DRUGDELIVERYASPECTS
EXPECTATIONSANDREALITIESOF MULTIFUNCTIONALDRUG DELIVERYSYSTEMS
VOLUME4
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RANJITA SHEGOKAR,PHD
CapnomedGmbH,Zimmern,Germany
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Contributors
HendAbd-Allah DepartmentofPharmaceutics andIndustrialPharmacy,FacultyofPharmacy, AinShamsUniversity,Cairo,Egypt
SaraM.AbdelSamie DepartmentofPharmaceutics andIndustrialPharmacy,FacultyofPharmacy,Ain ShamsUniversity,Cairo,Egypt
MonaM.A.Abdel-Mottaleb DepartmentofPharmaceuticsandIndustrialPharmacy,Facultyof Pharmacy,AinShamsUniversity,Cairo,Egypt; PEPITEEA4267,Univ.BourgogneFranche-Comte, Besanc ¸on,France
AbidRiazAhmed MerckHealthcareKGaA, Darmstadt,Germany
AkashChavrasiya DepartmentofPharmacy,Birla InstituteofTechnologyandScience,Pilani, Hyderabad,India
YanpingChen CenterofEmphasisinInfectious Diseases,DepartmentofMolecularandTranslationalMedicine,PaulL.FosterSchoolofMedicine, TexasTechUniversityHealthSciencesCenterEl Paso,ElPaso,TX,UnitedStates
JoaoDias-Ferreira DepartmentofPharmaceutical Technology,FacultyofPharmacy,Universityof Coimbra,Coimbra,Portugal
DianaDiaz-Arevalo ImmunologyFunctional Group,FoundationInstituteofImmunologyof Colombia-FIDIC,SchoolofMedicineandHealth Sciences,UniversidaddelRosario,Bogota,D.C., Colombia
SachinDubey Formulation,AnalyticalandDrug ProductDevelopment,GlenmarkPharmaceuticals, LaChauxdeFonds,Switzerland
AlessandraDurazzo CREA-ResearchCentrefor FoodandNutrition,Rome,Italy
ThomasD € urig R&DandInnovation,Ashland PharmaandHealth&Wellness,AshlandSpecialty IngredientsG.P.,Wilmington,DE,UnitedStates
RihamI.El-Gogary DepartmentofPharmaceutics andIndustrialPharmacy,FacultyofPharmacy, AinShamsUniversity,Cairo,Egypt
MuhammadIrfan DepartmentofPharmaceutics, FacultyofPharmaceuticalSciences,GCUniversity Faisalabad,Faisalabad,Pakistan
NirmalJayabalan DepartmentofPharmacy,Birla InstituteofTechnologyandScience,Pilani, Hyderabad,India
AnđelkaB.Kovacevic DepartmentofPharmaceuticalTechnology,FacultyofBiologicalSciences, InstituteofPharmacy,Friedrich-SchillerUniversity Jena,Jena,Germany
DharmeshMehta BusinessDevelopment,Gangwal Chemicals,Mumbai,India
JoanaPortugalMota Lecifarma—Laborato ´ rio Farmac^ eutico,Lda,Va ´ rzeadoAndrade—Cabec ¸o deMontachique,Lousa;CBIOS-ResearchCenter forBiosciencesandHealthTechnologies,Luso ´ fona University,Lisbon,Portugal
MostafaNakach SanofiR&D,VitrysurSeine, France
MahaNasr DepartmentofPharmaceuticsand IndustrialPharmacy,FacultyofPharmacy,Ain ShamsUniversity,Cairo,Egypt
RidahunlangNongkhlaw Department ofPharmacy,BirlaInstituteofTechnologyandScience, Pilani,Hyderabad,India
ParameswarPatra DepartmentofPharmacy,Birla InstituteofTechnologyandScience,Pilani, Hyderabad,India
AntonelloSantini DepartmentofPharmacy, UniversityofNapoli“FedericoII”,Napoli,Italy
AhmadAbdul-WahhabShahba KayyaliChairfor PharmaceuticalIndustries,DepartmentofPharmaceutics,CollegeofPharmacy,KingSaudUniversity,Riyadh,SaudiArabia
RanjitaShegokar CapnomedGmbH,Zimmern, Germany
