Understanding the adsorption of salmon calcitonin, antimicrobial peptide AP114 and polymyxin B onto lipid nanocapsules

Understanding the adsorption of salmon calcitonin, antimicrobial peptide AP114 and polymyxin B onto lipid nanocapsules

Anita Umerska*

^,1

, Nada Matougui, Anne-Claire Groo, Patrick Saulnier

INSERMU1066,MicroetNanomédecinesBiomimétiques,IBS-CHU,4RueLarrey,F-49933,Angers,France

ARTICLE INFO

Articlehistory:

Received4January2016

Receivedinrevisedform8April2016 Accepted13April2016

Availableonline22April2016

Keywords:

Lipidnanocapsules Nanoparticles Adsorption Peptide Calcitonin PolymyxinB Plectasin

Antimicrobialpeptide NZ2114

AP114

ABSTRACT

Theadsorptionoftherapeuticmolecules,e.g.,peptides,ontonanocarriersisinfluencedbytheproperties ofthecarrier,adsorbedmoleculeandcontinuousphase.Hence,throughchangesinthecompositionof thenanocarrierandthemedium,itshouldbepossibletotunethesystemtomakeitcapableofefficiently adsorbingpeptides.

Theadsorptionofcalcitonin,antimicrobialpeptideAP114andpolymyxinBontolipidnanocapsules wasinvestigated.TheadsorptiondatawerefittedtoaLangmuirisotherm.Dynamiclightscatteringand laserDopplervelocimetrywereusedtoinvestigatethechangesinthehydrodynamicdiameterandzeta potential,respectively,ofthenanocarrier.

Thepeptideadsorptionwasprimarilygovernedbyelectrostaticforces;however,evenwithoutthe presenceofanionisablesurfactant,asignificantamountofeachtestedmoleculewasadsorbedduetothe enormoussurfaceareaofthenanocarriersandtopeptide-nanocarrierinteractions.Theadditionofan ionisablelipophilicsurfactant,lecithin,improvedtheadsorptionyield,whichreachedvaluesofupto 100%. The adsorptionyield and the properties of the nanocarrier, particularly thezeta potential, dependedonthecarrierandpeptideconcentrationsandtheirmixingratio.Theadsorptionofalltested moleculesobeyedtheLangmuirmodeloveralimitedconcentrationrange.

ã2016ElsevierB.V.Allrightsreserved.

1.Introduction

Protein adsorption hasbeen extensively studied in thepast severalyears. Asaresultof theiramphipathic nature,proteins/

peptidescanadsorbandthusaccumulateatsolid-liquidinterfaces inawiderangeofbiologicalandnon-biologicalprocesses(Almeida and Souto, 2007). In addition to encapsulation and covalent attachment,surface adsorptionisonestrategyforincorporating therapeutic and diagnostic agents into drug delivery systems (Hartigetal.,2007).Thesmallerthecarrieris,themoredifficultit becomestoachievehighdrugencapsulationefficiencyandagood sustainedreleaseeffect.Tocircumventthisproblem,thedrugcan beadsorbedonto theparticlesurface ratherthan encapsulated within(AlmeidaandSouto,2007).Duetotheirsmallsizeandlarge surfacearea-to-massratio,nanoparticlesmayinteractwithmany biomolecules, includingpeptides and proteins (Saptarshi et al.,

2013).Interactionsofpeptideswithnanoparticlesoccurthrough severalforces,e.g.,hydrogenbondsandvanderWaalsinteractions, but electrostatic interactions seem to be the most important (Umerskaetal.,2014a,b,2015a).Theoverallnanoparticle-peptide complexformationisa multifactorialprocessthatisdependent notonlyonthecharacteristicsofthenanocarriersbutalsoonthe peptide/proteinproperties and concentrationsand the medium (AlmeidaandSouto,2007;Saptarshietal.,2013).Therehavebeen numerousstudiesontheadsorptionofnotonlyproteinsbutalso peptidesontopolymericorsolid lipidnanocarriers;however,to date, there is no study on the adsorption of proteins on lipid nanocapsules(LNCs).LNCshavemainlybeenusedascarriersfor lipophilic drugs (Huynh et al., 2009; Umerska et al., 2015b;

Valcourt et al., 2016). Lipophilic drugs could be incorporated within thelipid coreof the LNCs. However,due to theirsmall particlesizeandconsequentlyhighsurfacearea-to-volumeratio, LNCs couldpotentially offer a largearea for the adsorption of hydrophilicmoleculesthataredissolvedintheexternalaqueous medium. LNCs are obtained viaa phase inversion temperature method,whichemploysheatingandcoolingcycles.Theadsorption ontoLNCscouldbeparticularlyattractiveformoleculesthatare prone to thermal degradation, e.g., peptides. These molecules

* Correspondingauthor.

E-mailaddresses:[email protected],[email protected](A.Umerska).

1 Presentaddress:INSERMU1066,IBS-CHU,4rueLarrey,49933AngersCedex9, France.

http://dx.doi.org/10.1016/j.ijpharm.2016.04.028 0378-5173/ã2016ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

International Journal of Pharmaceutics

j o u r n a lh o m e p a g e : w w w . e l s e vi e r . c o m / l o c a t e/ i j p h a r m

couldbesafelyincorporatedintothesystemafterthesuccessful generationofLNCswithoutexposuretoelevatedtemperatures.

Mostofthestudiesconductedthusfarhavefocusedonlarge proteins, e.g., albumins. However, proteins and peptides vary greatly and have different physicochemical properties, e.g., molecularweight,isoelectric point, charge, charge density,and hydrophilicity, and all of these parameters may influence the adsorption.Onlyafewstudiesinvestigatedtheadsorptionofsmall peptides,e.g.,calcitonin(Calisetal.,1995;DaniandDeLuca,2001;

Garcia-Fuentesetal.,2005).Additionally,inmanystudies,amodel compound was selected, and the investigation focused on the influenceofthecarriersratherthantheadsorbedmolecule.There is a need for studies comparing the adsorption of different moleculesontothesamecarrierbecausedifferentmoleculesmight be adsorbed in differently by the same carrier, despite the similarity in their physicochemical properties. This study will focus on the adsorption of three positively charged peptide molecules.

Salmoncalcitonin(sCT)isa32aminoacidpolypeptidewitha molecularweightof3.4kDathatisfreelysolubleinwaterwithan isoelectricpointof8.86(Torres-LugoandPeppas,1999)andanet chargeofapproximately+2atpH7.4.Inadditionto

a

-aminoand

a

-carboxylic groups, sCT contains other amino acids with an ionisableside-chain:onecarboxylicgroupofglutamicacid(pKa 4.07),two

e

^{-aminogroupsoflysine(pKa10.53),one}guanidinium groupofarginine(pKa12.48)andoneimidazoleringofhistidine (pKa6.10).

