Sustained Release of Antibacterial Lipopeptides from Biodegradable Polymers against Oral

16 Pages·2016·1.34 MB·English

RESEARCHARTICLE
Sustained Release of Antibacterial
Lipopeptides from Biodegradable Polymers
against Oral Pathogens
LeaH.Eckhard1,YaelHouri-Haddad1,AsafSol2,RotemZeharia1,YechielShai3,
ShaulBeyth4,AbrahamJ.Domb5,GiladBachrach2☯,NuritBeyth1☯*
1 DepartmentofProsthodontics,theHebrewUniversity–FacultyofDentalMedicine,Jerusalem,Israel,
2 InstituteofDentalScience,theHebrewUniversity–FacultyofDentalMedicine,Jerusalem,Israel,
3 DepartmentofBiologicalChemistry,theWeizmannInstituteofScience,Rehovot,Israel,4 Orthopedic
SurgeryDepartment,HadassahMedicalCenter,Jerusalem,Israel,5 InstituteforDrugResearch,Schoolof
a11111 Pharmacology,FacultyofMedicine,theHebrewUniversity,Jerusalem,Israel
☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Thedevelopmentofantibacterialdrugstoovercomevariouspathogenicspecies,which
Citation:EckhardLH,Houri-HaddadY,SolA, inhabittheoralcavity,facesseveralchallenges,suchassalivaryflowandenzymaticactivity
ZehariaR,ShaiY,BeythS,etal.(2016)Sustained
thatrestrictdosageretention.Owingtotheiramphipathicnature,antimicrobialpeptides
ReleaseofAntibacterialLipopeptidesfrom
BiodegradablePolymersagainstOralPathogens. (AMPs)serveasthefirstlineofdefenseoftheinnateimmunesystem.Theabilitytosynthe-
PLoSONE11(9):e0162537.doi:10.1371/journal. sizedifferenttypesofAMPsenablesexploitationoftheiradvantagesasalternativestoanti-
pone.0162537
biotics.SustainedreleaseofAMPsincorporatedinbiodegradablepolymerscanbe
Editor:HélderA.Santos,HelsinginYliopisto, advantageousinmaintaininghighlevelsofthepeptides.Inthisstudy,fourpotentultra-short
FINLAND
lipopeptides,conjugatedtoanaliphaticacidchain(16C)wereincorporatedintwodifferent
Received:April2,2016 biodegradablepolymers:poly(lacticacidcocastoroil)(PLACO)andricinoleicacid-based
Accepted:August24,2016 poly(ester-anhydride)(P(SA-RA))forsustainedrelease.Thelipopeptideandpolymerfor-
mulationsweretestedforantibacterialactivityduringoneweek,byturbidometricmeasure-
Published:September8,2016
mentsofbacterialoutgrowth,anti-biofilmactivitybylive/deadstaining,biocompatibilityby
Copyright:©2016Eckhardetal.Thisisanopen
hemolysisandXTTcolorimetricassays,modeofactionbyfluorescence-activatedcellsort-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits ing(FACS)andreleaseprofilebyafluorometricassay.Theresultsshowthatanantibacte-
unrestricteduse,distribution,andreproductioninany rialandanti-biofilmeffect,aswellasmembranedisruption,canbeachievedbytheuseofa
medium,providedtheoriginalauthorandsourceare
formulationoflipopeptideincorporatedinbiodegradablepolymer.
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Funding:ThisworkwassupportedbytheLegacy Introduction
HeritageClinicalResearchInitiativeoftheIsrael
Theoralenvironmentcontainsvariousmicroorganismssomeofwhicharepathogenicspecies
ScienceFoundation(GrantNo.1764/11).Thefunders
involvedindentalcaries,periodontaldiseasesandendodonticlesions[1].Thesebacteria
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe includeStreptococcusmutans,Actinomycesnaeslundii,Porphyromonasgingivalis,Fusobacter-
manuscript. iumnucleatumandEnterococcusfaecalis.Theydifferonefromtheotherbutsharethecrucial
abilitytoformdentalbiofilm,adynamic,activemetabolicstructurethatpresentsachallenge
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. toantibiotics[2,3].
