IJN 27709 Improved Drug Loading and Antibacterial Activity of Minocycl 010912

7/31/2019 IJN 27709 Improved Drug Loading and Antibacterial Activity of Minocycl 010912

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InternationalJournalofNanomedicineDovepressopenaccesstoscientificandmedicalresearchOpenAccessFullTextArticleORIGINALRESEARCh

Improveddrugloadingandantibacterialactivityofminocycline-loadedPLGAnanoparticles(button)(button)preparedbysolid/oil/waterionpairingmethod

TaherehSadatJafarzadehKashi1SolmazEskandarion1,2MehdiEsfandyari-Manesh3,4SeyyedMahmoudAminMarashi5NasrinSamadi6SeyyedMostafaFatemi1,7FatemehAtyabi2,3SaeedEshraghi8RassoulDinarvand2,3

1DentalMaterialsDepartment,FacultyofDentistry,2Departmentof

Pharmaceutics,3NanotechnologyResearchCenter,FacultyofPharmacy,TehranUniversityofMedicalSciences,Tehran,4DepartmentofChemistry,AmirkabirUniversityofTechnology,Tehran,5DepartmentofMicrobiologyandImmunology,FacultyofMedicine,BabolUniversityofMedicalSciences,Babol,6DrugandFoodControlDepartment,FacultyofPharmacy,7ResearchCenterofScienceandTechnologyinMedicine,8Department

ofMicrobiology,TehranUniversityofMedicalSciences,Tehran,Iran

Background:Lowdrugentrapmentefficiencyofhydrophilicdrugsintopoly(lactic-co-glycolicacid)(PLGA)nanoparticlesisamajordrawback.TheobjectiveofthisworkwastoinvestigatedifferentmethodsofproducingPLGAnanoparticlescontainingminocycline,adrugsuitableforperiodontalinfections.Methods:Differentmethods,suchassingleanddoublesolventevaporationemulsion,ionpairing,andnanoprecipitationwereusedtopreparebothPLGAandPEGylatedPLGA

nanoparticles.Theresultingnanoparticleswereanalyzedfortheirmorphology,particlesizeandsizedistribution,drugloadingandentrapmentefficiency,thermalproperties,andantibacterialactivity.Results:Thenanoparticlespreparedinthisstudywerespherical,withanaverageparticlesizeof85424nm.Theentrapmentefficiencyofthenanoparticlespreparedusingdifferentmethods


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wasasfollows:solid/oil/waterionpairing(29.9%).oil/oil(5.5%).water/oil/water(4.7%).modifiedoil/water(4.1%).nanoprecipitation(0.8%).Additionofdextransulfateasanionpairingagent,actingasanionicspacerbetweenPEGylatedPLGAandminocycline,decreasedthewatersolubilityofminocycline,henceincreasingthedrugentrapmentefficiency.EntrapmentefficiencywasalsoincreasedwhenlowmolecularweightPLGAandhighmolecularweightdextransulfatewasused.DrugreleasestudiesperformedinphosphatebufferatpH7.4indicatedslowreleaseofminocyclinefrom3daystoseveralweeks.Onantibacterialanalysis,theminimuminhibitoryconcentrationandminimumbactericidalconcentrationofnanoparticleswasatleasttwotimeslowerthanthatofthefreedrug.Conclusion:Novelminocycline-PEGylatedPLGAnanoparticlespreparedbytheionpairingmethodhadthebestdrugloadingandentrapmentefficiencycomparedwithotherpreparednanoparticles.Theyalsoshowedhigherinvitroantibacterialactivitythanthe

freedrug.Keywords:nanoparticle,PEGylation,PLGA,ionpairing,minocycline,antibacterial

Correspondence:RassoulDinarvandFacultyofPharmacy,TehranUniversityofMedicalSciences,TehranPOBox14155-6451,[email protected]

Introduction

Manybiodegradablepolymerssuchaschitosan,gelatin,poly(lactic-co-glycolicacid)(PLGA),polymethylmethacrylate,polycaprolactone,andpoly(lacticacid)areusedforthepreparationofmicroparticlesandnanoparticles.15PLGAisoneofthemostbiocompatibleandbiodegradablepolymers,andithasbeenwidelystudiedforpreparingdrug-loadednanoparticles.6,7Severalmethods,suchasphaseseparationorcoacervation,emulsificationdiffusion,spray-drying,andemulsion-solventevaporationtechniqueshavebeenusedtopreparePLGAnanoparticles.811Usingemulsionsolven

tevaporationmethods,variousdrugmoleculeshavebeenencapsulatedintoPLGAnanoparticles.12,13Forpreparationofnanoparticles,thechoiceofaparticularmethodprimarilydependsonthehydrophilicity/hydrophobicityofthedrugmoleculeand

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2012Kashietal,publisherandlicenseeDoveMedicalPressLtd.ThisisanOpenAccessarticlehttp://dx.doi.org/10.2147/IJN.S27709whichpermitsunrestrictednoncommercialuse,providedtheoriginalworkisproperlycited.


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stabilityconsiderations.Thepatternofdrugreleasefromparticlesdependsonsomeoftheircharacteristics,includingsize,sizedistribution,entrapmentefficiency,anddrugloading.14Thesecharacteristicsareinfluencedbysomeofthepreparationparameters,suchaspoweranddurationofenergyapplied,organicandaqueousphasevolume,polymeranddrugconcentration,polymermolecularweight,andsolventvolume.Eachoftheseparametersinfluencesthesizeand/orthedrugloadingofthenanoparticles.

Inthefieldoforaldiseases,polymeric-baseddrugdeliverysystems,suchasfibers,strips,ormicroparticles,havebeenusedforlocaldrugdeliveryindentistrytoprovideadequatedrugconcentrationsdirectlyatthesiteofaction.Thesesystemsareusuallyinsertedintotheperiodontalpocketorinjectedinperiodontaltissuestobothenhancethetherapeuticeffectsofdrugsandreducethesideeffectsofdrugsrelatedtotheirsystemicuse.Localdeliverysystemshavebeensuggestedasanovelconceptfortreatmentofperiodontaldiseases,especiallyincasesthatarerecurrentandchronic.Severalspecializedlocal

deliverysystemshavebeendesignedforcontrolledreleaseofantibioticsinperiodontaltissues.15,16Thecomplexityofaccesstoperiodontaltissuesmakesallofthesesystemsonlypartiallysuccessful.17Thereareafewstudiesaboutpreparationofantibacterialnanoparticlesforperiodontaltherapy.18,19

Nanoparticlesprovideseveraladvantages.Forexample,becauseoftheirsmallsize,theypenetrateareas(extracellularandintracellularareas)thatmaybeinaccessibletootherdeliverysystems,suchasbacterialcells,alveolarbonetrabeculae,andfromthegingivalsulcusinwardtotheunderlyingconnectivetissueandtotheperiodontalpocketareasbelowthegumline.17,19,20Nanoparticlesprotectadrugagainstinvivodegradation

andreducesideeffects.Theyalsohavemorefavorabledrugpharmacokinetics.21Further,comparedwithmicroparticles,nanoparticleshavebetterstabilityinbiologicalfluids.