AP114,alsoknownasNZ2114,is a40amino acid(molecular weight of 4.4kDa) plectasin variant found in the saprophytic fungus,Pseudoplectanianigrella(Andesetal.,2009).Thenetcharge atpH7is+3,andtheisoelectricpointis8.62.Inadditiontothe

a

-amino and

a

-carboxylic groups, AP114 also contains other aminoacidswithanionisablesidechain:onecarboxylicgroupof glutamicacid(pKa4.07),two carboxylicgroupsofaspartic acid (pKa3.85),oneguanidinium groupof arginine(pKa12.48),five

e

^{-aminogroupsoflysine(pKa10.53)andtwoimidazoleringsof}

histidine(pKa6.10).

Although the therapeutic applicationsof AP114 and sCTare different,thestructuresofthetwomoleculesshowsimilarities:

similarisoelectricpointsandchargedensities.

PolymyxinB(PMB)isanantibioticcomposedofseveralclosely relatedpolypeptidesderivedfromBacilluspolymyxa.Themolecu- lar weight is 1301.56g/mol. The general structure comprises a cyclicheptapeptidemoietywithastraighttripeptidesidechain.

TheN-terminalaminogroupinthesidechainisacetylated(Orwa etal.,2001).Polymyxinshavefivenonproteogenicdiaminobutyric acidresidues,whichmakesthempolycationicatpH7.4.Similarto many other antimicrobial peptides, PMB has a mixture of hydrophilicandlipophilicgroupsthatmakePMBamphipathic,a physicochemical property that is crucial for the antibacterial activity(Velkovetal.,2013).

Peptides make a rapidly growing part of pharmaceutical market,but thedevelopment ofcommercialproducts islimited duetotheirphysicochemicalandbiologicalinstability.Lipid-based carrierscan offernumerousadvantages. Forinstance theyhave

beenshowntoprotectlabilemoleculesfromenzymaticdegrada- tion(Matouguietal.,2016).StudiesoftheadsorptionofAP114on LNCs are particularly interesting because the combination of AP114withmonolaurinlipidnanocapsuleshasasynergisticeffect againstmethicillin-resistantStaphylococcusaureus(Umerskaetal., 2015c).

Theaimofthisstudywastoexaminethefactorsaffectingthe adsorptionofcationicpeptidesontoLNCs.Differentfactorswere considered, e.g., composition of thenanocarrier, type of active pharmaceuticalingredient(API),concentrationsoftheAPIandthe nanocarrierandtheirmassmixingratio.Wheninvestigatingthe adsorption of anAPI onto the nanoparticles,it is important to characterisetheinfluenceoftheAPIadsorptiononthephysical characteristicsofthenanoparticlesandnanoparticle-APIinterac- tion. Therefore, dynamic light scattering and laser Doppler velocimetry were used to investigate the changes in the hydrodynamic diameterand zetapotential, respectively, of the NPsuponbindingtopeptides.Anotheraimofthispaperwasto determinewhethertheadsorptiondata followoneof themost commonly used isotherms, i.e., the Langmuir or Freundlich isotherms.

2.Materialsandmethods

2.1.Materials

Salmoncalcitoninand theAP114antimicrobialpeptidewere kindly provided by PolyPeptide Group (Sweden) and Adenium Biotech (Denmark), respectively. Polymyxin B sulphate was purchased fromSigmaAldrich(France).Labrafac¹CC (caprylic/

capric acid triglycerides;C8/C10-TG) and oleic Plurol¹ CC 497 (polyglyceryl-3 dioleate; HLB=3) were kindly provided by GattefosséS.A.(Saint-Priest,France).Lipoid¹S75-3(hydrogenated lecithinwithanaveragemolecularweightof780g/mol)waskindly provided by Lipoïd Gmbh (Ludwigshafen, Germany). Solutol¹ HS15(macrogol15hydroxystearate,polyoxyl15hydroxystearate;

HLB 14–16) was kindly provided by BASF (Ludwigshafen, Germany). All other chemicals and solvents were of analytical grade. Amicon Ultra-4 centrifugal filter devices were obtained fromMillipore(USA).

2.2.PreparationofLNCs

LNCswerepreparedataconcentrationof200mg/mlfollowing theproceduredescribedbyHeurtaultetal.(2002);thecomposi- tionofeachformulationispresentedinTable1.Thecomponentsof theLNCs(polyoxyl15hydroxystearate,hydrogenatedlecithinand C8/C10-TG) and optionallyNaCl (0.089g) wereweighed, mixed with2.1mlof water and heatedto95–100C. The samplewas cooled to60C. The sampleswere treated withthree heating- coolingcycles;duringthelastcoolingcycle,atthephaseinversion temperature(seeTable1),thesystemwasdilutedwithcold(4C) watertoafinalvolumeof10ml.TheLNCdispersionswerediluted with an aqueous solution of the API or water (control) and incubatedat4–8Cfor2h(Thistimewassufficienttoachievea

Table1

CompositionoftheLNCformulations(polyoxyl15hydroxystearate/oilmassmixingratio=0.81,LNC/APImassmixingratio=200).

Formulation F1 F2 F3 F4 F5

C8/C10-TG 55.00% 51.05% 51.05% 51.05% 57.89%

Polyoxyl15hydroxystearate 45.00% 41.77% 41.77% 41.77% 22.97%

Lecithin – 7.18% – 7.18% 14.35%

Polyglyceryl-3dioleate – – 7.18% – 4.78%

Continuousphase Water Water Water 0.9%aqueoussolutionofNaCl Water

Phaseinversiontemperature 92C 95C 86C 84C 95C

dynamicequilibriumbetweentheadsorbedmoleculesandthose inthesurroundingmedium).

Formulationswerestoredat4–8C.

2.3.APIloadingstudies

2.3.1.SeparationofnonadsorbedAPIandextractionofadsorbedAPI Nonadsorbed API was separated from the LNCs using a combinedultrafiltration-centrifugationtechniqueinvolvingAmi- conUltra-15centrifugalfilterdeviceswithamolecularweightcut off(MWCO)of 10kDa(Millipore,USA). Atotal of2mlof drug- loadedLNCs(orunloadedLNCsasa control)wereaddedtothe filterdeviceandcentrifugedfor30min(4500g).Then,thevolume of the sample remaining in the filter device (containing encapsulatedAPI) was increasedto 2ml with deionised water.