PLOSONE|DOI:10.1371/journal.pone.0162537 September8,2016 1/16
SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Antimicrobialpeptides(AMP's)aredevelopmentalcomponentsoftheinnateimmunesys-
temthatareproducedbydifferenttypesoforganisms.AMPsarepotentandefficientagainst
variouspathogensowingtotheirstructureandcharge[4].CationicAMPswereproposedfor
useaspotentialanti-infectivecompoundsagainstantibiotic-resistantvirulentstrainsofmicro-
organisms[5].Ingeneral,AMPactivitiesinclude:theabilitytokillawiderangeofpathogens,
aswellastheirpotentialtoactivateanti-inflammatorycellsandrecruitthemtotheinjuredtis-
suefromthebloodstream[6].AlthoughthecomplexmechanismofactionofAMPs,isnot
fullyclear,itisgenerallyacceptedthatthesepeptidesselectivelydisruptcellmembranesowing
totheiramphipathicstructuralarrangement[7].However,microbeshaveevolvedseveralresis-
tancestrategiestocircumventAMPfunction,suchasalterationofcellnetsurfacecharge[8],
intracellularAMPdegradation[9]andAMPexportviaeffluxpumps[10].
Anotherpotentfamilyofantibacterialfactorsisthenativelipopeptidesthatareproducedonly
bybacteriaandfungiduringtheircultivationonvariouscarbonsources.However,asnativelipo-
peptidesarenotcell-selective,theyaretoxictomammaliancells[11],although,thelipopeptide
daptomycinwhichisactiveonlyagainstGram-positivebacteria,wasapprovedbytheFDAfor
thetreatmentofskininfectionscausedbyStaphylococcusaureus[12].Mostnativelipopeptides
consistofashort(sixtosevenaminoacids)linearorcyclicpeptidesequence,withanetpositive
ornegativecharge,towhichafattyacidmoietyiscovalentlyattachedtotheN-terminus.Incon-
trasttotheAMPs,resistancetolipopeptidesisgenerallyrare[13].Aspreviouslydescribedin
detail,mostnativelipopeptides,similarlytotheAMPs,actviatwomajormechanisms:inhibition
ofthesynthesisofcellwallcompoundsandinductionofcellmembranelysis[14].
AttemptshavebeenmadetoproducesyntheticAMPsrecruitingallthestructuraladvan-
tagesofthenativeAMPstobuildimprovedantibioticagents[15,16].Ultra-shortlipopeptides
areamphiphilicmoleculesmimickingdetergents,inwhichthepeptidemoietyishydrophilic
andthefattyacidmoietyishydrophobic[17].Itwaspreviouslyshownthattheseultra-short
lipopeptidesareactiveagainstavarietyofmicroorganisms.LikenativeAMPs,theirmodeof
actioninvolvesdisturbanceofthemembrane[11].
Indentistrythereareseveralchallengesfacedbydrugdelivery,suchasmaintenanceofdrug
dosageinthepresenceofsalivaryflowandenzymaticactionthatcancleavecertainpeptides.
Consequently,asustainedreleasemechanismcouldallowhighconcentrationsoftherapeutic
agentsforprolongedperiodsoftime.
Acombinationofthestructuralandfunctionalpropertiesofpeptideswiththoseofsynthetic
polymershasgainedsignificantinterestinmaterialdesignandapplication.Thesesmartpoly-
mericsystemshaveseveraladvantagesoverconventionalmethods,suchaseaseofmanufac-
ture,andadministration,biodegradability,andtheabilitytoalterthereleaseprofilesofthe
incorporatedagents[18,19].Hybridmoleculesofpeptidesconjugatedtopolymerscanbeused
forvariousapplications,withtheadvantageofbeingresistanttoenzymaticcleavageandless
cytotoxictohumancells[20].