Accordingly,nanoparticlescanprovideapotentialperiodontalcarriersystemforthedeliveryofantibioticstoperiodontaltissues.Thesesystemsreducethedosageandfrequencyofantibioticusageandfurtherprovideanadequatesupplyofantibioticsoveranextendedperiodoftime.Differentpropertiesofnanoparticles,suchasparticlesize,entrapmentefficiency,minimuminhibitoryconcentration(MIC),minimumbactericidalconcentrations(MBC),anddrugreleaseinfluencetheclinicaloutcomeofdrugtherapy

viathefollowingmechanisms:

SmallerparticlesizefacilitatesthepenetrationofnanoparticlesandenhancestheirantibacterialpropertiesHigherentrapmentefficiencyofnanoparticlesincreasesthedrugcontentatthesiteofactionLowerMICandMBCachievedwithnanoparticles


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indicatesthatbetterantibacterialactivityisachievedwithasmalleramountofdrugInperiodontaltreatment,ahigherconcentrationofantibioticatinitialtimesandthereafteraconstantreleaseofantibioticwithlowerconcentrationisrequired,whichisachievedwithnanoparticles.Minocyclinehasabroaderspectrumofactivitythanothermembersofthetetracyclinegroupofantibiotics.Itisalong-actingandbacteriostaticantibiotic.Generallyithasserumlevelshigherthanthoseofsimpletetracyclinesbecauseofitslonghalf-life.Minocyclineisusedprimarilytotreatacneandothersimilarskindiseases,butinaccordancewiththebroaderspectrumofactivityofminocycline,italsoactsagainstperiodontalpathogens.Minocycline-loadedmicrocapsuleshavebeeninvestigatedinperiodontaltreatments.2224Ithasbeeneffectivelyusedfortreatmentofperiodontitisandrelatedinfectionsinperiodontaldiseases.2527Themajoradvantageofminocyclineisitsanticollagenasepropertiesandabilitytoreducesofttissuedestructionandboneresorptionwhichisveryimportantinthetreatmentofperiodontaldisease.19Inaddition,minocyclineisagoodcandidateforlocalantibioticdelivery.28,29Inthisstudy,minocyclinewaschosentobeincorporatedintoPLGAnanoparticles.Dueto

thehighhydrophilicityofminocyclineanditsrapidpartitioningtotheaqueousphase,preparationofminocyclinePLGAnanoparticlesremainsarealchallenge.

Developmentofnewmethodsforthedeliveryofhydrophilicdrugsisemergingasanimportantresearchfieldinpharmaceutics.30,31Incontrastwithlipophilicdrugs,therearesomeproblemswithencapsulationofhydrophilicagents.32,33Thecommonlyutilizedmethodsforpreparinghydrophilicdrugsinnanoparticlessufferfromlowdrugloadingbecauseduringpreparationthedrugrapidlydiffusestotheexternalaqueousphase.34However,anotherproblemwithhydrophilicdrugsisthatthedrugparticlesusually

encapsulatedintheformofsmallclustersonthesurfaceorwithinthepolymermatrixjustbelowthesurfaceoftheparticles,resultinginahighinitialburstrelease.35

Inthisstudy,weusedseveralpreparationstrategiestoimprovetheencapsulationefficiencyandloadingofminocyclineasahydrophilicdrug.Water/oil/waterandsolid/oil/wateremulsificationwereusedforthehydrochloridesaltformofminocycline.Theoil/oil,oil/wateremulsification,andnanoprecipitationmethodswereusedforthenon-saltformofminocycline.Wemadesomemodificationstothepreparationmethods,suchasaddinglecithinasanamphiphiliccompoundtothewaterphaseand/ororganic



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phase,orusingPEGylatedPLGA.36PEGcanbeusedasapromisingmaterialinbiomedicalapplications,becauseofitsgoodhydrophilicity,lowtoxicity,excellentbiocompatibility,andbiodegradability.37Boththesolid/oil/waterandionpairingtechniqueswerecombinedinonemethodtoencapsulatehydrophilicdrugs.WehypothesizedthatuseofPEGylatedPLGA(withdifferentmolecularweights)asanamphiphiliccopolymeranddextransulfate(withdifferentmolecularweights)asanionpairingagentcouldresultinabetterencapsulationyieldofcationicmoleculesinnanoparticles.Dextransulfatewasusedtoreducedrugsolubilitybycoacervatingmoleculesthatdiffuseslowlytotheexternalphaseandallowencapsulationofthecoacervateonthepolymericprecipitate.Dextransulfateisapolyanionicderivativeofdextran(apolymerofanhydroglucose)andhasbeenutilizedinvariouspharmaceuticalformulations.38,39Finally,nanoparticleswerecharacterizedandcomparedfortheirsize,morphology,andespeciallydrugloading,drugentrapmentefficiency,andinvitrodrugreleaseprofile.Inaddition,theantibacterialeffectofnanoparticlesagainststandardAggregatibacteractinomycetemcomitans,themost

importantpathogeninperiodontalinfections,wasinvestigatedinvitro.40

Materialsandmethods

Materials

PLGA(Resomer502Hand504H,withalactictoglycolicacidratioof50:50)waspurchasedfromBoehringerIngelheim(Ingelheim,Germany).Polyvinylalcohol(molecularweight30,00070,000),dextransulfatesodiumsalt(molecularweight6000and500,000Da),bifunctionalNH2-PEG-NH2(molecularweightof3350Da),sorbitanmonooleate

(Span80),N-hydroxysuccinimide,anddicyclohexylcarbodiimidewerepurchasedfromSigma-Aldrich(StLouis,MO).Lecithinandhighpressureliquidchromatography(HPLC)gradedimethylformamideandtetrahydrofuranwereobtainedfromMerck(Darmstadt,Germany).MinocyclinewaspurchasedfromKoutingChemicalCo,Ltd,(Songjang,China).Deionizedwaterwasusedthroughouttheexperiment.Brainheartinfusionagar(Merck)wasusedformicrobiologicaltests.Allotheragentsandsolventswereanalyticalorreagentgradeandusedasreceived.