VaibhavSihorkar Formulations,NCEandInnovation,SaiLifeSciencesLimited,ICICIKnowledge Park,GenomeValley,Hyderabad,Telangana, India
SarabjitSingh FormulationResearch,CIPLA, Mumbai,India
ElianaB.Souto DepartmentofPharmaceutical Technology,FacultyofPharmacy,Universityof Coimbra,Coimbra;CEB—CentreofBiological Engineering,UniversityofMinho,GualtarCampus,Braga,Portugal
HaiyanWang KeyLaboratoryofOralMedicine, GuangzhouInstituteofOralDisease,Stomatology HospitalofGuangzhouMedicalUniversity, Guangzhou,Guangdong,People’sRepublicof China;CenterofEmphasisinInfectiousDiseases,
DepartmentofMolecularandTranslationalMedicine,PaulL.FosterSchoolofMedicine,TexasTech UniversityHealthSciencesCenterElPaso,ElPaso, TX,UnitedStates
YongyongYan KeyLaboratoryofOralMedicine, GuangzhouInstituteofOralDisease,Stomatology HospitalofGuangzhouMedicalUniversity, Guangzhou,Guangdong,People’sRepublicof China;CenterofEmphasisinInfectiousDiseases, DepartmentofMolecularandTranslationalMedicine,PaulL.FosterSchoolofMedicine,TexasTech UniversityHealthSciencesCenterElPaso,ElPaso, TX,UnitedStates
MingtaoZeng CenterofEmphasisinInfectious Diseases,DepartmentofMolecularandTranslationalMedicine,PaulL.FosterSchoolofMedicine, TexasTechUniversityHealthSciencesCenterEl Paso,ElPaso,TX,UnitedStates
Versatilehyaluronicacidnanoparticles forimproveddrugdelivery
MonaM.A.Abdel-Mottaleb a,b,HendAbd-Allah a , RihamI.El-Gogarya,MahaNasra
aDepartmentofPharmaceuticsandIndustrialPharmacy,FacultyofPharmacy,AinShamsUniversity, Cairo,Egypt
bPEPITEEA4267,Univ.BourgogneFranche-Comte,Besanc ¸ on,France
1Introduction
Althoughmorethan80yearshavepassedsince thediscoveryofhyaluronicacid(HA),itstillsurprisesresearcherswithitsuniquephysicochemical propertiesandphysiologicalrolesinthehuman body.HAwasfirstdiscoveredbyKarlMeyer andJohnPalmerin1954 [1].Theyisolatedan unknownmaterialfromthevitreousbodyofa bovineeye,containingtwosugarmolecules including“uronicacid.”So,byconnectingthesubstitutenameforthevitreous—“hyaloid”—with thenameofacomponentofthatpolysaccharide—“uronicacid”—thenameofHAwas adoptedforthismaterial.HAwasfirstusedcommerciallyasasubstituteforeggwhiteinbakery products.Lateron,itsfirstmedicalapplication forhumanswasinitiatedasavitreousreplacement duringeyesurgeryinthelate1950s [2]
HAbelongstoagroupofsubstancescalled mucopolysaccharidesbelongingtotheglycosaminoglycans(GAGs)family [1,2].HAincludes
severalthousandrepeatingdisaccharidesmoleculesinthebackbone.Themolecularweightof HAmoleculesdiffersowingtothevariable numberoftheserepeatingdisaccharideunits ineachmolecule,itsmolecularweightranges from1to10,000kDa [3].HAhasanunusual mechanismofbiosynthesisandexceptional physicalproperties.Sodiumhyaluronateisthe predominantformofHAatphysiologicalpH. SodiumhyaluronateandHAarecollectively referredtoashyaluronan.Duetothefactthat HAexistsasapolyanion,itcanself-associate andcanalsobindwatermoleculesgivingita stiff,viscousqualitywithjelly-likeconsistency thatcausesittobehavelikealubricant [2].