Next,0.3mlwastransferredtoa2mlEppendorftubecontaining 0.1ml of a 10% aqueoussolutionof NaCl,and themixturewas dilutedwith1.6mlofmethanoltorupturetheparticlesandextract theencapsulatedAPI.Centrifugation(16,000g)wasthenapplied for30mintoremoveaggregatedparticles.

The adsorption efficiency (AE) and drug loading (DL) were calculatedusingthefollowingequations:

AE¼ AB A

100% ð1Þ whereAisthetotalamount(mass)oftheAPI(sCT,AP114orPMB), andBisthemassofthenonadsorbedAPI.

DL¼ AB C

100% ð2Þ

whereC isthetotalweightofallcomponentsof theLNCs(the associatedAPIand themassofsurfactants andoil usedfor the preparationoftheLNCs).

2.3.2.QuantificationofAPIbyHPLC

ThesCT,AP114andPMBcontentswereanalysedusinganHPLC systemcomposedofanAFD92Watersin-linedegasser,Waters 717plusautosampler,Waters600HPLCpumpandcontroller,and Waters 2487 Dual

l

Absorbance detector. Briefly, standard solutionsoftheAPIs(1–1000

m

g/ml)werepreparedindeionised water,and20

m

lofthestandardorsamplewasinjectedintothe C18Uptisphere¹5

m

m2504.6mmcolumn(Interchim,France).

A flow rate of 1.2ml/min was employed usingmobile phase A composedof0.1%trifluoroaceticacid(TFA)inwaterand mobile phase B composed of 0.085% trifluoroacetic acid (TFA) in acetonitrile/water (4:1). A linear gradient was run: 10% B for 5min,35%Bfor9min,60%Bfor0.1min,100%Bfor2min,and10%

Bfor10.1min.TheAPIpeakshadretentiontimesofapproximately 16,13and12minforsCT,PMBand AP114,respectively.TheUV detectionwasperformedat215nm.Datacollectionandintegra- tionwereaccomplishedusingEmpower¹3software.

Themethodshowedgoodlinearity(R²0.9998)foralltested molecules.The quantification limits were2

m

g/ml forPMB and 1

m

g/ml for sCTand AP114, whereas the detection limits were 1

m

g/ml, 0.5

m

g/ml and 0.5

m

g/ml for PMB, AP114 and sCT, respectively.89–101%ofpeptidewasrecoveredeachtime.

2.4.CharacterisationofLNCs

Theintensity-averagedparticlediameterandthepolydispersity indexof theLNCs weredeterminedbydynamiclight scattering (DLS) using 173 backscatter detection. The electrophoretic mobility values measured by laser Doppler velocimetry (LDV) wereconvertedtozetapotentialsbytheSmoluchowskiequation.

Both,DLSandLDVmeasurementswereconductedonaZetasizer

nanoseriesNano-ZSfittedwitha633nmlaser(MalvernInstru- ments, UK). The measurements were performed at an LNC concentration of 3mg/ml obtained after dilution with MilliQ water.Eachanalysis wasperformedat25C. Thereadingswere carried outat leastthree timesforeach batchand theaverage valuesofatleastthreebatchesarepresented.

2.5.Adsorptionisothermfitting

ThedatawerefittedtotheLangmuirequation:

x m¼ abc

1þbc ð3Þ

wherexistheamountofsolute(API)adsorbedbyaweight,m,of adsorbent(LNCs),cistheconcentrationofsolutionatequilibrium (nonadsorbed API), b is a constant related to the enthalpy of adsorption,andaisrelatedtothesurfaceareaofthesolid.

TheLangmuirequationcanbearrangedintoalinearform:

c x=m¼ 1

abþc

a ð4Þ

Valuesaandbweredeterminedfromtheinterceptandslopeof plotsofc/(x/m)againstconcentration.

2.6.Statisticalanalysis

Thestatisticalsignificanceofthedifferencesbetweensamples was determined using one-way analysis of variance (ANOVA).

Differenceswereconsideredsignificantatp<0.05.

3.Resultsanddiscussion

3.1.FactorsaffectingtheadsorptionofsCT,AP114andPMBontoLNCs– influenceofthecompositionofthesurfactantshellofLNCs

LNCs, varyinginsize from25to100nm,arecomposed ofa hydrophilic surfactant, polyoxyl 15 hydroxystearate, and an oil (e.g., caprylic/capric acid triglycerides). The presence of the PEGylatedsurfactantisnecessaryforphaseinversion.Additionally, alipophilicsurfactant,lecithin,maybeusedinsmallquantitiesto significantlyincreasethestabilityoftheLNCs(Minkovetal.,2005;

Vonarbourg et al., 2005). The presence of lecithin or another lipophilicsurfactantisnecessarytoobtainlargerLNCs,especially 100nmLNCs.AlltestedAPIshadgoodsolubilityinwaterandcould beconsideredhydrophilicmolecules;thus,itisveryunlikelythat theAPIswouldpenetrateintotheoil coreof thenanocapsules.

Instead,the APIsareexpectedtobe localisedwithin oronthe particle shell. Therefore, in the beginning, experiments were performed toexamine the influence of the composition of the surfactantshellontheadsorptionefficiencyofsCT,AP114andPMB.

The polyoxyl 15hydroxystearate/oil mass ratio (MR) was kept constant(MR=0.81),andoptionallyeitherpolyglyceryl-3dioleate (anonionicW/Osurfactant)orlecithin(anamphotericsurfactant) wasadded.

The properties of the LNCs depended on their composition (Table2).TheLNCscomposedsolelyofpolyoxyl15hydroxystearate and C8/C10-TG were approximately 85nm and had a neutral surfacecharge(aZPofapproximately2mV).Theincorporationof polyglyceryl-3dioleateresultedinanimportantdecreaseinthe particlesizeto60nm,andthezetapotentialbecameslightlymore negative (12mV). The incorporation of lecithin yielded even smaller LNCs with a size of 46nm. Lecithin is an ampholytic surfactant containing phosphateand amino groups and hasan isoelectric point of 4.15 (Petelska and Figaszewski, 2002).

Therefore, the highly negative zeta potential value (54mV) indicates that the negative charge of the phosphate groups

predominatedundertheexaminedconditions(pH=6)(Valcourt et al., 2016), providing a good opportunity for electrostatic interactionwithpositivelychargedmolecules.Indeed,soylecithin dispersionsinNaClsolutionshavebeenshowntobear negative zeta potentials (Manconi et al., 2003). Similarly, the negative chargeofnanoemulsionswasattributedtothelecithinmolecules (Garcia-Fuentesetal.,2005).

