetal.,2006) andhigherpercentageofPEGinitscomposition(duetopresence of Kolliphor1 HS15 and DSPE-mPEG2000) (Jeon et al., 1991)
compared totheotherNDDSs.Although,mean diameterof CL-AG,AL-AGandPNC-AGweresimilar,theirCH50unitconsumption wasdifferent.ComplementconsumptionofCL-AGwas compara-ble with AL-AG up to 600cm2/mL of NHS, but augmented
comparatively faster then, possibly due toits positive surface-charge(Capriottietal.,2012).ComplementactivationofPNC-AG was higher at surface area<600cm2 compared to otherthree
NDDSs, but reached only 23.7% at 832cm2/mL of NHS. The
differencecanbeduetoseveralfactorsi.e.composition,PEGchain conformation,PEGdensityorpresenceofsurfactantcoating(PS80) (GaoandHe,2014).Furtherstudywouldbenecessarytovalidate theprecisereason.Overall,theNDDSsdidnotshowanystrong complement consumption and should not be rapidly removed fromsystemiccirculationbyMPS.
Toxicity of the NDDSs on a human endothelial cell line (EAhy926) was evaluated to assess their injectability. The commonlyusedcytotoxicassaysworksbydifferentmechanisms andtheirsensitivitycanbedissimilarwithalterationofcelllines (Fotakis and Timbrell, 2006; Lappalainen et al., 1994; Lobner, 2000).Moreover,presenceofnanoparticles(Holderet al.,2012; Krolletal.,2009)orthedrugmolecule(Wangetal.,2010)may interfere with the assay procedures and provide misleading results.Therefore,twocommoncytotoxicityassaysi.e.MTSand LDH assays were used for evaluation and comparison of the possibletoxiceffectsoftheNDDSsontheendothelialcells.Thetwo methods for cytotoxicity assessment provided similar results. AmongtheNDDSs,onlyCL-AGshowedsignificanttoxicityinadose dependentmannerinbothassaysatconcentrationsabove171
m
g/ mL.Thisisprobablyduetothepresenceofthetertiarynitrogen groupcontainingcationiccholesterolderivative(DC-Chol)in CL-AG,whichcanactasproteinkinaseCinhibitorandresulttoxicity (Lvetal.,2006).Incomparison,theAL-AG,LNC-AGandPNC-AG werenontoxicattheirmaximumtestconcentrations(420,436and 189m
g/mLrespectively).Overall,theNDDSswerenontoxicupto highconcentrationsandcanbeconsideredsuitableasinjectable AG-loadednanovectors.5.Conclusion
Inthisstudy,novelAG-loadedNDDSs,i.e.liposomes,LNCand PNCweredevelopedaspotentialinjectabledosageformsofAG. Thenanovectorswerecharacterizedbytheirsize,surfacecharge, EE,massyieldanddrugloadingcapacity.Moreover,drugrelease property,drugleakagepossibilityandstabilityduringstoragewere evaluated. Furthermore, stability of the NDDSs in serum at physiological temperatureand cytotoxicityon ahuman macro-vascular endothelialcelllinewas evaluated.The size ofall the NDDSswaswithintheacceptablelimitforinjectablenanocarriers.
The surface of the nanocarriers was positively (CL-AG) or negativelycharged(AL-AG, LNC-AGand PNC-AG) whichhinder particle aggregation and provided stability during storage. PresenceofDC-CholshowedsignificantincreaseinAGentrapment at physiological pH, and application of such cationic lipids to improve AG encapsulation can be utilized in future NDDS developmentofthedrug.Although,toxicityduetocationiclipids andchemicalstabilityofthedrughavetobecarefullyconsidered. Presence of oily core in the NDDSs was beneficial for AG encapsulation, and the LNC-AG and PNC-AG showed high EE. Moreover,thisisthefirststudyreportingthesuitabilityanduseof thetocopherolgraftedPEG-b-polyphosphateamphiphilic block-copolymerPEG120-b-(PBP-co-Ptoco)9forstablenanocapsule
prep-aration.