HAwasfoundtobeabundantlydistributed incellularsurfaces,inthebasicextracellular substancesoftheconnectivetissuesofvertebrates,inthesynovialfluidofjoints,inthevitreoushumoroftheeye,andinthetissueofthe umbilicalcord;allthisattractedsignificant attentionregardingitsmedicalapplications
[4].AlthoughHAhasaverysimplestructure, almosteverythingelseconcerningthemolecule isunusual.Sometimesitsroleismechanicaland structural,suchasinsynovialfluid,thevitreous humor,ortheumbilicalcord.Inothercases,it caninteractinlowconcentrationswithcellsto triggerimportantcellularresponses.HA’scharacteristics,includingitsconsistency,biocompatibility,andhydrophilicity,havemadeitan excellentmoisturizerincosmeticdermatology andskin-careproducts.Moreover,itsunique viscoelasticityandlimitedimmunogenicity haveledittobeusedforviscosupplementation inosteoarthritistreatment,asasurgicalaidin ophthalmology,andforsurgicalwoundregenerationindermatology.Inaddition,HAhascurrentlybeenexploredasadrugdeliveryagentfor differentroutessuchasnasal,pulmonary,ophthalmic,topical,andparenteral [2].Hencethe useofnanotechnologywouldcombinetheoutstandingpropertiesofHAbeingbiocompatible, biodegradable,nontoxic,andabletobindspecificreceptorswiththedifferentadvantagesof nanoparticlessuchasenhancedtherapeutic effectsandtargetability.Inthischapter,the useofHAnanoparticlesasaversatiledrug deliverysystemwillbediscussedbyhighlightingthedifferentproductiontechniquesbased onthechemicalandbiologicalpropertiesofHA.
2Hyaluronicacid
2.1Chemistry
TheexactchemicalstructureofHAwas determinedbyWeissmanandMeyerin1954. Asalreadynoted,HAbelongstoagroupofsubstancescalledmucopolysaccharidesbelonging totheGAGsfamily.ItisanunbranchednonsulfatedGAGcomposedofrepeatingdisaccharides [β-1,4-D-glucuronicacid(knownasuronicacid) and β-1,3-N-acetyl-D-glucosamide],asshown in Fig.1.1.Bothsugarsarespatiallyrelatedto glucoseinthebetaconfiguration,thusallowing
allitsbulkygroups(thehydroxyls,thecarboxylatemoietyandtheanomericcarbononthe adjacentsugar)tobeinstericallyfavorableequatorialpositions,whileallthesmallhydrogen atomsoccupythelessstericallyfavorableaxial positions.Thus,thestructureofthedisaccharide isenergeticallyverystable [3].
TheHAbackboneisstiffenedinphysiological solutionviaacombinationofinternalhydrogen bonds,interactionswithsolvents,andthechemicalstructureofthedisaccharide.HAmolecular investigationssuggestedthattheaxialhydrogen atomsformanonpolarface(relativelyhydrophobic)andtheequatorialsidechainsformamore polarface(hydrophilic)whichleadstoatwisted ribbonstructureforHAcalledacoiled structure [4]
Owingtothisconformationalbehavioras wellasitshighmolecularweight,thesolutions ofHAareveryviscousandelastic.Atvery lowconcentrations,chainsentanglewitheach other,leadingtoamildviscosity(molecular weightdependent).However,HAsolutionsat higherconcentrationshaveahigherthanexpectedviscosityduetogreaterHAchainentanglementthatisshear-dependent.Forinstance,a1% solutionofhighmolecularweightHAcan behavelikejelly,butwhenshearstressis applied,itwilleasilyshearthinlyandcanbe administeredviaafineneedle [2].HAisthereforea“pseudo-plastic”material.Thisrheologicalproperty(concentrationandmolecular weightdependent)ofHAsolutionshasmade
FIG.1.1 Chemicalstructureofhyaluronicacidunit.
itidealasalubricantinbiomedicalapplications. Thereisevidencethathyaluronanseparates mosttissuesurfacesthatslidealongeachother. Theextremelylubriciouspropertiesofhyaluronan,meanwhile,havebeenshowntoreduce postoperativeadhesionformationfollowing abdominalandorthopedicsurgery.
HAhasseveralinterestingmedical,pharmaceutical,food,andcosmeticusesinitsnaturally occurringlinearform.However,chemicalmodificationsoftheHAstructurerepresentastrategytoextendthepossibleapplicationsofthe polymer,obtainingbetterperformingproducts thatcansatisfyspecificdemandsandcanbe characterizedbyalongerhalf-life.Duringthe designofnovelsyntheticderivatives,particular attentionispaidtoavoidthelossofnativeHA propertiessuchasbiocompatibility,biodegradability,andmucoadhesivity [5].HAcanbechemicallymodifiedbycrosslinkingorconjugation reactions.Thesechemicalmodificationsmainly involvetwofunctionalsitesofthebiopolymer: thehydroxyl(probablytheprimaryalcoholic functionofthe N-acetyl-D-glucosamine)andthe carboxylgroups [6].Furthermore,synthetic modificationscanbeperformedafterthedeacetylationofHAN-acetylgroups [7].