NanoparticlepreparationO/Wemulsiontechnique

OilinwateremulsificationwasperformedaccordingtothemethoddevelopedbyEsmaeilietal,withalittlemodification.41Drugandpolymersolutionwerepreparedbydissolving

150mgminocyclineand350mgofPLGA504Hin20mLofdichloromethaneatroomtemperatureusingamagneticstirrer(Heidolph,Germany).Theorganicphasewasinjectedthroughasyringeequippedwitha20-Gangiocatheterinto75mLofanaqueouspolyvinylalcoholsolutionandhomogenized


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(Ultra-turrax,IKA,Germany)at24,000rpmfor5minutes.Theemulsionwasthensonicated(Misonix,USA)for2minutes(30W).Theresultingnanoemulsionwasmaintainedunderamechanicalstirrer(IKA)undergentlemixingfor4hourstoevaporateofftheorganicsolvent.Afterevaporation,nanoparticleswerecollectedbycentrifugation(Sigma3K30,Germany)at20,000rpmfor20minutesandwashedthreetimeswithdeionizedwatertoremovenonencapsulateddrugandtheremainingsolvent.Thenanoparticledispersionwasfreeze-driedat-40Cfor48hours(Christ,Alpha2-4LD,Germany)toobtainafinepowder.

ModifiedO/Wemulsiontechnique

Phospholipidlecithin20mgwasaddedtothewaterphase,oilphase,andoilandwaterphaseasanamphiphiliccompoundaccordingtothemethodreportedbyCheowandHadinoto.36TheO/Wprocesswascarriedoutaccordingtotheprevioussectionwith100mgofminocyclineand400mgofPLGA504H.

O/Oemulsiontechnique

Oilinoilemulsificationwasperformedaccordingtothemethod

developedbyMahdavietal,withalittlemodification.42Drugandpolymersolutionwaspreparedbydissolving7.5mgofminocyclineand37.5mgPLGA504Hin3mLofacetonitrile.Thissolutionwasaddedinto40mLofviscousliquidparaffincontaining200LSpan80andcontinuouslystirred,yieldingafinelydisperseddrugsuspension.Thesuspensionwasheatedto55Candstirredfor2hourstoensurecompleteevaporationofacetonitrile.Nanoparticleswerecollectedbycentrifugationat20,000rpmfor20minutesandwashedthreetimeswithn-hexanetoremoveresidualmineraloilandSpan80.Thenanoparticledispersionwasthenfreeze-driedfor48hours.

W/O/Wemulsiontechnique

AdoubleemulsionprocesswasperformedaccordingtothemethoddevelopedbyDillenetal,withalittlemodification.43Anaqueoussolutionofminocyclinewaspreparedbydissolving25mgdrugin3mLofdeionizedwater(innerwaterphase).Thesolutionwassonicatedfor30secondsat20Wandthenfor45secondsat35W.Theinnerwaterphasewasinjectedduringsonicationforoneminuteat20Winanorganicphasewhichconsistedof250mgofPLGA504H



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dissolvedin10mLofdichloromethane.TheresultingW/Oemulsionwasdispersedin12.5mLofthefirstouterwaterphase,a0.5%w/vpolyvinylalcoholsolution,andthensonicatedfor30secondsat15WtoobtainamultipleW/O/Wemulsion.Theresultingemulsionwasaddedtothesecondouterphase,consistingof60mLof0.2%w/vpolyvinylalcoholinordertominimizecoalescenceoftheemulsion.Theorganicsolventwasallowedtoevaporateduring4hoursatroomtemperatureundergentlemixing.Finally,thenanoparticleswerecollectedbycentrifugationandfreeze-driedafterthreetimeswashingwithdeionizedwater.

S/O/Wemulsiontechniquebyionpairingmethod

ThesynthesisschemeforPEGylatedPLGAisshowninFigure1.PEGylatedPLGAwaspreparedaccordingtothemethoddevelopedbyEsmaeilietalwithalittlemodification.8Briefly,2gPLGAdissolvedindichloromethanewasactivatedbydicyclohexylcarbodiimide(207mgforPLGA502Hand115mgfor504H)andN-hydroxysuccinimide(414mgforPLGA502Hand230mgfor504H)atroomtemperature

underanitrogenatmospherefor24hours.Theresultingmixturewasfilteredandprecipitatedbyadditionofice-colddiethylether.TheactivatedPLGAwasdriedundervacuumandthenreactedwithbis-aminePEG(200mgforPLGA504Hand600mgfor502H)in16mLofdichloromethane.Stoichiometricmolarratiosof1/1.5and1/1.1wereusedforPLGA504H/bis-aminePEGandPLGA502H/bis-aminePEG,respectively.Thereactionwasperformedfor6hoursundernitrogenatmosphereatroomtemperature.Theresultingmixturewasprecipitatedbydroppingitintoice-colddiethylether.TheprecipitatedPEGylatedPLGAwasfiltered,dialyzed,anddried.H-NMRspectraforPEGylatedPLGA

O

wereobtainedusingtheBrukerAvence500MHz,inCDCL3.(Figure2).TheconjugationpercentageofterminalcarboxylicacidofPLGAwithbis-aminePEGwas100%(H-chemicalshiftCH,CH2,andCH3ofPLGAandCH2ofPEGare5.2,4.8,1.6,and3.7cm-1,respectively).

ThemethodusedforpreparationofPEGylatedPLGAnanoparticleswasacombinationofS/O/Wandtheionpairingtechnique.WepreparedfournanoparticleformulationsusingtwokindsofPLGA(502Hand504H)anddextransulfatewithtwomolecularweights(6000and

500,000).A0.7mLaqueoussolutionofminocyclineHClwasinjectedinto10mLofacetonesolutioncontaining200mgPEGylatedPLGAduringsonication(40W)for60seconds.A0.3mLaqueoussolutioncontaining24mgdextransulfatewasaddedandsonicatedfor30seconds.Theresultingsolidinoilphasewasaddedto60mLofcontinuousphasecontaining0.5%polyvinylalcoholassurfactantunderhomogenation(24,000rpmfor5minutes).Theresultingnanoemulsionwasmaintainedundergentlemixingfor3hours.Consequently,thenanoparticleswere


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collectedbycentrifugationandthenfreeze-driedafterthreetimeswashingwithdeionizedwater.