Thepresentstudyfocusedonantibacterialevaluationoflipopeptidesaspartofasustained
releasetherapeuticmeans.Thehypothesiswasthatlipopeptidesmixedwithbiodegradable
polymerswouldmaintainaneffectiveconcentrationandbeeffectiveagainstvariousoralpath-
ogenicbacteria.
MaterialsandMethods
Testmaterials
Syntheticantimicrobialpeptides. Fourdifferentultra-shortlipopeptides:C16-KGGK,
C16-KKK,C16-KAAKandC16-KLLK,synthesizedandpurifiedaspreviouslydescribed[11,
21],weretested.
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SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Biodegradablepolymersynthesis. Poly(lacticacidcocastoroil30:70)(PLACO)and
ricinoleicacid-basedpoly(ester-anhydride)(P(SA-RA))weresynthesizedaspreviously
described[22–26].Inbrief,PLACOwassynthesizedbyringopeningpolymerizationofDLlac-
tide(6g)witha1%w/wsolutionofstannushexanoateascatalystincastoroil(14g)ina20mL
ampule.Theampulewasheatsealedandkeptat140oCfor48htoformthedesiredpastypoly-
mer(MW2300).FTIRand1H-NMRspectralanalysisconfirmedthestructureandthe3:7w/w
ratio.Thepoly(ester-anhydride)copolymerofsebacicacid(SA)andricinoleicacid(RA)ata
weightratioof3:7[P(SA-RA)3:7]wassynthesizedbytransesterification,followedbyanhy-
dridemeltcondensation.Inthefirststep,sebacicacid(SA)ispolymerizedtoPSAwithaMW
of20000orhigherbytheuseofaceticanhydrideasactivationagent.TheformedPSAwas
reactedwithricinoleicacid(preparedfromthehydrolysisofcastoroil)ata3:7w/wratio.The
formeddimersandtrimersofRA-SAorRA-SA-RAwerereactedwithaceticanhydridetoacti-
vatethecarboxylicacids,followedbypolymerizationintoapolyanhydrideat160oCundera
vacuumof20mmHgfor7hrs.Theobtainedpolymerwaspastyatroomtemperature,witha
MWof13000.FTIRand1H-NMRspectralanalysisconfirmedthestructureandthe3:7w/w
ratio.Theactiveagentpowderwasgentlymixedwiththepastypolymersatroomtemperature
andloadedinasyringeforfurtherexperimentaluse.
FormulationofAMP-basedbiohybridmedia. Thepeptidepowdersweremixedwiththe
pastypolymertoformahomogeneouspasteataratioof100μgpeptide:100mgpolymer,as
previouslydescribed[27].
Bacterialstrains,celllinesandgrowthconditions
Preparationofbacterialsuspensions. E.faecalis(ATCC#v583),wasculturedovernight
in5mLbrain-heartinfusion(BHI)(Difco,Detroit,MI,USA)brothsupplementedwith2mg/
mLvancomycin(Sigma-Aldrich),at37°Cunderaerobicconditions.S.mutans(ATCC#
27351)wasculturedsimilarlyinBHIbrothsupplementedwith2.77μg/mLbacitracin(Sigma-
Aldrich)and5%glucose(Sigma-Aldrich),A.naeslundii(ATCC#17233)wasculturedinWil-
kins-Chalgrenanaerobebroth(OxioidLtd.,Basingstoke,Hampshire,England)supplemented
with2%sucroseunderanaerobicconditions.P.gingivalis(ATCC#33277)andF.nucleatum
(ATCC#1594)wereculturedinWilkins-Chalgrenbrothunderanaerobicconditions.Thetop
4mLofeachbacterialtubeweretransferredtoafreshtesttubeandtheopticaldensity(OD)
wasdeterminedaccordingtothespecificexperiment.