The adsorptionyieldofalltestedAPIsrangedfrom9 to33%

(Table2)forformulationscontainingeitherpolyoxyl15hydrox- ystearatealoneorpolyoxyl15hydroxystearateandpolyglyceryl- 3dioleateasa surfactantand canbeconsideredrelativelylow.

Suchlow values arenot surprising becausemany studieshave demonstratedthatPEG-coatedsurfacesdisplayproteinresistance (i.e.,theymarkedlydecreasetheadsorptionofproteins)(Leeetal., 1989;Jeonet al.,1991;Malmstenetal.,1998;Herrwerth etal., 2003;Welschetal.,2013).Theprotein-resistantcharacterofPEGis probablycausedbyastericstabilisationeffect(Jeonetal.,1991).

Stericrepulsionisanentropiceffectcausedbytheunfavourable change in free energy associated with the dehydration and confinement of a polymer chain that had highconformational freedom(Herrwerthetal.,2003).However,incontrasttothedata ontheresistanceofPEG-coatedsurfacestoproteinadsorption,in ourstudy,despitethepresenceofaPEGylatedsurfactantattheLNC surface,a significant amountof each API(45–155

m

g/ml, being 9–31% of theAPI) was adsorbed onto theLNCs. Therefore, the adsorptionoftheAPIwasnotfullyprevented.Inmanyadsorption studies,proteinswereusedrather thanpeptides, andthemost commonly examined proteins, e.g., albumin (Lee et al., 1989;

Wittemannetal.,2003)orfibrinogen(Malmstenetal.,1998)have properties,e.g.,isoelectricpoint,thataredifferentfromthoseof sCTorAP114.Theisoelectricpointofalbuminsisapproximately 4.7,andthatoffibrinogenis5.1-6.3;therefore,atneutralpH,these moleculesarenegativelycharged,whereasAP114,sCTandPMBare expectedtobearanetpositivecharge.Furthermore,thepresence ofPEGdipolesmaycontributetotheslightlynegativechargeofthe LNCs(Vonarbourgetal.,2005).Thepresenceofanegativedipole onPEGandapositivechargeontheAPIsmoleculesmayprovidean opportunityfordipole-ioninteractions.Furthermore,inaddition toelectrostatic interactions, hydrophobic interactions mayalso driveproteinadsorption.Beingasurfactant,polyoxyl15hydrox- ystearate contains a hydrophobic moiety that is covalently attachedtothehydrophilicPEG-containingmoiety.Peptidesand polymyxinareknowntopossesssurfaceactiveproperties;thus,it ispossiblethatsomeoftheirmoleculesarealsolocalisedatthe nanocapsule surface together with the PEGylated surfactant.

Moreover,whentheflatsurfacesincontactwiththebulkliquids arecoatedwithPEGs,theircontactareaismuchsmallerthanthat

ofthenanocapsules,whichpresentanenormoussurfaceareaand therefore provide much more possibilities for peptide-PEG interaction.

Incontrasttotheeffectforpolyglyceryl-3dioleate,theaddition of lecithin was found to increase the adsorption of the drug markedly.Lecithincontainsionisable groups;therefore,electro- static interactions are likely to occur between drug molecules bearinganetpositivechargeandthephosphategroupoflecithin.It hasbeenshown that denselayersofchargedpolymers leadto strong adherence of proteins to colloidal particles and planar surfaces.Layersofcrosslinkedpolymersattachedtocolloidalcores (core-shell microgels)also attractproteins andmaybe usedto immobiliseenzymes(BallauffandBorisov,2006;Ballauff,2007;

Wittemann et al., 2003; Welsch et al., 2013). Polyelectrolyte complexeshavebeenshowntoencapsulatesalmoncalcitoninvery efficiently,andtheprocesswasdrivenbyelectrostaticinteractions (Umerskaetal.,2014a,b,2015a).Therefore,thefactthatlecithin LNCshadthehighestadsorptionefficiencymaydemonstratethe predominantroleofelectrostaticinteractionsbetweenoppositely chargedionsoverothertypesofinteractions(e.g.,vanderWaals forces,ion-dipoleinteractions).

However,thesCTadsorptionyieldwassmallerfortheLNCsthan for polyelectrolyte complex nanoparticles or lecithin-coated nanoemulsions(Garcia-Fuentesetal.,2005),wheretheobserved efficiency was closeto 100%.This findingmight be due tothe presenceoftwotypesofsurfactantmoleculesattheLNCsurface:

lecithin and PEGylated surfactant,thelatter beingpresent in a markedlylargerquantity.Despitethepresenceofalargeamountof polyoxyl 15hydroxystearate, thehighly negative zetapotential valuesoflecithin-LNCssuggestthatthephosphategroupsofthe lecithin contact theexternal aqueousenvironment and are not completelyscreenedbyPEGs.Therefore,when anAPImolecule approachesanLNC,thestericrepulsionbyPEGchainsmightbe compensatedbytheelectrostaticattractionbetweenthepeptide and phosphategroups of lecithin. Theinhibition of theprotein adsorptionon thesurfacesby thePEGcoating depends onthe densityandlengthofthePEGchains(Jeonetal.,1991;Malmsten etal.,1998;Herrwerthetal.,2003).TheattractivevanderWaals componentoftheinteractionwiththesubstratedecreaseswith increasing surface density and chain length of the terminally attachedPEGchains,anda highsurfacedensityof PEGis more importantthanalongchainlength(Jeonetal.,1991).Theaddition oflecithindecreasesthedensityofPEGchainsonthesurfaceofthe LNCs,asindicatedbythedecreaseinthezetapotentialoflecithin- LNCs.

TheparticlesizeoftheLNCswasnotaffectedbytheadsorption ofthedrug,withtheonlyexceptionbeingAP114-loadedlecithin- Table2

Compositionandpropertiesofdrug-loadedLNCs.LNCconcentration=100mg/ml,initialdrugconcentration=500mg/ml.*p<0.05,**p<0.01and***p<0.001versusAPI-free LNCs.