Nanoprecipitationtechnique

NanoprecipitationprocesswasperformedaccordingtothemethoddevelopedbyBilatietal,withalittlemodification.44PLGA504H50mgandminocycline20mgweredissolvedin2mLofdimethylsulfoxidetoformthediffusingphase.Thisphasewastheninjectedto10mLofethanolasanon-solventundergentlemixing.Theresultingnanoparticleswerethencentrifugedthreetimesforcyclesof20minutesat20,000rpmandwashedwithdeionizedwater.

HO

OOx-1y-1PEG-bis(amine)PLGADCCNHSOO

OOOOnNHOCH3OHnNH2H2NxyO

O+NH2

CH3OCH3OH

PEGylatedPLGA

Figure1SynthesisofPEGylated-PLGAcopolymer.Abbreviations:PEG,poly(ethylene)glycol;PLGA,poly(lactic-co-glycolicacid).



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Particlesizeandmorphologycharacterization

Particlesizeandsizedistributionofthenanoparticleswereinvestigatedbylaserlightscattering(MalvernZetasizerZS,Malvern,Worcestershire,UK),aftersuspending5mgofthenanoparticlesin20mLofdeionizedwater.Threedeterminationswerecarriedoutforeachformulation.Morphologicalcharacterizationwasconductedusingscanningelectronmicroscopy(30XLFEG,Philips,Eindhoven,TheNetherlands).Scanningelectronmicroscopywasemployedtodeterminetheshapeandsurfacemorphologyofthenanoparticles,andtheparticleswerecoatedwithgoldusingasputtergoldcoater(BAL-TEC,Switzerland)undervacuumbeforehand.

Differentialscanningcalorimetry

Differentialscanningcalorimetry(DSC)ofminocycline,andemptyandminocycline-loadednanoparticleswereperformedonaMettlerDSC823(MettlerToledo,

Switzerland)equippedwithaJulaboThermocryostateModelFT100Y(JulabolabortechnikGmbH,Germany).AMettlerStarsoftwaresystem,version9.0wasusedfordataacquisition.DSCmeasurementswereperformedataheatingrateof10C/minuteinthe20C350Ctemperaturerange.Thedrynanoparticleswereweighed,putintoanaluminumpan,andsealedcarefully.Duringmeasurement,thesamplecellwaspurgedwithnitrogengas.Calibrationoftheinstrumentwasperformed.

Determinationofdrugloading

andentrapmentefficiency

TheamountofminocyclineentrappedinthenanoparticleswasdeterminedbyHPLCanalysis.Theminocycline-loadednanoparticles(20mg)weredissolvedin5mLacetonitrileand10mLofmethanolwasthenaddedtoprecipitatethepolymer.Thisprocedurewasperformedusing10mLofdeionizedwaterand10mLofdimethylformamidefortheminocycline-loadednanoparticles.Thesampleswerepassedthrougha0.22mMilliporemembraneandtheamountofdrugwasdeterminedbyHPLCanalysis.Thedrugencapsulationefficiencywasdeterminedfromthemassratiooftheencapsulateddrugtotheamountofdruginitiallyadded.The

amountofdrugloadingandencapsulationefficiencywerecalculatedusingthefollowingequations:42

Drugloading(%)=(minocyclineweightinsample/totalweightofsample)100%

Encapsulationefficiency=(actual/theoreticalminocycline


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loading)100%

Invitrominocyclinereleasefromnanoparticles

Drugreleasefromthenanoparticleswasstudiedusingadialysistechnique.A20mgsampleofnanoparticleswasresuspendedin5mLofphosphatebuffersolutionatpH7.4andplacedinadialysisbag(Spectra/Por,molecularweightcutoff2000Da)sealedatbothendswithclips(Spectra,Torrance,CA).Thedialysisbagwassoakedin40Lofphosphatebuffersolution(pH7.4)andmaintainedat37C0.5Cand1005rpmshakinginashaker(HeidolphUnimax1010,Germany).Atpredeterminedtimeintervals,individualsamplesweretakenandthewholeofthemediumwasreplacedwith40mLoffreshphosphatebuffersolution.TheamountofminocyclinereleasedintoeachmediumwasquantifiedbyHPLCandcomparedwithastandardcalibrationcurvegeneratedusingknownconcentrationsofminocycline.

Theconcentrationofminocyclinewasanalyzedusinga

modifiedUSPHPLCmethod.HPLCanalysiswasperformedatroomtemperatureusingaKnauerapparatusmodelK-1001,WellChrom(Berlin,Germany),equippedwithamodelPDAK-2700ultravioletdetector(Knauer,Germany).TheanalyticalcolumnwasNucleodurC18(250.46cminternaldiameter,poresize5m;Macherey-Nagel,Dren,Germany).Themobilephaseconsistedof0.1Mammoniumoxalate,0.005Medetatedisodium,dimethylformamide,andtetrahydrofuran

(600:180:240:160v/v)andadjustedwithammoniumhydroxidetoapHof7.2.Theflowratewasfixedat1.5mLperminuteandultravioletdetectionwasperformedat280nm.Theretention

timeofminocyclinewasabout7.460.2minutes.Theconcentrationofminocyclinewascalculatedforeachsampleusingacalibrationcurveofknownamountsofdrug(withalinearregressioncoefficientofR=0.999).Antibacterialpropertiesofminocycline-loadednanoparticles

TheantibacterialactivityofthenanoparticleswascomparedwiththatoffreeminocyclinebythewelldiffusionmethodusingAggregatibacteractinomycetocomitans(43718,AmericanTissueCultureCollection,Vanassus,VA).

Thesurfaceofthebrainheartinfusionagarplatessupplementedbyhemin,vitaminK,bovineserumalbumin,andsheepbloodwereseededbythebacterialsuspensionatacelldensityequivalentto0.5McFarland(1.5108CFU/mL).Theantibacterialagentwassterilizedbyfiltrationusing

0.22mMilliporemembranes.Wellswith8mmdiameterswerepreparedbypunchingasterilecorkborerontotheagarplatesandremovingtheagartoformawell.Aliquots


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of80Lofeachtestcompoundsolution(3.5g/mLsubmityourmanuscript|www.dovepress.com


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5.75421.0000-CHofPLGA-CH2ofPLGA-CH2ofPEG-CH3ofPLGA2.50624.91929.04477.63155.24615.19904.87584.86053.50453.43813.40453.36833.33263.29702.75542.63462.59042.53942.50622.50332.49992.49642.49282.36092.0840147371.46524.90915.26011050[ppm]

Figure2h-NuclearmagneticresonancespectrumofsynthesizedPEGylatedPLGAinCDCL3.