Antibacterialactivity
Minimalinhibitoryconcentration. Theantibacterialactivityofthelipopeptideswas
determinedbymicrodilutionassayasdescribedbefore[27,28].Inbrief,thebacterialsuspen-
sionwasdilutedandaliquotswereaddedtopeptidedilutionsinphosphate-buffered-saline
(PBS)(Sigma-Aldrich)(intriplicateforeachconcentration)inwellsofa96-wellplate(Nunc
96-wellmicrotiterplates,Roskilde,Denmark).OD(595nm)wasrecordedwithamicroplate
reader(VERSAmaxtunablemicroplatereader,MolecularDevices,Sunnyvale,CA,USA)at
37°Cfor18–24hrs.Theminimalinhibitoryconcentration(MIC)wasdeterminedasthecon-
centration,whichpreventedvisiblegrowthafter18–24hrs.
Antibacterialactivityofsustainedreleaselipopeptides. Theantibacterialactivityofthe
formulationwastestedforoneweekbyaturbidometricassayasdescribedpreviously[27].In
brief,atotal10mgofformulationwasplacedonthesidewallsofeachof6wellsina96-well
microtiterplateand270μlofmediumwereadded.Every24hrsthemediumwascollectedand
transferredtoanewsetof6wellsandfreshmediumwasaddedtothe6originalwellscontain-
ingthetestedformulation.Afteroneweek,a10μlvolumeofE.faecalissuspensionwasadded
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SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
toeachofthe6wellsandbacterialoutgrowthwasrecorded.Theplatewasincubatedat37°Cin
aVERSAmaxmicroplatereaderandturbidity(OD nm)changeswererecorded,every20
650
minfor18–24hrs.
Antibiofilmactivity
Antibiofilmactivitywastestedagainst72hrsbiofilmsformedin96-wellmicrotiterplatesas
previouslydescribed[27].Chlorhexidine(CHX)servedascontrol.Confocallaserscanning
microscopy(CLSM)wasusedtoverifythevitalityofthebacteriainthedifferentbiofilmlayers.
Thebacteriawerestainedwithalive/deadkit(Live/DeadBacLightviabilitykit,Molecular
Probes,OR,USA)asdescribedpreviously[29].
Biocompatibility
HemolysisofRBC. Thetestwasperformedasdescribedpreviously[30],inafinalvolume
of100μLPBScontainingdifferentconcentrationsofthelipopeptidesand100μLsheepred
bloodcells(RBCs)[finalconcentration4%(vol/vol)].Hemoglobinreleasewasmonitoredby
measuringtheabsorbanceofthesupernatantat540nm.Thecontrolsfor0%hemolysis
(blank)consistedofRBCssuspendedinPBS,thatfor100%hemolysiswereRBCssuspendedin
1%TritonX-100.
ColorimetricXTTassay. Cellviabilitywastestedaspreviouslydescribed[31].Inbrief,
mousemacrophagesRAW-246wereculturedovernightinDulbecco'sMinimumEssential
Medium(DMEM,Sigma-Aldrich)supplementedwith10%inactivatedfetalcalfserum(FCS,
BiologicalIndustries,Beit-Ha’emek,Israel),1%L-glutamine(BiologicalIndustries)and1%
streptomycin(BiologicalIndustries),at37°Cin5%CO .Eachformulationofbiodegradable
2
polymerandlipopeptidewithinplasticinserts(Rosenshein,Israel)thatwerepreviouslysand-
blasted,wasaddedtoeightwellsofa96-wellmicrotiterplate.Then200μLofcellsuspension
wereaddedtothewellsandafter24hrstheXTTassay(BiologicalIndustries)wasinitiatedby
theadditionof50μLactivatedXTTsolutiontoeachwell.Themicrotiterplatewasincubated
for2–4hrsandthenmonitoredbymeasuringtheabsorbanceofthesupernatantat450nmina
VERSAmaxmicroplatereader.