ConjugationreactionsusuallyconsistofaddingamonofunctionalmoleculeontooneHA chainbyasinglecovalentbond,whilecrosslinkingemployspolyfunctionalcompoundstolink togetherdifferentchainsofnativeorconjugated HAbytwoormorecovalentbonds.Crosslinked hyaluronancanbepreparedfromnativeHA (directcrosslinking)orfromitsconjugates. Crosslinkingisnormallyintendedtoimprove themechanical,rheological,andswellingpropertiesofHAandtoreduceitsdegradationrate, todevelopderivativeswithalongerresidence timeinthesiteofapplicationandcontrolled releaseproperties [5]
2.2Sources
HAisanaturalpolymerbiologicallysynthesizedbycellsinthebodybyanenzymaticprocess.HAproductionisaunique,highly controlled,andcontinuousprocess.Approximatelyhalfofourbody’sHAisdistributedin thecutaneousregion.Itisproducedand secretedbycellsincludingfibroblasts,keratinocytes,orchondrocyteswithvaryingmolecular weightsbetween50and3000kDa.TheGolgi networkistheproductionsiteformostGAGs. IntissuessuchasskinandcartilagewhereHA comprisesalargeportionofthetissuemass, HAissynthesizedinlargeamounts.Itisnaturallysynthesizedbyhyaluronansynthases (HAS1,HAS2,andHAS3),aclassofintegral membraneproteins [10].Thethreeenzymes arelocatedondifferentchromosomes,producingHAwithdifferentmolecularweights. HAS1andHAS2proteinsareresponsiblefor thesynthesisofhighmolecularweightHA ( 2 106 Da)withthelattermoreactivecatalyticallythantheformer,whereastheenzyme HAS3isthemostactivebutcanonlysynthesize shortHAchainsfrom200,000to300,000Da.The differentmolecularweightsofHAchainscan leadtodifferenteffectsoncellbehavior.HAperformsitsbiologicalactionsaccordingtotwo basicmechanisms:itcanactasapassivestructuralmoleculeandasasignalingmolecule.Both mechanismsofactionhavebeenshowntobe size-dependent [11].
Asmentionedabove,HAhasanessential functionalcomponentofalmostalltissuesin thevertebrateorganism.Thus,variousanimal
ConjugationofdrugstoHAwasreportedas earlyas1991.ThisapproachaimedtoformaprodrugbycovalentlybindingadrugtotheHA backbonethroughabondthatideallyshould bestableduringthebloodcirculationand promptlycleavedataspecifictargetsite [8]. OwingtoHAsolubility,itispossibletoperform thereactioninwater.However,intheaqueous phase,somereactionsarepH-dependentand needtobeperformedinacidicoralkalineconditions,whichhavebeenshowntoinducesignificantHAchainhydrolysis [8,9].
tissues,forexample,inroostercombs,sharkskin, andbovineeyeshavebeenusedassourcesofisolationandproductionofhighmolecularweight HA.SinceHAinbiologicalmaterialsisusually presentinacomplexlinkedtootherbiopolymers, severalseparationproceduresmustbeappliedto obtainapurecompound,suchasproteasedigestion [10].HAwasinitiallyisolatedfrombovine vitreoushumorandlaterfromroostercombs andhumanumbilicalcords [11].Themean molecularweightofthecommerciallyavailable “extractive”HApreparationsobtainedfromanimaltissuesismostlyintherangefromseveral hundredthousandDauptoapproximately2.5 MDa [12].However,ithasbeenobservedthat theHAproductsobtainedfromroostercombs causedsomeallergicresponses.Further,thetechnologyhasbeendevelopedrecentlytowardbacterialfermentationtoreducetheproductioncost andcomplexpurificationprocesses.Suchalternativesourcesincludeattenuatedstrainsof Streptococcuszooepidemicus and Streptococcusequi for theproductionofHA.Thebacteriumsecretes theHAintofermentationbrothandthisbehavior isanadditionaladvantageforisolationoftheHA directlyfrombrothwithouttheneedforhomogenizingbacterialcells [13,14].However,therisk ofmutationofthebacterialstrains,andpossible co-productionofvarioustoxins,pyrogens,and immunogens,decreasestheapplicationoffermentativeHAinclinicalpractice.Thisisalso whyHAsamplesoriginatingfromroostercombs arestillcurrentlypreferredforhumantreatment incaseswhentheHAmaterialisdesignatedfor injection,intheeyesorsynovialjoints.However, thesearealsonotidealsourcesofHA,asallHA productsobtainedfromroostercombsareobligatedtocarrywarningsforthosewhoareallergic toavianproducts.Thus,alternativesourcesfor productionofHAarepresentlyasubjectof research [12]
Oneofthepromisingpotentialcandidatesis ageneticallymodifiedbacterialstrain, Bacillus subtilis ,carryingtheAgenefrom Streptococcus equisimilis encodingtheenzymeHAsynthase.