Abbreviations:PEG,poly(ethylene)glycol;PLGA,poly(lactic-co-glycolicacid).

minocyclinepowderindeionizedwater)weredeliveredintothewells.After48hoursofincubationat37C,theinhibitionzonesaroundthewellsweremeasuredinmillimetersusingacaliper.

TheMICandMBCofthetestcompoundsweredeterminedusingthebrothmacrodilutionmethod.Astockconcentration

offreedrugwaspreparedindeionizedwaterthatwasfurtherdilutedinbrainheartinfusionbrothtoreachaconcentrationrangeof0.125to32g/mL.Basedontheactualdrugloadingofthenanoparticles,theamountofnanoparticlesinbrainheartinfusionbrothwasusedtoprovidetheequivalentconcentrationofminocyclinesimilartothatoffreedrug.Thefinalconcentrationofbacteriaintheindividualtubeswasadjustedtoabout5105CFU/mL.ControltubescontainedPEGylatedPLGAnanoparticleswithoutdrugandwithnoantibacterialagent.After48hoursofincubationat37C,thetesttubeswereexaminedforpossiblebacterialturbidity,andtheMICofeachtestcompound

wasdeterminedasthelowestconcentrationthatcouldinhibitvisiblebacterialgrowth.AfterMICdetermination,analiquotof10Lfromalltubesinwhichnovisiblebacterialgrowthwasobservedwasseededinbrainheartinfusionagarplatesnotsupplementedwithanyfreeminocyclineorminocyclineloadednanoparticles.Theplateswerethenincubatedfor48hoursat37C.TheMBCendpointisdefinedasthelowestconcentrationofantimicrobialagentthatkills.99.9%oftheinitialbacterialpopulationwherenovisiblegrowthofthebacteriawasobservedontheplates.16,41

Statisticalanalysis

Resultswereexpressedasthemeanstandarddeviation.One-wayanalysesofvariancewereperformedforevaluationoftheresults.Pvalueslessthan0.05wereconsideredtobestatisticallysignificant.

Results

Thebasiccharacteristicsoftheminocycline-loadednanoparticlespreparedusingvariousmethodsinthisstudyare


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presentedinTables1and2.

Particlesizeandmorphology

AllofthenanoparticlespreparedfromPLGAandPEGPLGAundervariousconditionshadanacceptablesize(lessthan500nm)andweresuitableforourfinalclinicalpurposesexceptforthenanoparticlespreparedusingtheoilinoilemulsionmethod(OO5).Themeanparticlesizes(z-average)ofallthesamplesareshowninTable1.Therangeofnanoparticlesizewas857070nm.ItcanbeseenthatthesizeofthenanoparticleswasthesmallestandlargestwhentheywerepreparedusingnanoprecipitationandtheO/Oemulsionmethod,respectively.AtypicalscanningelectronmicrophotographofnanoparticlesisshowninFigure3.Theminocycline-PLGAnanoparticleswerewelldefinedandspherical,andhadasmoothsurfacewithoutpores.Nanoparticlespreparedbythenanoprecipitationmethodhadthenarrowestsizedistributionrangewithapolydispersityindexof0.08(Figure4A).Nanoparticlespreparedbythe



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Table1PreparationofPLGA-basednanoparticleswithdifferentmethodsandtheirparticlesize

SamplePreparationmethodMeandiameter(nm)OW1Oil/water19314OW2ModifiedLecithininwater22523OW3oil/waterLecithininoil21516OW4Lecithinintwophases24221OO5Oil/oil7070208WOW6Water/oil/water42417SOW7Solid/oil/waterPLGA(MW48,000)+20919byionpairingDS(MW6000)SOW8PLGA(MW48,000)+22615DS(MW500,000)SOW9PLGA(MW12,000)+13912DS(MW6000)

SOW10PLGA(MW12,000)+18619DS(MW500,000)NPC11Nanoprecipitation857

Abbreviations:DS,dextransulfate;PLGA,poly(lactic-co-glycolicacid);MW,molecularweight.

S/O/Wionpairingmethodshowedasomewhatgoodsizedistributionwithapolydispersityindexof0.2(Figure4B).

Drugloadingandentrapmentefficiency

Table2displaysthedrugloadingandentrapmentefficiencyforallthesamplespreparedusingvariousmethods.Drugloadingrangedfrom2.6to19.2g/mg.Theleastdrugloading(0.26%)andentrapmentefficiency(0.81%)wasfoundinnanoparticlespreparedbythenanoprecipitationmethod.SignificantlyincreaseddrugloadingandentrapmentefficiencywasobtainedbytheS/O/Wionpairingmethod(1.92%and29.95%).Inclusionofdextransulfateintothepreparations(SOW710)significantlyloweredthepercentageoffreeminocyclinecomparedwiththosewithoutdextransulfate

Table2Drugloadingandentrapmentefficiency

SampleDrugloading(%)SDEntrapmentefficiency(%)SDOW10.410.023.850.16OW20.910.034.140.03OW30.310.011.480.28OW40.480.022.150.13OO50.980.045.480.30WOW60.440.014.690.21SOW70.460.0157.691.32SOW80.780.03513.092.40


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SOW91.320.1222.234.70SOW101.920.1929.952.47NP110.260.060.810.18

Abbreviation:SD,standarddeviation.

500nm

ACCVSpotMagnDetWD

17.0kV2.030,000xSE9.5SI200nm

ACCVSpotMagnDetWD

17.0kV2.060,000xSE9.6SIFigure3AtypicalscanningelectronmicrophotographofnanoparticlespreparedbyS/O/Wionpairingmethod,SOW10(upperandlowerimageispresentedat30,000and60,000magnification,respectively).

(WOW6)andimproveddrugloadingcomparedwithotherformulations.NanoparticlespreparedbytheS/O/Wionpairing

method,withthehighestentrapmentefficiency,wereusedforotheranalyses.

DSCanalysis

DSCthermogramsofminocyclineandtheemptyandminocycline-loadednanoparticleswereobtainedtodefinethephysicalstateofthedruginthenanoparticlesandthermalpropertiesofthepolymernanoparticles.Figure5showstheDSCthermogramsforminocycline,PLGA-PEGnanoparticles,andminocycline-PLGA-PEGnanoparticles(SOW10),respectively.Pureminocyclineshowedanexothermicpeakat200C.Therewasnopeakobservedatthistemperatureforthe

nanoparticles.DSCstudiesdidnotdetectanyfreeminocyclineinthenanoparticlesamples.Thisconfirmedthemoleculardispersionofminocycline.Afterpreparationthenanoparticles,minocyclinecouldbeinanamorphousphaseofamoleculardispersionorinasolidsolutionstateinthepolymermatrix.