Bacterialmembranedisruption
Toevaluatetheeffectofalipopeptideonthebacterialcellmembrane,cytoplasmicmembrane
depolarizationwasmeasuredbytheDiOC (3)assayandfluorescence-activatedcellsorting,as
2
describedbelow.TheBacLightbacterialmembranepotentialkit(MolecularProbes,Invitrogen,
Eugene.OR,USA)providesafluorescentmembranepotentialindicatordye,3,30-diethyloxa-
carbocyanineiodide[DiOC (3)],alongwithcarbonylcyanide3-chlorophenylhydrazone
2
(CCCP).Atlowconcentrations,DiOC (3)exhibitsgreenfluorescenceinallbacterialcells.As
2
itbecomesmoreconcentratedinhealthycellsthataremaintainingamembranepotential,the
dyeself-associatesandthefluorescenceemissionshiftstored.CCCPisincludedinthekitfor
useasapositivecontrolbecauseitisaprotonionophoreanditeliminatesthebacterialmem-
branepotential.Allbacterialsuspensionsamples(1mL)wereincubatedinEppendorftubes
for1hr.Thenthesampleswerefilteredthroughacellstrainer70μL(SPLlifescientific,Korea).
A10μLvolumeofCCCPwasaddedtothecontrolgroupand10μLofDiOC (3)wereadded
2
toeachsample.Thesampleswerekeptatroomtemperaturefor30minbeforeanalysisbyflow
cytometry(BDaccuriC6FlowCytometer,BDBioscience,Becton,DickinsonandCompany).
Whereastherelativeredandgreenfluorescenceintensityvariesaccordingtocellsizeand
aggregation,theratioofredtogreenfluorescenceintensitycanbeusedasasize-independent
indicatorofmembranepotential.ThedatawereanalyzedwithDeNovoFCSExpresssoftware.
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SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Sustainedreleasekinetics
Thereleaseprofileofthelipopeptidefromthebiodegradablepolymerwastestedbythefluores-
camineassayduetoitshighsensibilityandspecificityforprimaryaminogroups,asdescribed
previously[32,33].Inbrief,2mLofassaysolutioncontaining1800μLpeptidereleasedfrom
thepolymerinboricacidbuffer(Sigma-Aldrich)togetherwith200μLoffluorescaminereagent
wereintroducedintoa12X75mmglasstube.A200μLvolumeofformulationwasaddedto
eachassay.Fluorescamine(AldrichChemical,Milwaukee.WI)waspreparedinacetone(Fisher
Scientific)toafinalconcentrationof0.1mg/mLinaglassscrewcaptube.Theassaybufferwas
vortexeduntilthesolutionswerecompletelymixed.Samplesweretransferredtoapolystyrene
cuvettethatwaspreviouslycleanedwithnitricacidfollowedbyseveralrinseswithdeionized
water.FluorescencewasmeasuredatambienttemperaturewithaSpex1680spectrofluorome-
ter(λ =390,λ =460)andatime-basedscanmodewith2secintegrationtime.Themea-
ex em
surementswerecorrectedforlampintensityfluctuationsandforthebackgroundfluorescence
fromasolutioncontainingbufferandfluorescaminesolution.Thefinalamountofpeptide
releasedfromthepolymerwascalculatedaccordingtocalibrationcurvesmadebeforethe
experiment.
Statisticalanalysis
Thedataarepresentedasthemeanandstandarddeviationofarepresentativeexperimentper-
formedintriplicate.MultiplecomparisonswerecalculatedwithStudent'st-test.Thelevelof
significancewasp<0.01.
Results
Antibacterialactivity
Minimalinhibitoryconcentration. TheMICforeachofthetestedlipopeptidesagainst
thetestedmicroorganismsarepresentedinTable1.Thedifferentbacteriashowedvaryingsus-
ceptibilitytothevariouslipopeptides.ThemostpotentlipopeptideagainstE.faecaliscausing
completegrowthinhibitionwasKGGKaspreviouslyshown[27].Themosteffectivelipopep-
tidesagainstS.mutanswereC16-KGGKandC16-KLLK.KKKexhibitedantibacterialactivity
atlowconcentrationsagainstF.nucleatum,P.gingivalisandA.naeslundii.
Sustainedreleaseandantibacterialactivity. Thesustainedreleaseeffectofthelipopep-
tidesobtainedfromthebiodegradablepolymerisshowninFig1.Theantibacterialactivitywas
examinedforthemostpotentlipopeptidesagainsteachpathogen,asshownintheMICexperi-
ment.Lowerfinalopticaldensitiesandmildergrowthcurveslopesforallthebacteriatreated
withtheformulatedpeptideswererecorded.Themostsignificantantibacterialeffectwas
observedbetween24–48hrs,exceptfortheKKKandP(SA-RA)formulation,whereitwasevi-
dentbetween0–24hrs.