Formulation Drug AE[%] DL[%] PS[nm] PDI ZP[mV]

F1 – 851 0.0860.015 1.80.4

F2 – 462 0.1960.020 54.05.9

F3 – 571 0.0710.010 12.42.6

F4 – 473 0.0340.012 22.64.1

F1 sCT 255 0.1250.025 851 0.0830.016 1.60.4

F2 sCT 593 0.2940.015 451 0.1250.010* 24.02.8**

F3 sCT 193 0.0950.015 571 0.0680.009 8.72.7

F4 sCT 332 0.1650.010 482 0.0540.014 20.24.1

F1 AP114 3113 0.1550.065 851 0.0850.015 0.90.4*

F2 AP114 795 0.3930.025 602*** 0.0880.014** 11.61.4***

F3 AP114 3112 0.1550.060 571 0.0570.017 7.32.1

F4 AP114 3410 0.1700.050 535 0.0560.013 5.91.1**

F1 PMB 94 0.0450.020 851 0.0870.015 0.90.6

F2 PMB 427 0.2100.035 471 0.0650.013** 6.21.0***

F3 PMB 154 0.0750.020 581 0.0500.010 7.21.0**

F4 PMB 177 0.0850.035 493 0.0500.009 5.81.0**

LNCs (F2),where asignificantincreasein theparticle sizewas observed.The size distributionof theLNCs was not influenced otherthanforlecithin-LNCs,whereasignificantdecreaseinPDI was observed for all tested APIs. The adsorption of the drug producedadecreaseinthesurfacecharge,resultinginasignificant increaseinthezetapotentialvalues.Thestrongesteffectof the adsorptiononthezetapotentialdecreasedinthefollowingorder:

F2(lecithinLNCs)>F3(polyglyceryl-3dioleateLNCs)>F1.Changes inzetapotential wereobservedeven fortheLNC formulations, which did not contain an ionised surfactant; this result may indicatethattheinteractionoftheAPIswiththeLNCs,although dominatedbyelectrostaticinteractions,alsoinvolvesdipole-ion, vanderWaalsorhydrophobiccomponents.

3.2.FactorsaffectingtheadsorptionofsCT,AP114andPMBontoLNCs –influenceoftheionicstrengthofthemedium

A significant decrease in adsorption efficiency values was observedforalltestedAPIswhenNaClsolutionwasusedasthe continuous phase instead of water (Table 2). The decreased adsorption yield at higher ionic strength confirmed that the electrostaticinteractionswerethemainfactorresponsibleforthe association of the drug with the LNCs. Similarly, at low ionic strength,astrongadsorptionofbovineserumalbuminwasfound on spherical polyelectrolyte brushes, and this adsorption de- creaseddrasticallywiththeadditionofincreasingamountsofsalt (Wittemannetal.,2003).Ithasbeendemonstratedthattherelease ofsCTfromchondroitin/protaminenanoparticleswasnegligiblein environmentswithlowionicstrength(i.e.,inwaterandin0.01M acetatebuffer);howevertheincreaseintheionicstrengthofthe medium(0.1Macetatebufferor0.15MPBS)triggeredareleaseof thismoleculefromthenanoparticles(Umerskaetal.,2015a,b,c).

Moreover, even higher amounts of sCT were released when hyaluronicacid/chitosan or hyaluronic acid/protamineparticles weredilutedwithPBS(Umerskaetal.,2014a,b).

Theincreaseintheionicstrengthofthecontinuousphasedue todissolvingNaCldecreasedthesurfacecharge–anincreasein

zeta potential from 54mV to approximately 22mV was observed whenwaterand anaqueousNaClsolutionwereused as diluents, respectively(Table 2).A significantdecreasein the polydispersityindexfrom0.196to0.034wasalsoobservedwhen 0.9%NaClwasusedasadispersant.Thisbehaviourissimilartothat ofthepolyelectrolytecomplexnanoparticlesdescribedpreviously, where decreases in both the zeta potential and polydispersity index werealsoobserved withincreasingionic strengthof the medium (Umerska et al., 2015a). However, in contrast to polyelectrolytecomplexnanoparticles,theionicstrength ofthe mediumdidnotaffecttheLNCsize.

TheadsorptionoftheAPIdidnotaffecteithertheparticlesize or thepolydispersity indexof the LNCs dispersed in 0.9%NaCl solution(Table2).Theadsorptiondidinfluencethezetapotential in 0.9%NaClsolution;however, theeffectwasless pronounced thantheeffectinwater.

Lecithin-LNCsaresimilartopolyelectrolytesinthesensethat eachnanocapsulehasmultiplenegativeandpositivechargesdue tothepresenceoflecithinmolecules.PeptidesandpolymyxinB mayalsobeconsideredpolyelectrolytes.Itiscommonlyaccepted thatpolyelectrolytecomplexformationismainlycausedbystrong electrostatic interactions between oppositely charged polyions, whereby the gain in entropy due to the release of the low molecularcounterions,whichareinitiallyboundtopolyelectro- lytes,playsadecisiverole(Schatzetal.,2004).Counterionshave been incorporated within nanoparticles (Umerska et al., 2012, 2015a).Insalt-freesolution,onlyasmallfractionofthecounter- ions can evade the polyelectrolyte brush layer; most of the counterionsareconfinedwithinthelayer(Welschetal.,2013).The noticeable osmotic pressure within the brush determines the propertiesoftheparticlesinaqueoussolutionandtheirinteraction withproteins.Ifthesaltconcentrationisraisedsufficiently,this effectis stronglydiminished,andthesystemsbehavenearlyas unchargedsystems(Welschetal.,2013).Indeed,thebehaviourof lecithin-LNCsdispersedin0.9%NaCl(F4)resemblesthatofF1and F3, which do not containanionisedsurfactant. Theadsorption

Fig.1.ThepropertiesofsCT-loadedLNCs:PS–hydrodynamicdiameter,PDI–polydispersityindexandZP–zetapotential.*p<0.05,**p<0.01and***p<0.001versussCT- freeLNCs(whitebars).

efficiencyofeachAPIissimilarforF4andF1orF3,andtheyall differmarkedlyfromF2(lecithinLNCsdispersedinwater).

3.3.FactorsaffectingtheadsorptionofsCT,AP114andPMBontoLNCs –influenceoftheLNC/APImassmixingratio

Successfulnanocarriersshouldhaveahighloadingcapacityto reducethenumber of theparticles thatmust beadministered.

High associationefficiencyis alsoa very important parameter, especiallyforexpensivedrugs.

Insummary,lecithin-LNCs(F2)providedthebestadsorptionof alltested drugs (Section3.1). Additionally,to achieve effective adsorptionofthedrugontotheLNCs,acontinuousphasewithlow ionicstrengthshouldbeused(Section3.2).