Invitrodrugrelease

TheinvitroreleasebehaviorofallnanoparticlesisascumulativepercentagesinFigure6.Releaseover5days



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Intensity(%)

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B

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110100Size(d.nm)100010,000110100100010,000

Intensity(%)

14

121086420

Size(d.nm)

Figure4Particlesizedistributionsofnanoparticlespreparedby(A)S/O/Wionpairingmethod(SOW10)and(B)nanoprecipitationmethod(NP11).

wasmeasured.Releaseprofilesfornanoparticlesinphosphatebuffersolution(pH7.4)wereaffectedbythepreparationmethodsandformulationsused.Theinitialburstreleasewasdetectedforallformulationsduringthefirst7hours(morethan20%)exceptforsamples1,2,5,and6.TheSOW9formulationshowedasignificantlyhigherburstrelease(72%in7hours)comparedwiththeotherformulations.Therateofdrugreleasegraduallydecreasedafterabout12daysandremainedconstantevenafter5days(exceptforsample9).


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AsshowninFigure6,drugreleasefromthenanoparticlespreparedbyO/Owasslowerthanfromtheothernanoparticles(approximately10%in5days).

Antibacterialpropertiesofminocyclinenanoparticles

Theminocycline-loadednanoparticles,freeminocycline,andemptynanoparticlesweretestedagainstAggregatibacteractinomycetemcomitansinvitrotocomparetheirantibacterialactivity.Inthecaseofminocycline-loadedPLGA-PEGnanoparticles,theSOW10samplehadthehighestentrapmentefficiency.Awelldiffusionassayshowedthattheinhibitionzoneofminocycline-loadednanoparticles(9.2mm)wasgreater

thanthatoffreeminocycline(3.5mm)andemptyPLGAnanoparticles,whichdidnotshowanyinhibitoryeffect.TheMICsofthesamplesareshowninTable3.TheMICofminocycline-loadednanoparticles(4g/mL)wastwo-foldlessthanforfreeminocycline(8g/mL).TheMBCofminocyclineloadednanoparticles(8g/mL)wasalsotwo-foldlessthanthatforfreeminocycline(16g/mL).Therefore,itcanbeseenthattheantibacterialactivityofminocycline-loadednanoparticleswasgreaterthanforthefreedrug.Emptynanoparticles

dilutedwithbrainheartinfusionbrothasacontroldidnotshowanyantibacterialeffectagainstthebacteriatested.HenceitcanbeconcludedthattherewasnoantibacterialinteractionbetweenPLGAandthetestedbacteria.

Discussion

Thechoiceofasuitablenanoparticlepreparationmethodisdependentonthephysicochemicalpropertiesofthedrugtobeencapsulated.Differentmethodshavebeensuccessfullyusedfortheentrapmentoflipophilicdrugsintonanoparticles.33,45Themainprobleminthepreparationofminocycline-loadednanoparticleswasthelackofdrug

loadingandentrapmentefficiencybecauseminocyclineisa



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051015mW20MinocyclinePLGANPsMinocycline-PLGANPs20406080100120140160180200220240260280300320340

Temperature(C)

Figure5DSCthermogramsofminocycline,PLGA-PEG,andminocycline-PLGA-PEGnanoparticles.Abbreviations:DSC,differentialscanningcalorimetry;PEG,poly(ethylene)glycol;PLGA,poly(lactic-co-glycolicacid).

AB

Releaseofminocycline(%)

10080604020000

OW1OW2OW3OW4OO5WOW6100806040200Releaseofminocycline(%)..

..................

..

..

..

..

................

......

....

....

..

..

..

..

..

..0204060801001201400


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20406080100120140

Time(hours)Time(hours)

C

Releaseofminocycline(%)

100

80

60

40

20

0

SOW7SOW8SOW9SOW10020406080100120140..................

....

..

..

..

..

..

..Time(hours)

Figure6Invitrocumulativereleaseofminocyclinefromnanoparticles(A)OW1,OO5,andWOW6;(B)modifiedO/W(OW2,OW3,andOW4),and(C)S/O/Wionpairingmethod(SOW710),inphosphatebuffersolution(ph7.4,37C).



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Table3MICandMBCoffreedrugandminocycline-loadednanoparticlesagainstAggregatibacteractinomycetemcomitansbymacrodilutionmethod

Freedrug(g/mL)Minocycline-loadednanoparticles(g/mL)MIC84MBC168

Abbreviations:MIC,minimuminhibitoryconcentration;MBC,minimumbacterialconcentration.

hydrophiliccompoundandthereisaconcernaboutincreasingthedrugloadingofhydrophilicagents.35However,inpractice,preparationofnanoparticleswiththedesiredproperties(adequateentrapmentefficiencyanddrugloading,suitablereleaseprofile,andparticlesizedistribution)canbedifficultduetothelargenumberoffactorsinfluencingtheoutcomeofnanoparticlepreparation.Inthisstudy,severalexperimentswerecarriedouttodeterminethebestmethodandtheformulawiththehighestantibioticloadingofPLGAnanoparticles.

Nanoparticlespreparedbysolventdiffusionmethods(OW1andWOW6)showedlowentrapmentefficiencyanddrugloadingbecauseminocyclinerapidlydiffusedfromthehydrophobicmatrixintotheexternalaqueousphaseduringpreparation.46Comparedwiththesolventdiffusionmethod,betterentrapmentefficiencyanddrugloadingwereobtainedbytheO/Oemulsionevaporationmethod(OO5).Thiswascontributedtobytheliquidparaffinusedasacontinuationphasewiththismethod.42Thelimitedsolubilityofminocyclineandnildiffusionoffirstoilintoliquidparaffinwereadvantageoustorestrictminocyclineleakageincomparisonwithotheraqueoussolventdiffusionmethods.Polymeric

particlesobtainedbythismethodhadthelargestparticlesize.AnincreaseofparticlesizewiththeO/Omethodmighthavebeenduetothehighviscosityofthecontinuousphasewhichhindersthehomogenousdispersionofpolymersolutionintothecontinuousphase.Particlesizeiskeytothebiologicalfateofnanoparticlecarriers.Decreasingsizeimprovesthepermeationandpenetrationofnanoparticlecarriers.Therefore,nanoparticlesshouldhaveanappropriatesizetopenetratethebacterialcellwall.Inclusionoflecithinintotheaqueousphase(OW2)usingtheO/Wmethodledtoimprovementinentrapmentefficiencyanddrugloadingcomparedwiththestandardmethod(OW1).Inthisregard,addinglecithinhasbeenshowntoresultinsurfaceadsorptionofamphiphilic

lecithinontonanoparticlesthroughhydrophobicinteractions.36Minocyclineinteractswiththehydrophobicphospholipidtailsoflecithinsimilartolecithin-PLGAinteractions.Incorporatinglecithinontothenanoparticlesurfacecanenhanceentrapmentefficiencybytrappingminocyclinein

thephospholipidlayer.However,inclusionoflecithinintheoilphase(OW3)andbothphases(oilandwater)(OW4)hadthereverseeffectonentrapmentefficiencyanddrugloading.Lecithinadsorptionontothenanoparticlesurfacecauseda