Table1. MICsoftheAMPstested[μg/mL].
Aminoacidsequence E.faecalis S.mutans F.nucleatum P.gingivalis A.naeslundii
C16-KGGK CH (CH ) CO-KGGK-NH 4–5 6–12.5 12.5–25 12.5–25 12.5–25
3 214 2
C16-KKK CH (CH ) CO–KKK-NH 6–12.5 12.5–25 4–5 6–12.5 6.25–12.5
3 214 2
C16-KAAK CH (CH ) CO—KAAK—NH 12.5–25 >25 12.5–25 >25 >25
3 214 2
C16-KLLK CH (CH ) CO–KLLK—NH 6.25–12.5 6–12.5 12.5–25 6–12.5 >25
3 214 2
TheunderlinedaminoacidsareD-enantiomers.
doi:10.1371/journal.pone.0162537.t001
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SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Fig1.GrowthinhibitionofbacteriabylipopeptidesreleasedfromP(SA:RA)orfromPLACO.Thetestedformulation100μgpeptide+100
mgpolymer,ratio1:1000wasevaluatedforitsantibacterialeffectevery24hrsduring1week.(A,B)KGGKformulationsagainstE.faecalis.(C,D)
C16-KKKformulationsagainstF.nucleatumand(E,F)KLLKformulationsagainstS.mutans.
doi:10.1371/journal.pone.0162537.g001
Antibiofilmeffect
Theanti-biofilmeffectagainstuni-strainbiofilmsisshowninFig2.P(SA:RA)waseffective
againstalltheuni-strainbiofilms.Formulationscontainingboththelipopeptidesandthebio-
degradablepolymersexhibitedahigherantibacterialeffectthanthenon-formulatedlipopep-
tidesandCHX.
Biocompatibility
HemolysisofRBC. TheresultsofthehemolysisassayarepresentedinFig3.Lipopeptides
C16-KGGK,C16-KKKandC16-KLLKcausedhigh-levelhemolysisatthehigherconcentra-
tionsandlow-levelhemolysisoccurredatlowerconcentrations.
ColorimetricXTTassay. PLACOandP(SA-RA)wereanalyzedwiththeXTTtest.The
viabilityoftheRAWcellsdecreasedsignificantlyfollowingP(SA-RA)exposure,whereasthe
PLACOpolymerdidnotaffectcellviabilitycomparedwiththatofthecontrol(seeFig4A).
Applicationsofallfourformulations(peptideswithPLACO)tothecellsresultedinhighper-
centagesofcellssurvivalwithaminimaldecreaseinviabilityvsthatofthecontrol.The
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SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Fig2.Biofilmgrowthinhibitionbylipopeptideincorporatedinbiodegradablepolymers.Theantibiofilmeffectwasevaluatedwiththeuseof
adead/livedyingkitagainsta72hrformedbiofilm.Thelivebacteriawerestainedwithagreendye,thedeadbacteriawerestainedwithareddye.
ResultsareshownforE.faecalis,F.nucleatumandS.mutans.Thecontrolgroupincluded0.025%CHX.
doi:10.1371/journal.pone.0162537.g002
C16-KKK,C16-KGGKandC16-KAAKformulationsledtolowercellsurvivalthantheformu-
lationcontainingC16-KLLK(seeFig4B).
Bacterialmembranedisruption
Comparedwiththeuntreatedbacteria,contactwithC16-KGGK,increasedbacterialstaining
withDiOC2seenasashifttotheleft(redemission)intheflowcytometrypresentedinFig5A
andindicatingmembranedisruption(seeFig5A).Theratiobetweentheredandgreenemis-
sionwascalculatedforeachtestgroup(seeFig5B).Thetreatedbacteriapresentedlowerratios
vsthecontrol,indicatingthatthebacterialmembranewaspermeated.CCCP,whichwasdesig-
natedtodisruptthecellmembrane,didnotshowdepolarizationactivityagainstthetest
bacteria.