Finally, all thenanovectors werestable in FBS for extended periods,showedweak complement systemactivationand were non-toxicto humanmacrovascularendothelialcells up tohigh concentrations,and therefore weresuitable asinjectable nano-carriersofAG.Duetolesspronouncedbursteffectandextended releasecharacteristics,thenanocapsuleformulationsi.e.LNC-AG andPNC-AGcouldbefavorableapproachforachievingprolonged pharmacologicalactivityortumor-targeteddeliveryofAGusing injectableNDDS.
Funding
Thisworkwas supportedbytheNanoFarConsortiumof the Erasmus Mundus program; and Fonds Léon Fredericq, CHU, University of Liege, Liege, Belgium. CERM is indebted to the Interreg Euregio Meuse-Rhine IV-A consortium BioMIMedics (2011–2014) and IAP VII-05 (FS2) for supporting research on newdegradablepolymers.
AppendixA.Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in the online version, at http://dx.doi.org/10.1016/j.ijpharm.2017. 04.064
References
Allard,E.,Passirani,C.,Garcion,E.,Pigeon,P.,Vessieres,A.,Jaouen,G.,Benoit,J.P., 2008.Lipidnanocapsulesloadedwithanorganometallictamoxifenderivative asanoveldrug-carriersystemforexperimentalmalignantgliomas.J.Control. Release130,146–153.
Baeten,E.,Vanslambrouck,S.,Jérôme,C.,Lecomte,P.,Junkers,T.,2016.Anionicflow polymerizationstowardfunctionalpolyphosphoestersinmicroreactors: polymerizationandUV-modification.Eur.Polym.J.80,208–218.
Bellavance,M.A.,Poirier,M.B.,Fortin,D.,2010.Uptakeandintracellularrelease kineticsofliposomeformulationsingliomacells.Int.J.Pharm.395,251–259.
Cajot,S.,Lautram,N.,Passirani,C.,Jerome,C.,2011.Designofreversiblycore cross-linkedmicellessensitivetoreductiveenvironment.J.Control.Release152,30– 36.
Capriotti,A.L.,Caracciolo,G.,Cavaliere,C.,Foglia,P.,Pozzi,D.,Samperi,R.,Lagana,A., 2012.Doplasmaproteinsdistinguishbetweenliposomesofvaryingcharge density.J.Proteomics75,1924–1932.
Caracciolo,G.,2015.Liposome-proteincoronainaphysiologicalenvironment: challengesandopportunitiesfortargeteddeliveryofnanomedicines. Nanomedicine11,543–557.
Clement,B.,Grignard,B.,Koole,L.,Jérôme,C.,Lecomte,P.,2012.Metal-free strategiesforthesynthesisoffunctionalandwell-definedpolyphosphoesters. Macromolecules45,4476–4486.
DeMelo,N.F.,DeAraujo,D.R.,Grillo,R.,Moraes,C.M.,DeMatos,A.P.,dePaula,E., Rosa,A.H.,Fraceto,L.F.,2012.Benzocaine-loadedpolymericnanocapsules: studyoftheanestheticactivities.J.Pharm.Sci.101,1157–1165.
Eavarone,D.A.,Yu,X.,Bellamkonda,R.V.,2000.TargeteddrugdeliverytoC6glioma bytransferrin-coupledliposomes.J.Biomed.Mater.Res.51,10–14.
Engelmann,C.,Blot,E.,Panis,Y.,Bauer,S.,Trochon,V.,Nagy,H.,Lu,H.,Soria,C.,2002. Apigenin-strongcytostaticandanti-angiogenicactioninvitrocontrastedby lackofefficacyinvivo.Phytomedicine9,489–495.
Favaro,G.,Clementi,C.,Romani,A.,Vickackaite,V.,2007.Acidichromismand ionochromismofluteolinandapigenin,themaincomponentsofthenaturally
occurringyellowweld:aspectrophotometricandfluorimetricstudy.J.Fluoresc. 17,707–714.
Felgner,P.L.,Ringold,G.M.,1989.Cationicliposome-mediatedtransfection.Nature 337,387–388.
Fotakis,G.,Timbrell,J.A.,2006.Invitrocytotoxicityassays:comparisonofLDH, neutralred,MTTandproteinassayinhepatomacelllinesfollowingexposureto cadmiumchloride.Toxicol.Lett.160,171–177.