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minorincreaseinparticlesize(about10%20%).

Nanoparticlespreparedbythenanoprecipitationmethodwereconsiderablysmaller(807nm)thannanoparticlespreparedbytheemulsionmethod.ThesizedistributionofnanoparticlespreparedusingthismethodisshowninFigure4A,whereahighlyuniformsizedistributionisobserved(polydispersityindex0.08).Briefly,therearetwomisciblesolventswhenusingthismethod.Drugandpolymerdissolveinthefirstoneasthesolvent.Nanoprecipitationoccursbyrapiddiffusionandaprecipitateofthepolymerwhenthefirstpolymersolutionisaddedtothesecondphaseasanonsolvent.44Precipitationinvolvesimmediatedrugentrapment.Nanoparticlespreparedbythismethodshowthelowestdrugloadingandentrapmentefficiencycomparedwiththeothermethods(Table2).Becauserapiddiffusionofdrug-polymersolutionintothesecondphaseincreasedtheamountoffreedruginthesecondphase,thismethodwasunabletoretainsubstantialamountsofminocyclinetoincorporateintothepolymermatrix.

HighentrapmentefficiencyusingtheS/O/Wionpairingmethodoccurredasfollows.Thismethodwasacombination

ofS/O/Wemulsionandionpairingtechniques(Figure7).AnaqueoussolutionofminocyclinewasaddedtoanorganicsolutionofPEGylatedPLGA.Additionofaqueousdextransulfatesolutionproducedaminocycline-dextransulfateprecipitate(solidinoil).Dextransulfatewasusedasanionpairingagenttoreducedrugsolubilitybycoacervation.Theresultingsolidintheoilphasewasthenaddedtotheaqueouspolyvinylalcoholsolution.Theorganicsolventwasremovedbyevaporationunderstirringtoproducenanoparticles.Theelectrostaticforcesformedbetweenoppositeions,cationicminocyclineandanionicdextransulfate,andcationicPEGylatedPLGAandanionicdextransulfate.Dextransulfateasanionpairingagentplayedaroleasanionicspacerbetween

PEGylatedPLGAandminocycline.Ifionpairingformationisfasterthanpolymerprecipitation,theionpairscouldbetrappedduringparticulateprecipitation.Intheabsenceofdextransulfate(WOW6),theinteractionsbetweentheterminalgroupofthepolymerandminocyclineweremostlydirectedbyweakvanderWaalsforces.AccordingtotheH-NMRresults,thePEGcontentinlowmolecularweightPLGA(502H,molecularweight12,000)wasapproximatelyfourtimesmorethanthehighmolecularweightPLGA(504H,molecularweight48,000).Soahighamountofion



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Solventdiffusion-evaporation

Minocycline

PEGylatedPLGAS/O/WemulsificationMECHANICALSTIRRER300rpmULTRADextransulphateTURRAXON24,000rpmOFFONOFFSolidificationS/OemulsionW/OemulsificationULTRASOUNDONPVAaqueoussolutionDSOFF

ULTRASOUNDaqueoussolutionMinocyclineONOFFW/OemulsionaqueoussolutionPEGylatedPLGAoilphase

Figure7SchemeofcombinationofS/O/Wemulsionandionpairingtechniques.

pairswasformedinlowmolecularweightPEGylatedPLGA,andhencedrugentrapmentwasincreased(SOW9andSOW10).

Thesignificantlyincreasedentrapmentefficiencyanddrugloadingusinghighmolecularweightdextransulfate(500,000)indicatesthattheadditionalnegativechargesofdextransulfatehavefurtherincorporatedminocycline(about2.32.9times).Thiswasduetoanionpairingmechanismwhichwasmentionedearlier.ImprovedinteractionbetweenthedrugandpolymerandthusentrapmentefficiencywasobtainedbyincreasingPEGylationandahighmolecularweightofdextransulfate.

Thesizeofthenanoparticlespreparedbydifferentdoubleemulsionmethodswasasfollows:WOW6(424nm).SOW7(209nm).SOW9(139nm).ThereductioninparticlesizecouldbelargelyduetothedecreaseinPLGAmolecularweight(SOW9)andincreaseinamphiphilicpropertiesofthepolymerwithincreasingPEGcontent(SOW7and9).ThesepolymerswithhigherPEGcontentreduceinterfacial


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tensiontofacilitatenanoparticleformation,andresultina

smallerparticlesize.47Anapproximatelynarrowsizedistributionwasalsoobserved(Figure4B)forthenanoparticlespreparedbyS/O/Wmethod(polydispersityindex0.2).

Thereleasekineticsofminocyclinefromthenanoparticleswasideal,asusuallyaninitialburstreleaseinthefirst24hoursfollowedbyacontrolledpatternofdrugreleaseisclinicallyfavorable.25,48,49Inmostofthenanoparticlespreparedinthiswork(OO5,WOW6,SOW79,NP10),weobservedlinearminocyclinereleaseratesafterthebursteffectformorethan5days,andthisprofilecouldbeidealforperiodontaldisease.50Theinitialburstcouldbeascribedtotheminocyclinedistributedatorjustbeneaththesurfaceofthenanoparticles.Thelaterconstantreleaseismainlyduetodrugdiffusionandmatrixerosionmechanisms.Theminocyclinereleaseratefromnanoparticlesin24hourswasasfollows:

OW1(18%).WOW6(14%).OO5(6%).FromthesustaineddrugreleaseofOO5,itcanbeassumedthat

minocyclinewaswelldispersedinthepolymermatrix,whichallowedregularreleaseofminocyclinewithpolymerdegradation.Thelargesizeoftheparticlessubmityourmanuscript|www.dovepress.com


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(OO5.WOW6.OW1)decreasedthesolid-liquidcontactanddrugdiffusionthroughthepolymericwall.