Sustainedreleasekinetics
ThereleaseprofileofC16-KGGKlipopeptidefromP(SA:RA)andPLACOpolymersduring
oneweekisshowninFig6.ThesustainedreleaseofC16-KGGKincorporatedinPLACO
peakedafterabout72hrs,whereasC16-KGGKincorporatedinP(SA:RA)wasreleasedcontin-
uouslythroughoutthisperiod.
Discussion
Thepresentstudyfocusedonantibacterialevaluationoflipopeptidesaspartofasustained
releasetherapeuticmeans.Twobiodegradablepolymerswere:poly(lacticacidcocastoroil)
(PLACO)andricinoleicacid-basedpoly(ester-anhydride)P(SA:RA)(Fig7).Lipopeptides
incorporatedinthebiodegradablepolymerswereeffectiveagainstvariousoralpathogenicbac-
teria.Thedeliveryofpeptidesandproteinsbypolymericcarriersforextendedperiodsoftime
hasbeenachallengebecauseoftheirinstability.Althoughtherearemorethan200peptides
andproteinsinclinicaluseandclinicaldevelopment,thereareonlyafewlong-actingdrug
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SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Fig3.Lipopeptidehemolysisassay.Allfourlipopeptides:C16-KGGK,C16-KKK,C16-KAAKandC16-KLLKweretestedforhemolysisinsheep
RBC,atconcentrationsof5,10,20,50and100μg/ml.InsignificanthemolysiswasdetectedattheMICs.Thecontrolgroupwasconsidered
completehemolysis.
doi:10.1371/journal.pone.0162537.g003
deliverysystems.Luteinizinghormone-releasinghormone(LHRH)andsomatostatindelivery
systemsbasedonpoly(lacticacid),whichdelivertheseagentsformonthsafterasingleinjec-
tion,arestillthemaindeliveryformulations,developedthreedecadesago.Thechallengesof
peptidedeliveryhavebeenreviewedextensively[34,35].Thesenovelformulationshavebroad
applications,fromcancerimmunotherapytodentistry.Thereisawiderangeofcarriers,
includinglipids,liposomes,nanoparticlesandmicelles.Intheoralcavity,modificationofpep-
tidesbyhydrophobicfattyacidresiduesoramphiphilicblockcopolymershasbeenacknowl-
edgedasausefulstrategyforproteindelivery.Inthefieldofdentistry,polymericparticlesand
micellesareapplicableforbindingmineralstothetoothsurface,deliveringAMPsoverapro-
longedperiodoftimeandthusinhibitingthegrowthoforalpathogenbiofilminthepresence
ofthesalivapelliclelayer[36].Thefirstpolymerforthedeliveryherewassynthesizedbyring
openingpolymerizationofDL-lactideontocastoroilthatservedasco-catalystforalcohol
groups.Thesecondpolymerwassynthesizedbyinsertionpolymerizationprocessthatguaran-
teedalternatingester-anhydridepolymerstructure.Thesetwopolymersarepastyandthe
incorporationofvulnerablepeptidesisbygentlemixingwithoutanysolvent,heatorsheer
stress.Consequently,theactivityofthesepeptidicantimicrobialagentswasnotaffectedwhen
incorporatedintothedeliverysystem.
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SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Fig4.PLACOformulationbiocompatibilitybytheXTTassay.MicemacrophagesRAW-246werecultivatedinwellsofa96wellmicrotiterplate.Each
polymerwastested.PLACOexhibitedthehighestcellsurvivability(A).EachformulationcontaininglipopeptideandPLACOpolymerwastested.Allthe
formulationsshowedhighcellsurvivability.C16-KLLKexhibitedthehighestcellsurvivalvstheC16-KKK,C16-KGGKandC16-KAAKformulations(B).
doi:10.1371/journal.pone.0162537.g004
Theworld-widesearchforalternativetherapeuticsagainstantibiotic-resistantvirulent
strainsofmicroorganisms,hasledtothenotionofusingcationicAMPsaspotentialanti-infec-
tivecompounds[5].AMPsareknowntobetheimmunesystem’sfirstdefenselineinmany
organisms[4].Lipopeptides,whicharesynthesizedbybacteria,sharesimilarcharacteristics
suchascationiccharge,amphipathicdesignandtheabilitytopermeatemembranes[11].