Fu,J.Y.,Blatchford,D.R.,Tetley,L.,Dufes,C.,2009.Tumorregressionaftersystemic administrationoftocotrienolentrappedintumor-targetedvesicles.J.Control. Release140,95–99.
Gao,H.,He,Q.,2014.Theinteractionofnanoparticleswithplasmaproteinsandthe consequentinfluenceonnanoparticlesbehavior.ExpertOpin.DrugDeliv.11, 409–420.
Gradolatto,A.,Canivenc-Lavier,M.C.,Basly,J.P.,Siess,M.H.,Teyssier,C.,2004. MetabolismofapigeninbyratliverphaseIandphaseiienzymesandbyisolated perfusedratliver.DrugMetab.Dispos.32,58–65.
Gradolatto,A.,Basly,J.P.,Berges,R.,Teyssier,C.,Chagnon,M.C.,Siess,M.H., Canivenc-Lavier,M.C.,2005.Pharmacokineticsandmetabolismofapigenininfemaleand maleratsafterasingleoraladministration.DrugMetab.Dispos.33,49–54.
He,C.,Hu,Y.,Yin,L.,Tang,C.,Yin,C.,2010.Effectsofparticlesizeandsurfacecharge oncellularuptakeandbiodistributionofpolymericnanoparticles.Biomaterials 31,3657–3666.
Heurtault,B.,Saulnier,P.,Pech,B.,Venier-Julienne,M.-C.,Proust,J.-E.,Phan-Tan-Luu, R.,Benoît,J.-P.,2003.Theinfluenceoflipidnanocapsulecompositionontheir sizedistribution.Eur.J.Pharm.Sci.18,55–61.
Holder,A.L.,Goth-Goldstein,R.,Lucas,D.,Koshland,C.P.,2012.Particle-induced artifactsintheMTTandLDHviabilityassays.Chem.Res.Toxicol.25,1885–1892.
Iyer,A.K.,Khaled,G.,Fang,J.,Maeda,H.,2006.Exploitingtheenhancedpermeability andretentioneffectfortumortargeting.DrugDiscov.Today11,812–818.
Jeon,S.I.,Lee,J.H.,Andrade,J.D.,DeGennes,P.G.,1991.Protein–surfaceinteractions inthepresenceofpolyethyleneoxide.J.ColloidInterfaceSci.142,149–158.
Karim,R.,Palazzo,C.,Evrard,B.,Piel,G.,2016.Nanocarriersforthetreatmentof glioblastomamultiforme:currentstate-of-the-art.J.Control.Release227,23–37.
Karim,R.,Somani,S.,AlRobaian,M.,Mullin,M.,Amor,R.,McConnell,G.,Dufes,C., 2017.Tumorregressionafterintravenousadministrationoftargetedvesicles entrappingthevitaminEa-tocotrienol.J.Control.Release246,79–87.
Kroll,A.,Pillukat,M.H.,Hahn,D.,Schnekenburger,J.,2009.Currentinvitromethods innanoparticleriskassessment:limitationsandchallenges.Eur.J.Pharm. Biopharm.72,370–377.
Lainé,A.-L.,Clavreul,A.,Rousseau,A.,Tétaud,C.,Vessieres,A.,Garcion,E.,Jaouen,G., Aubert,L.,Guilbert,M.,Benoit,J.-P.,2014.Inhibitionofectopicgliomatumor growthbyapotentferrocenyldrugloadedintostealthlipidnanocapsules. Nanomedicine10,1667–1677.
Laine,A.L.,Gravier,J.,Henry,M.,Sancey,L.,Bejaud,J.,Pancani,E.,Wiber,M.,Texier,I., Coll,J.L.,Benoit,J.P.,Passirani,C.,2014.Conventionalversusstealthlipid nanoparticles:formulationandinvivofatepredictionthroughFRET monitoring.J.Control.Release188,1–8.
Lamprecht,A.,Bouligand,Y.,Benoit,J.P.,2002.Newlipidnanocapsulesexhibit sustainedreleasepropertiesforamiodarone.J.Control.Release84,59–68.
Lappalainen,K.,Jaaskelainen,I.,Syrjanen,K.,Urtti,A.,Syrjanen,S., 1994.Comparison ofcellproliferationandtoxicityassaysusingtwocationicliposomes.Pharm. Res.11,1127–1131.