OW3(59%).OW4(49%).OW1(18%).OW2(14%).Theeffectoflecithinonthereleaseratewassimilartoitseffectonentrapmentefficiency.InsampleOW2entrapmentefficiencywasimprovedandthereleaseratewasreduced.Conversely,insamplesOW3andOW4entrapmentefficiencywasdecreasedandthereleaseratewasenhanced.SOW9(91%).SOW10(72%),andSOW7(38%).

SOW8(29%).Nanoparticlespreparedusingdextransulfate(molecularweight500,000)showedaslowerreleaseratethandextransulfatealone(molecularweight6000).Thismaybeduetoahighernegativechargerelatedtodextransulfate500,000thatinducedbetterinteractionwiththecationicdrugandwhichcouldincorporatefurtheramountsofdrugontheinsideaswellasthesurfaceofthenanoparticles.SOW10(72%).SOW8(29%),andSOW9(91%).SOW7(38%).Useoflowmolecularweight

PLGAinSOW9andSOW10increasedthediffusionofminocyclinethroughthenanoparticlewall.51NanoparticleswiththehighestPEGcontenthavemoreamphiphilicandhydrophilicproperties.Thesepropertiesenhancemoleculardiffusionintoaqueoussolution.SOW7(38%).WOW3(14%).Asmentionedearlier,nanoparticlespreparedbytheS/O/Wionpairmethodhavemoreamphiphilicandhydrophilicpropertiesandsmallerparticlesize.Thesepropertiesenhancediffusionandsurfacedesorptioninaqueoussolution.Thermalstudieswereusedtoinvestigatethephysical

stateofminocyclineinthenanoparticles,becausethispropertycouldinfluencedrugreleasefromthenanoparticles.Drugmaybepresenteitherasamorphousorcrystallineinanamorphousorcrystallinepolymer.Figure3showstheDSCthermogramsforpureminocycline,andforemptyandminocycline-loadednanoparticles.Polymershowsaglasstransitiontemperatureat50Canddoesnothaveameltingpointtemperature,meaningthatitisanamorphouspolymer.Twopeaksinthetemperaturerangeof150C170Cmayberelatedtoresidenceofpolyvinylalcohol.Pureminocycline


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showedanexothermicpeakat200Crelatedtothemeltingpointwithdecompositiontransition.Minocyclinemeltingwithadecompositionpeakwasdepletedinthethermogramfortheloadednanoparticles,indicatingthepresenceofamorphousminocyclineinthenanoparticles.Itmaybehypothesizedthatcrystallizationofminocyclineisinhibitedduringproductionofnanoparticles.Therefore,minocycline

inthenanoparticlesisintheamorphousphaseofamoleculardispersion.Theantibacterialpropertiesofminocyclineloadednanoparticlesweredeterminedinvitro;theantibacterialactivityofthenanoparticleswasremarkableandmainlyattributedtothehighantibacterialeffectofthenanosizedparticles(Table3).EncapsulationofminocyclineintonanoparticlesimprovedtheantibacterialefficiencyofminocyclineagainststandardAggregatibacteractinomycetemcomitansbytwo-fold.Becausefreedrugandnanoparticleswereinvestigatedusingthesameconcentrationofminocycline,theimprovementinantibacterialeffectactivitybeduetobetterpenetrationofthenanoparticlesintobacterialcellsandbetterdeliveryofminocyclinetoitssiteofaction.7Nanoparticlesarecapableofbeingendocytosedbyphagocyticcellsandreleasingdrugintothosecells.52,53Theminocycline-loadednanoparticlescouldbe

suitablefordeliveryofminocyclinetophagocyticcellstoachievebettertreatmentofinfectioncomparedwithtreatmentusingfreeminocycline.Thisindicatesthatthenewlydesignedantibiotic-releasingnanoparticlesmaybeappropriateforperiodontaltreatment.

Conclusion

Effectiveentrapmentofdrugsthatarehighlysolubleinbothaqueousandorganicsolventsisdifficulttoachieveusingstandardapproaches,suchassingleanddoubleemulsificationsolventevaporationandnanoprecipitationmethods.Inthisstudyweusedanovelmethodforthepreparationofminocycline-

loadednanoparticlesbyapplyinganionpairingtechniqueusingsolid/oil/wateremulsification.OurresultsdemonstratethatusingPLGA-PEG-dextransulfateforionpairingsignificantlyincreasesminocyclineloading,andproducesnanoparticleswiththedesiredsize,sizedistribution,andmorphologicalproperties.BydecreasingthemolecularweightofPLGAandincreasingthemolecularweightofdextransulfate,thedesireddrugcontentcanbeobtainedbecauseofthehighPEGcontentandlowdruginsolubility,respectively.PEGylatedPLGA(molecularweight12,000)anddextransulfate(molecularweight500,000)wasthepreferredchoiceforionpairingbecauseitallowedthehighestdrugloadingandentrapmentefficiency.Aninvestigationof

differentpreparationmethods,physicochemicalcharacterization,invitroreleasetesting,andantibacterialpropertiesofthenanoparticleswascarriedout.Releaseratesvarieddependingonthepreparationmethodandnanoparticlecharacteristics,suchasamphiphilic,polymermolecularweight,particlesize,andpolymercomposition.Invitroreleaseindicatedthataftertheinitialburstrelease,controlledrelease



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ofminocyclinecontinuedformorethan5days;thisreleaseprofilecouldbetheidealforperiodontaldisease.Nanoparticlespreparedbynanoprecipitationhadagoodsizeandmorphology;however,thisisnotanefficientmethodforencapsulationofhighlywater-solubledrugssuchasminocycline(reverseofnanoparticlespreparedbyoil/oilmethod).TheinvitroantibacterialresultsshowedthattheminocyclineloadednanoparticlesareremarkablymoreeffectivethanthefreedrugagainstAggregatibacteractinomycetemcomitans.

Acknowledgments

ThisstudywasfundedandsupportedbytheTehranUniversityofMedicalSciences(Grant10221).TheauthorsaregratefultotheNanotechnologyResearchCenterofTehranUniversityofMedicalSciences,Tehran,Iran,fortheirfinancialsupportofthiswork.TheyalsothankMrsFKhosraviforhertechnicalassistanceintheexperiments.

Disclosure

Theauthorsreportnoconflictsofinterestinthiswork.

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