Recentstudiesshowthattheyalsohavetheabilitytopenetratethecell,bindtointracellular
molecules(DNA,RNAandvariousproteins)andtherebyinhibitthesynthesisofcell-walls,
nucleicacidsandproteins,aswellastoinhibitenzymaticactivity[37,38].
ToexploittheadvantagesofAMPs,improvedmimeticAMPsweresynthesized[15,16].
Industrialconsiderationsrequirethatthepeptidesbesmallandofsimplestructure.Therefore,
considerableresearchhasbeendevotedtooptimizepeptidelengthcombinedwithasimple
design,suchas:ultra-shortlipopeptides.Theultra-shortlipopeptidesdescribedhere,
C16-KGGK,C16-KKK,C16-KAAKandC16-KLLK,composedofonlyfouraminoacidscon-
jugatedtoanaliphaticacidchain(16C,palmitate),weresynthesizedandtested.Studieshave
revealedthatfattyacidsareabletocompensateforthelengthofashortpeptidechain.Acyla-
tionofsyntheticornaturalAMPswithfattyacidshasprovedtobeausefulapproachfor
improvingtheirantimicrobialandantifungalactivity.Thiseffectisduetochangesintheover-
allhydrophobicityofthesemolecules,whichaffectstheiroligomerization,organizationinsolu-
tionandaffinityformembranes[39].Theabilityofthelipopeptidestooligomerizeandturn
intoaggregatesinsolutionprotectsthemfromproteolyticdegradationwhichcanaffectthe
half-lifeofthepeptideanditsefficacy.Asdescribedindetailpreviously,theinvivoactivityof
theultra-shortlipopeptideswasanalyzedinmousemodelsoffungalinfection.Moreover,one
ofthelipopeptidesprovedmoreefficientthantheknownamphotericinB,atnontoxicdoses
[14].Thepresentstudyfocusedmainlyontheantibacterialeffectoftheselipopeptidesagainst
oralpathogens.
Inthisinvestigation,firsttheMICforeachtestedlipopeptideagainstthebacterialpatho-
genswasdetermined.Eachbacteriumwassensitivetoadifferentlipopeptide.Certain
PLOSONE|DOI:10.1371/journal.pone.0162537 September8,2016 9/16
SustainedReleaseofAntibacterialLipopeptidesfromBiodegradablePolymers
Fig5.Thebacterialmembraneisdisruptedbylipopeptideaftera1hrexposure.Arepresentativeexperimentshowinglipopeptide
C16-KGGKanditsinteractionwiththeE.faecalisbacterialmembrane.Fluorescence-activatedcellsortingwasusedtomeasure
cytoplasmicmembranedepolarizationandtodeterminemembranedisruption.AthighcytoplasmicconcentrationstheDiOC (3)self-
2
associatesandthegreenfluorescenceemission(FL1-Aaxis)shiftstored(FL3-Aaxis).ThebacteriawerestainedwithDiOC (3),
2
exhibitinggreenfluorescence(FL1-A)withashifttoredemissionshiftasthedyemoleculesself-associateatthehighercytosolic
concentrationscausedbythelargermembranepotential(FL3-A).LeftshiftofthebacteriaexposedtoKGGKisshowninthedotplot(A).
Thered/greenratioislowerforthebacteriaexposedtoC16-KGGK,indicatingthatthebacterialmembranewasdisruptedandthus
revealingthelipopeptideantibacterialmechanism(B).
doi:10.1371/journal.pone.0162537.g005
PLOSONE|DOI:10.1371/journal.pone.0162537 September8,2016 10/16