Lee,J.H.,Zhou,H.Y.,Cho,S.Y.,Kim,Y.S.,Lee,Y.S.,Jeong,C.S.,2007.Anti-inflammatory mechanismsofapigenin:inhibitionofcyclooxygenase-2expression,adhesion ofmonocytestohumanumbilicalveinendothelialcells,andexpressionof cellularadhesionmolecules.Arch.Pharm.Res.30,1318–1327.
Lertsutthiwong,P.,Noomun,K.,Jongaroonngamsang,N.,Rojsitthisak,P., Nimmannit,U.,2008.Preparationofalginatenanocapsulescontainingturmeric oil.Carbohydr.Polym.74,209–214.
Li,B.,Robinson,D.H.,Birt,D.F.,1997.Evaluationofpropertiesofapigeninand[G-3H] apigeninandanalyticmethoddevelopment.J.Pharm.Sci.86,721–725.
Li,F.,Zhao,X.,Wang,H.,Zhao,R.,Ji,T.,Ren,H.,Anderson,G.J.,Nie,G.,Hao,J.,2015. Multiplelayer-by-layerlipid-polymerhybridnanoparticlesforimproved FOLFIRINOXchemotherapyinpancreatictumormodels.Adv.Funct.Mater.25, 788–798.
Lim,L.Y.,Koh,P.Y.,Somani,S.,AlRobaian,M.,Karim,R.,Yean,Y.L.,Mitchell,J.,Tate,R. J.,Edrada-Ebel,R.,Blatchford,D.R.,Mullin,M.,Dufes,C.,2015.Tumorregression followingintravenousadministrationoflactoferrin-andlactoferricin-bearing dendriplexes.Nanomedicine11,1445–1454.
Lobner,D.,2000.ComparisonoftheLDHandMTTassaysforquantifyingcelldeath: validityforneuronalapoptosis?J.Neurosci.Methods96,147–152.
Lopalco,A.,Ali,H.,Denora,N.,Rytting,E.,2015.Oxcarbazepine-loadedpolymeric nanoparticles:developmentandpermeabilitystudiesacrossinvitromodelsof theblood-brainbarrierandhumanplacentaltrophoblast.Int.J.Nanomed.10, 1985–1996.
Lv,H.,Zhang,S.,Wang,B.,Cui,S.,Yan,J.,2006.Toxicityofcationiclipidsandcationic polymersingenedelivery.J.Control.Release114,100–109.
Meerasa,A.G.,Huang,J.X.,Gu,F.,2011.CH50:Arevisitedhemolyticcomplement consumptionassayforevaluationofnanoparticlesandbloodplasmaprotein interaction.Curr.DrugDeliv.8,290–298.
Moore,T.L.,Rodriguez-Lorenzo,L.,Hirsch,V.,Balog,S.,Urban,D.,Jud,C., Rothen-Rutishauser,B.,Lattuada,M.,Petri-Fink,A.,2015.Nanoparticlecolloidalstability incellculturemediaandimpactoncellularinteractions.Chem.Soc.Rev.44, 6287–6305.
Mora-Huertas,C.E.,Fessi,H.,Elaissari,A.,2010.Polymer-basednanocapsulesfor drugdelivery.Int.J.Pharm.385,113–142.
Mora-Huertas,C.E.,Garrigues,O.,Fessi,H.,Elaissari,A.,2012.Nanocapsules preparedviananoprecipitationandemulsification-diffusionmethods: comparativestudy.Eur.J.Pharm.Biopharm.80,235–239.
Mosqueira,V.C.,Legrand,P.,Morgat,J.L.,Vert,M.,Mysiakine,E.,Gref,R., Devissaguet,J.P.,Barratt,G.,2001.Biodistributionoflong-circulating PEG-graftednanocapsulesinmice:effectsofPEGchainlengthanddensity.Pharm. Res.18,1411–1419.
Nag,O.K.,Awasthi,V.,2013.Surfaceengineeringofliposomesforstealthbehavior. Pharmaceutics5,542–569.