To improvetheadsorptionof theAPIsontotheLNCs,anew optimisedformulationwasdesignedthatcontainedanincreased quantityoflecithinanddecreasedquantityofpolyoxyl15hydrox- ystearate.Itwasalsonecessarytoaddpolyglyceryl-3dioleateto theformulation becausewithout this surfactant,the optimised formulation did not undergo phase inversion. The LNCs had a particlesizeof63nm,ahomogenoussizedistribution(PDIvalueof 0.26)and ahighlynegativezetapotentialvalueof64mV.The zetapotentialdidnotdiffersignificantly(p=0.0734)fromthatof theF2formulation;however,significantincreasesinparticlesize andpolydispersityindexwereobserved(p=0.006and0.0074for PSandPDI,respectively).TheparticlesizeofLNCsdependsonthe quantityof the polyoxyl15 hydroxystearatein the formulation (Heurtaultetal.,2002);therefore,theincreaseintheparticlesize ofF5fromthatofF2isnotsurprising.

IncorporationofsCTresultedinasignificantdecreaseinparticle sizecomparedwiththatofthesCT-freesampleatlowerLNC/sCT MMRs(50–200)andhighersCTconcentrations(500–100

m

g/ml).

AthigherLNC/sCTMMRs(300–800)andlowersCTconcentrations (125

m

g/ml),theparticlesizeremainedthesameas thatofthe control(Fig.1).Theincorporationofcalcitoninintheshellofthe LNCs maycause partial dehydration of the shell and therefore decreasethedegreeofswelling.Similarly,asignificantdecreasein

PDIwasobservedatlowerMMRs(50–200),whereasatMMRsfrom 300to800,thesizedistributionwasnotaffectedbythepresenceof calcitonin.TheadsorptionofsCTalsodecreasedthesurfacecharge oftheparticles.Significantchangesinzetapotentialwereobserved athigherLNC/SCTMMRsincontrasttothePSorPDIchanges–a decreaseinabsolutevalueofzetapotentialwasobservedevenat LNC/sCT MMR of 400 for less concentrated samples. A linear correlationhasbeenfoundbetweenthezetapotentialandsCT/LNC MMRfortheMMRsbetween50and200(R²=0.9855).

Theinfluenceof sCTonthepropertiesofthepolyelectrolyte complexnanoparticleshasbeeninvestigated.Theparticlesizewas dependent on the type of nanoparticle and the nanoparticle/

peptidemixingratio.sCTdecreasedthehydrodynamicdiameterof thehyaluronicacid/chitosannanoparticleswithahighnanoparti- cle/peptidechargemixingratio,andanincreasewasobservedin formulations containing larger amount of sCT and chitosan (Umerskaetal.,2014b).Anincreaseddiameterofthehyaluronic acid/protamine nanoparticles was observed after the incorpo- ration of calcitoninfor allmixing ratiostested(Umerskaet al., 2014a).Interestingly,theformernanoparticlesareexpectedtobe composedofahydrophobiccoreandhydrophiliccorona(Boddohi etal.,2009;Umerskaetal.,2012)similarlytotheLNCs,whereas the behaviour of the latter nanoparticles indicated that the structuremightbedifferent(Umerskaetal.,2014a).

Atlowconcentrations(125

m

g/ml)andhighLNC/AP114MMRs (300–800), AP114 did not affect the particle size of the LNCs (Fig.2).AtMMRsof150–200andAP114concentrationsof250– 500

m

g/ml, the particle size reached the minimum and was significantlylowerthanthatofthepeptide-freesamples,andwith further increases in the AP114 concentration, the particle size increased markedly. The particle size at MMRs of 50–75 was markedlylargerthanthatathigherMMRs.Noimportantchanges wereobservedinthesizedistributionaftertheincorporationof AP114,exceptforthesampleswiththelowestLNC/AP114MMRs (50 and 75),where a dramatic decrease in thePDIvalues was observed. Interestingly, this decrease corresponded to a major increaseintheparticlesize.Asignificantreductionintheabsolute

Fig.2.ThepropertiesofAP114-loadedLNCs:PS–hydrodynamicdiameter,PDI–polydispersityindexandZP–zetapotential.*p<0.05,**p<0.01and***p<0.001versus AP114-freeLNCs(whitebars).

value of the zeta potential compared with that for the LNCs withoutAP114wasobservedatLNC/AP114MMRsbetween50and 200,withastronglinearcorrelation(R²=0.9192).

TheincorporationofPMBstronglyinfluencedtheparticlesize oftheLNCs(Fig.3).Theparticlesizewassignificantlysmallerthan beforetheincorporationofPMBevenatlowPMBconcentrations (125

m

g/ml)andhighPMB/PMBMMRs(200–600).WhentheMMR wasfurtherdecreased(50–150),asignificantincreaseinparticle sizewas observed. The PDIdecreased significantlyat LNC/PMB MMRsof200–300,andthenasignificantincreasewasobservedat MMRsof50–100.Asignificantdecreaseintheabsolutevalueofthe zetapotentialwasobservedevenatLNC/PMBMMRssmallerthan orequalto600.FortheMMRsbetween200and400,averystrong linearcorrelationwasobserved(R²=0.99939);then,thedecrease waslesssharp,andthezetapotentialwasthesameforthetwo lowestMMRs(50and75).

Thereductioninparticlesizeafterpeptideadsorptionmaybe attributedtoosmoticdeswellingofthenanocapsuleshellanda consequentdecreaseoftheshellvolume.

In conclusion, changes in the properties of the particles (particlesize and sizedistribution,zetapotential) indicatethat the APIs interact with the LNCs. Therefore, monitoring these parametersmayprovideinformationabouttheadsorptionofthe API.Thezetapotentialchangesoccurredforsystemswithlower APIquantities thanfor theparticlesizechanges. Moreover,the linear correlation between the zeta potential and the LNC/API MMR(whichisproportional tothequantity ofAPI)mightbea usefultool toprovidenotonlyqualitativebut alsoquantitative information ontheadsorption.Similarly toLNCs, in hyaluronic acid/chitosan/sCT nanoparticles, a linear relationship has been found betweenthe zeta potential and the mixing ratio of the componentsofthenanoparticles(Umerskaetal.,2014b).

sCTwasveryefficientlyadsorbedontheLNCsathighLNC/sCT MMRs(Table3).ForhighLNC/sCTMMRs(300–800),theAEvalues

were constant (98–100%). A slight decrease in AE could be observed at MMRS of 200(98 and 93% for samplescontaining 0.25 and 0.5mg/ml of sCT, respectively) and 150 (90%). This decrease became more pronounced at lower MMRs (50–100), whereaverystronglinearcorrelationbetweentheMMRandAE wasobserved(R²=0.9973).AP114wasalsoadsorbedontheLNCs veryefficiently(Table4).TheAEreachedthevaluecloseto100%at LNC/AP114MMRsequaltoorgreaterthan150.AtMMRsof50–100, adecreaseinAEwithdecreasingAP114/AP114MMRwasobserved withthemaximumpositivecorrelation(R²=1).Similarbehaviour wasobservedforPMB–atLNC/PMBMMRvaluesbetween300and 800, the AE was maximal (98–100%), and at lower MMRs, a decreaseinAEcorrespondingtodecreasingLNC/PMBMMRwas observed withastronglinear correlation(R²=0.9464)between theseparameters(Table5).Thisbehaviourwas observedforall 3 APIs,indicating that a limitedamount of drugthat couldbe adsorbed ontheLNCs. Thisresultmay beobserved becauseat higherdrugconcentrations,thebindingplacesontheLNCsbecame saturatedandfurtherincreasesinthedrugconcentrationdidnot increasetheamountofthedrugadsorbedontheLNCs.