Nag,O.K.,Yadav,V.R.,Hedrick,A.,Awasthi,V.,2013.Post-modificationofpreformed liposomeswithnovelnon-phospholipidpoly(ethyleneglycol)-conjugated hexadecylcarbamoylmethylhexadecanoicacidforenhancedcirculation persistenceinvivo.Int.J.Pharm.446,119–129.
Nel,A.E.,Madler,L.,Velegol,D.,Xia,T.,Hoek,E.M.,Somasundaran,P.,Klaessig,F., Castranova,V.,Thompson,M.,2009.Understandingbiophysicochemical interactionsatthenano-biointerface.Nat.Mater.8,543–557.
Palchetti,S.,Colapicchioni,V.,Digiacomo,L.,Caracciolo,G.,Pozzi,D.,Capriotti,A.L., LaBarbera,G.,Lagana,A.,2016.TheproteincoronaofcirculatingPEGylated liposomes.Biochim.Biophys.Acta1858,189–196.
Papay,Z.E.,Sebestyen,Z.,Ludanyi,K.,Kallai,N.,Balogh,E.,Kosa,A.,Somavarapu,S., Boddi,B.,Antal,I.,2016.Comparativeevaluationoftheeffectofcyclodextrins andpHonaqueoussolubilityofapigenin.J.Pharm.Biomed.Anal.117,210–216.
Patel,D.,Shukla,S.,Gupta,S.,2007.Apigeninandcancerchemoprevention: progress,potentialandpromise(review).Int.J.Oncol.30,233–245.
Peer,D.,Karp,J.M.,Hong,S.,Farokhzad,O.C.,Margalit,R.,Langer,R.,2007. Nanocarriersasanemergingplatformforcancertherapy.Nat.Nanotechnol.2, 751–760.
Rabanel,J.M.,Hildgen,P.,Banquy,X.,2014.AssessmentofPEGonpolymeric particlessurface,akeystepindrugcarriertranslation.J.Control.Release185, 71–87.
Romanova,D.,Vachalkova,A.,Cipak,L.,Ovesna,Z.,Rauko,P.,2001.Studyof antioxidanteffectofapigenin,luteolinandquercetinbyDNAprotective method.Neoplasma48,104–107.
Senior,J.,Crawley,J.C.,Gregoriadis,G.,1985.Tissuedistributionofliposomes exhibitinglonghalf-livesinthecirculationafterintravenousinjection.Biochim. Biophys.Acta839,1–8.
Sharma,A.,Sharma,U.S.,Straubinger,R.M.,1996.Paclitaxel-liposomesfor intracavitarytherapyofintraperitonealP388leukemia.CancerLett.107,265– 272.
Shukla,S.,Gupta,S.,2010.Apigenin:apromisingmoleculeforcancerprevention. Pharm.Res.27,962–978.
Straubinger,R.M.,Sharma,A.,Murray,M.,Mayhew,E.,1993.Noveltaxol formulations:taxol-containingliposomes.J.Natl.CancerInst.69–78.
Tripodo,G.,Pasut,G.,Trapani,A.,Mero,A.,Lasorsa,F.M.,Chlapanidas,T.,Trapani,G., Mandracchia,D.,2015.Inulin-D-alpha-tocopherolsuccinate(INVITE) nanomicellesasaplatformforeffectiveintravenousadministrationof curcumin.Biomacromolecules16,550–557.
Tungjai,M.,Poompimon,W.,Loetchutinat,C.,Kothan,S.,Dechsupa,N., Mankhetkorn,S.,2008.Spectrophotometriccharacterizationofbehaviorand thepredominantspeciesofflavonoidsinphysiologicalbuffer:determinationof solubility,lipophilicityandanticancerefficacy.OpenDrugDeliv.J.2,10–19.
Vanslambrouck,S.,2015.Polyphosphate-basedAmphiphilicCopolymers:Synthesis andApplicationtoDrugNanocarriers.UniversityofLiège,Liège,Belgium.
Vonarbourg,A.,Passirani,C.,Saulnier,P.,Simard,P.,Leroux,J.C.,Benoit,J.P.,2006. Evaluationofpegylatedlipidnanocapsulesversuscomplementsystem activationandmacrophageuptake.J.Biomed.Mater.Res.78,620–628.