F5-LNCs provided better adsorption of the APIs than did F2-LNCs.Thisfindingmightbeattributedtothehigherquantityof lecithinintheformerbecauseitseemsthatelectrostaticbinding with lecithin molecules is the main driving force for API adsorption.ThelowerdensityofPEGchainsontheLNCsurface ofF5comparedwithF2mayalsofacilitatetheadsorption.

Thestabilityofadsorbedmoleculeshasbeenexaminedafter 1weekofstorageat4–8C.Nodegradationpeakswereobservedin thechromatogram,andtheadsorptionefficiencydidnotchange significantlyafterstorage.Althoughtheinteractionsbetweenthe peptidesandnanocapsulesingredients,particularlyPEGmoieties, are weakand reversible, thedynamic equilibrium betweenthe LNCsandsurroundingmediumwasestablishedandthedesorption didnotoccur.Thephysicalpropertiesoftheformulations(particle Table3

CompositionandpropertiesofsCT-loadedLNCs.AE:adsorptionefficiency,DL:drugloading.

LNCconcentration[mg/ml] Totaldrugconcentration[mg/ml] LNC-associateddrugconcentration[mg/ml] AE[%] DL[%] LNC/sCTmassmixingratio

100 125 1250 1000 0.1250.000 800

100 250 2461 981 0.2450.001 400

100 500 4656 931 0.4630.006 200

100 1000 81035 813 0.8030.034 100

75 125 1250 1000 0.1660.000 600

75 250 2461 991 0.3270.002 300

75 500 4489 902 0.5930.011 150

75 1000 72035 723 0.9510.046 75

50 125 1250 1000 0.2490.000 400

50 250 2441 981 0.4850.003 200

50 500 40512 812 0.8030.023 100

50 1000 60529 613 1.1960.057 50

Table4

CompositionandpropertiesofAP114-loadedLNCs.AE:adsorptionefficiency,DL:drugloading.

LNCconcentration[mg/ml] Totaldrugconcentration[mg/ml] LNC-associateddrugconcentration[mg/ml] AE[%] DL[%] LNC/AP114massmixingratio

100 125 1250 1000 0.1250.000 800

100 250 2500 1000 0.2490.000 400

100 500 4956 991 0.4930.006 200

100 1000 85832 863 0.8500.032 100

75 125 1250 1000 0.1660.000 600

75 250 2500 1000 0.3320.000 300

75 500 48414 973 0.6410.019 150

75 1000 80849 815 1.0650.065 75

50 125 1250 1000 0.2490.000 400

50 250 2500 1000 0.4980.000 200

50 500 43013 863 0.8530.026 100

50 1000 75832 763 1.4920.063 50

size, zeta potential) did not change during short-term storage (1week)at4–8C.Nevertheless,inaqueousmediapeptidesand proteinscanundergodegradationreactionssuchashydrolysis.To minimisethesereactionsthefutureworkistodesignsolidstate, lyophilisedformulations.

3.4.FittingtotheLangmuirisotherm

Anadsorptionisothermisagraphicalrepresentationshowing therelationshipbetweentheamountadsorbedbyaunitweightof adsorbent and the amount of adsorbate remaining in a test mediumatequilibrium(Desta2013).Themostcommonlyused modelsto describe the distribution of the solute betweenthe liquidandsolidphasesatvariousequilibriumconcentrationsare theLangmuirandFreundlichmodels.Attemptsweremadetofit theadsorption data tothe Freundlich equation, but they were

unsuccessful.Thisresultisinagreementwiththedataobtainedby Garcia-Fuentesetal.(2005),whoshowedthattheadsorptionof sCTontoPEG-orchitosan-coatedlipidnanoparticlesdidnotfitthe Freundlichequation.

Thelinearplotofthespecificadsorptionagainsttheequilibri- umconcentrationshows that theadsorptionof sCT,AP114and PMBobeyedtheLangmuirmodel(Fig.4)overalimitedrange.The Langmuirisothermassumesmonolayeradsorptionontoasurface containingafinitenumberofadsorptionsitesthatareidenticaland equivalent, with no lateral interaction and steric hindrance betweentheadsorbedmolecules(FooandHameed,2010).Once asiteisfilled,nofurtheradsorptioncantakeplaceatthatsite.This condition indicatesthat thesurface reaches a saturationpoint wherethemaximumadsorptionofthesurfacewillbeachieved.

Thechitosan-orPEG-coatedlipidnanoparticlesdescribedby Garcia-Fuentesetal.(2005)followedtheLangmuirisothermunder Table5

CompositionandpropertiesofPMB-loadedLNCs.AE:adsorptionefficiency,DL:drugloading.

LNCconcentration[mg/ml] Totaldrugconcentration[mg/ml] LNC-associateddrugconcentration[mg/ml] AE[%] DL[%] LNC/PMBmassmixingratio

100 125 1250 1000 0.1250.000 800

100 250 2500 1000 0.2490.000 400

100 500 39314 793 0.3910.014 200

100 1000 39349 395 0.3910.049 100

75 125 1250 1000 0.1660.000 600

75 250 2447 983 0.3250.009 300

75 500 33618 674 0.4460.024 150

75 1000 36838 374 0.4880.050 75

50 125 1250 1000 0.2490.000 400

50 250 21812 875 0.4330.025 200

50 500 25318 524 0.5020.037 100

50 1000 34076 348 0.6750.151 50

Fig.3. ThepropertiesofPMB-loadedLNCs:PS–hydrodynamicdiameter,PDI–polydispersityindexandZP–zetapotential.*p<0.05,**p<0.01and***p<0.001versus PMB-freeLNCs(whitebars).

the experimental conditions tested. However, it has been demonstrateddeviationsfromthetypicalLangmuirplotcanoccur at high concentrations, and these deviations are then usually attributedtotheformation ofmultilayers (El-Masry andKhalil, 1974). In another study, Calis et al. (1995) showed that the adsorption of sCT onto PLGA microspheres follows a mixed Langmuir and Freundlich model, depending on the peptide concentrationandtheadsorptionmedium.