Wan,L.,Guo,C.,Yu,Q.,Li,Y.,Wang,X.,Wang,X.,Chen,C.,2007.Quantitative determinationofapigeninanditsmetabolisminratplasmaafterintravenous bolusadministrationbyHPLCcoupledwithtandemmassspectrometry.J. Chromatogr.B855,286–289.
Wang,P.,Henning,S.M.,Heber,D.,2010.LimitationsofMTTandMTS-basedassays formeasurementofantiproliferativeactivityofgreenteapolyphenols.PLoS One5,e10202.
Watnasirichaikul,S.,Rades,T.,Tucker,I.G.,Davies,N.M.,2002.Effectsofformulation variablesoncharacteristicsofpoly(ethylcyanoacrylate)nanocapsulesprepared fromw/omicroemulsions.Int.J.Pharm.235,237–246.
Wei,X.,Gao,J.,Zhan,C.,Xie,C.,Chai,Z.,Ran,D.,Ying,M.,Zheng,P.,Lu,W.,2015. Liposome-basedgliomatargeteddrugdeliveryenabledbystablepeptide ligands.J.Control.Release218,13–21.
Wolfram,J.,Suri,K.,Yang,Y.,Shen,J.,Celia,C.,Fresta,M.,Zhao,Y.,Shen,H.,Ferrari, M.,2014.Shrinkageofpegylatedandnon-pegylatedliposomesinserum. ColloidsSurf.BBiointerfaces114,294–300.
Xu,X.,Yu,L.,Chen,G.,2006.DeterminationofflavonoidsinPortulacaoleraceaLby capillaryelectrophoresiswithelectrochemicaldetection.J.Pharm.Biomed. Anal.41,493–499.
Yilmaz,Z.E.,Jerome,C.,2016.Polyphosphoesters:newtrendsinsynthesisanddrug deliveryapplications.Macromol.Biosci.16,1745–1761.
Yilmaz,Z.E.,Vanslambrouck,S.,Cajot,S.,Thiry,J.,Debuigne,A.,Lecomte,P.,Jérôme, C.,Riva,R.,2016.Corecross-linkedmicellesofpolyphosphoestercontaining amphiphilicblockcopolymersasdrugnanocarriers.RSCAdv.6,42081–42088.
Yuan,J.-L.,Liu,Z.-G.,Hu,Z.,Zou,G.-L.,2007.Studyoninteractionbetweenapigenin andhumanserumalbuminbyspectroscopyandmolecularmodeling.J. Photochem.Photobiol.A:Chem.191,104–113.
Zanotto-Filho,A.,Coradini,K.,Braganhol,E.,Schroder,R.,deOliveira,C.M., Simoes-Pires,A.,Battastini,A.M.,Pohlmann,A.R.,Guterres,S.S.,Forcelini,C.M.,Beck,R. C.,Moreira,J.C.,2013.Curcumin-loadedlipid-corenanocapsulesasastrategyto improvepharmacologicalefficacyofcurcuminingliomatreatment.Eur.J. Pharm.Biopharm.83,156–167.
Zhang,L.,Gu,F.X.,Chan,J.M.,Wang,A.Z.,Langer,R.S.,Farokhzad,O.C.,2008. Nanoparticlesinmedicine:therapeuticapplicationsanddevelopments.Clin. Pharmacol.Ther.83,761–769.
Zhang,J.,Liu,D.,Huang,Y.,Gao,Y.,Qian,S.,2012.Biopharmaceuticsclassification andintestinalabsorptionstudyofapigenin.Int.J.Pharm.436,311–317.
Zhang,J.,Huang,Y.,Liu,D.,Gao,Y.,Qian,S.,2013.Preparationofapigenin nanocrystalsusingsupercriticalantisolventprocessfordissolutionand bioavailabilityenhancement.Eur.J.Pharm.Sci.48,740–747.
Zhao,L.,Zhang,L.,Meng,L.,Wang,J.,Zhai,G.,2013.Designandevaluationofa self-microemulsifyingdrugdeliverysystemforapigenin.DrugDev.Ind.Pharm.39, 662–669.
Zheng,P.-W.,Chiang,L.-C.,Lin,C.-C.,2005.Apigenininducedapoptosisthrough p53-dependentpathwayinhumancervicalcarcinomacells.LifeSci.76,1367– 1379.