ThecalculatedLangmuirconstantsareshowninTable6.The valueofaisameasureoftheadsorptivecapacityoftheadsorbent for the particular adsorbate under examination. When mass concentration is considered, PMB had the lowest degree of adsorption,whichwasmarkedlysmallerthanthoseoftheother peptides.ThevaluesofAEforF2alsoindicatethatthedegreeof adsorptiondecreasesinthefollowingorder: AP114sCT>PMB.

However,when the adsorbedquantity was considereda molar concentration, the tendencies were inversed PMB showed markedlyhigheraffinityforLNCsthandidsCTorAP114.

ThevalueofbisconsiderablysmallerforsCTthanforAP114or PMB.Interestingly,theadsorptionofPMBorAP114hadastronger effectonthesizeorsizedistributionoftheLNCsthanthatofsCT (seeSection3.3).

Thevalueof9.27

m

g/mgforsCTwassignificantlysmallerthan that of PEG-coated tripalmitin nanoparticles (23.4–25.6

m

g/mg) (Garcia-Fuentesetal.,2005).Thedifferencemightbeduetothe differentcompositionofthecarriers–theconcentrationofthePEG derivativewascomparablebetweenboth systems,buttheLNCs containedasmallerquantityoflecithin(14versus25%).Although thephaseinversiontechniqueusedforthepreparationoftheLNCs yielded smaller nanocarriers (50–60nm versus 200nm), the disadvantageisthesmalleramountofthelecithinthatcouldbe incorporatedintothenanocarriercomparedwiththeamountfor thedouble emulsionsolventevaporation method.Coatingwith chitosandramaticallydecreasedthesCTadsorptioncapacityfor lipidnanoparticles(1.9

m

g/mg)(Garcia-Fuentesetal.,2005).Inthe

caseofPLGAmicrospheres,thebindingcapacitydependedonthe concentrationofthepeptideandthemodelused–themultilayer peptideadsorption(Freundlichmodel)showedhigheradsorption capacity (24

m

g/mg) compared with that of the monolayer peptide-polymeradsorption(Langmuirmodel–5.1

m

g/mg)(Calis etal.,1995).DaniandDeLuca(2001)producedPLGAmicrospheres capableofadsorbing20

m

gofsCTpermgoftheparticlesin0.1M PBS.

Proteinshavepositivelyandnegativelychargedareasontheir surface(Ballauff,2007).AtaparticularpH,onetypeofchargemay prevail; however, the areas bearing opposite charge are still present in the molecule. Despite the net positive charge, the peptides,e.g.,sCTorAP114,alsocontainnegativelychargedamino acids,e.g.,thecarboxylendofthepeptide,anditispossiblethat these amino acids interact with the amino group of lecithin molecule while the negatively charged phosphate groups of lecithin interact with the positively charged amino acids. In contrasttosCTorAP114,PMBdoesnothavemoietiescapableof providinganegativechargeandthereforeisnotabletointeract electrostaticallywiththeaminogroupoflecithinmolecules.Each mgofF5lecithin-LNCsshouldcontainapproximately184nmoles of lecithin, which is equivalent to 184nmoles of phosphate or amino groups. AP114 has 7–9 positively charged amino acids capable of interacting with the phosphate group of lecithin molecules(twohistidinemoleculesarenotionisedatpH7.4,but they may be approximately 50% ionised at pH 6); this value corresponds to approximately 16.8–21.6nmoles, making an approximately10-foldstoichiometricexcessofphosphategroups oflecithinovertheaminogroupsofthepeptide.Asimilarsituation occursinthecaseofsalmoncalcitonin–therearefivepositively chargedaminoacids,includinghistidine,thatgiveapproximately 13.5nmoles, causing an even higher stoichiometric excess of lecithinphosphategroups.Moreover,PMBhasfive

g

-aminogroups of diaminobutyric acid, leading to 18.5nmoles of positively chargedgroupscapableof reactingwith184nmolesoflecithin phosphategroups.Therefore,evenatthepointwheretheLNCsare saturated with the adsorbed molecules, most of the lecithin phosphategroupsremainunbound.Thisbehaviourmayoccurdue tothestericrepulsionbetweenthepeptidesandthePEGmoieties ofthepolyoxyl15hydroxystearatemoleculesbecausethelatterare present at much higher quantities in thesurface layer and are associated with the triglycerides to form the oil core of the nanocapsulesviahydrophobicbonds.

4.Conclusions

The properties of the obtained LNCs (particle size, size distributionandzetapotential)dependedontheircomposition.

AsignificantamountofthedrugswasadsorbedontheLNCsinall experimentalconditionstestedduetotheenormoussurfacearea of theLNCsand theinteractionsbetweenthepeptides andthe nanocarriers.Theadsorptionyieldwasimprovedbytheadditionof an ionisablelipophilic surfactant,lecithin. PolymyxinBshowed noticeablyloweradsorptionontheLNCswhenconsideringtheAPI mass adsorbed per mg of the LNCs; however, because the molecular weight of PMB is considerably lower than those of sCTorAP114,theamountofPMBmoleculesadsorbedwashigher thanthoseofsCTorAP114.

Thequantity ofthedrugadsorbedand thepropertiesofthe LNCs, particularly the zetapotential, depended onthe LNC/API massmixingratioandtheLNCandAPIconcentrations.Atlower concentrations(250

m

g/ml)andhighLNC/drugmassmixingratios, alldrugswereeffectivelyadsorbedontheLNCs;however,when thedrugconcentrationwasincreased,theadsorptionefficiency decreased,anddifferencesbetweenthedrugswereobserved.The adsorptionofsCT,AP114andPMBobeyedtheLangmuirmodelover Fig.4.LinearfittingoftheadsorptiondatatotheLangmuirequation;bluesquares-

sCT, red circles-AP114, and green triangles-PMB. (For interpretation of the referencestocolourinthisfigurelegend,thereaderisreferredtothewebversion ofthisarticle.)

Table6

IsothermparametersfortheadsorptionofsCT,AP114andPMBontheLNCs.

API a(mg/mg) a(nmol/mg) b(ml/mg) R²

sCT 9.27 2.7 0.089 0.9775

AP114 10.48 2.4 0.336 0.991

PMB 4.78 3.7 0.282 0.9886