SuchanengineeredstraincouldproduceHA withthemolecularweightinthe1MDarange. Theadvantageofusing B.subtilis isthatitis easilycultivatedonalargescaleanddoes notproduceexotoxinsorendotoxins,and manyproductsmanufacturedbythismicroorganismhavereceivedaGRAS(generallyrecognizedassafe)designationintheearly 1960s.Atpresent,microbiologicallyproduced HAhasbeenapprovedfortreatmentofsuperficialwoundsaswellasforuseinthecosmetic industry [12] .
2.3Physiologicalrole
HAdiffersfromothersyntheticpolymersin thatitisbiologicallyactive.Togetherwith HA’soutstandingviscoelasticnature,itsbiocompatibilityandnonimmunogenicityhave ledtoitsuseinseveralclinicalapplications. HAwasdescribedasanubiquitouscarbohydratepolymerthatispartoftheextracellular matrix [15].Ahumanbodyweighing70kgcontains15gofHA.ThegreatestamountofHAis presentintheskin,followedbythesynovial fluid,thevitreousbody,andtheumbilicalcord. Itcanalsobefoundinplaceswherefriction occurs:thejoints,tendons,sheaths,pleura,and pericardium [16].
Inthehumanbody,HAoccursinmany diverseforms,circulatingfreely,decoratedwith avarietyofHA-bindingproteins(hyaladherins), tissue-associated,intercolatedintotheextracellularmatrixbyelectrostaticorcovalentbindingtoothermatrixmolecules.Itcomprisesa majorportionoftheintimateglycocalyxthat surroundsallcells.HAcanbetetheredtocell surfacesbyanyofthemembrane-associated receptors.RecentevidenceindicatesthatHA alsoexistswithincells,thoughlittleisknown oftheformorfunctionofsuchHA [17]
HAisamajorcomponentofthesynovial fluid,andwasfoundtoincreasetheviscosity ofthefluid.Alongwithlubricin,itisoneof thefluid’smainlubricatingcomponents.Itis
consideredanimportantcomponentofarticular cartilage,presentingacoataroundindividual chondrocytesandprovidingitsresistanceto compression.Themolecularweight(size)of HAincartilagedecreaseswithage,butthe amountofitincreases [18].HApossessesanumberofprotectivephysiochemicalfunctionsthat mayprovidesomeadditionalchondroprotectiveeffectsinvivo,explainingitslonger-term effectsonarticularcartilage.HAdecreasesthe nerveimpulsesandnervesensitivityassociated withpain.Inexperimentalosteoarthritis,HA hasprotectiveeffectsoncartilage [19].ExogenousHAenhancesitssynthesistogetherwith proteoglycaninchondrocyte,reducestheproductionandactivityofproinflammatorymediatorsandmatrixmetalloproteinases,andalters thebehaviorofimmunecells.Thesefunctions aremanifestedbythescavengingofreactive oxygen-derivedfreeradicals,theinhibitionof immunecomplexadherencetopolymorphonuclearcells,andtheinhibitionofleukocyteand macrophagemigrationandaggregation [20]
Alubricatingroleofhyaluronaninmuscular connectivetissuestoenhanceslidingbetween adjacenttissuelayershasalsobeensuggested. Aparticulartypeoffibroblasts,embeddedin densefascialtissues,hasbeenproposedasbeing cellsspecializedforthebiosynthesisofthe hyaluronan-richmatrix.Theirrelatedactivity couldbeinvolvedinregulatingthesliding abilitybetweenadjacentmuscularconnective tissues [18].
HAisalsoamajorcomponentofskin.More thanhalfofthetotalbody’sHAispresentin theskin [8],whereitplaysastructuralrolethat dependsonitsuniquehydrodynamicproperties anditsinteractionswithotherextracellular matricesmolecules(ECM)components.HA excellentconsistencyandtissue-friendliness andbeingoneofthemosthydrophilicmolecules innaturehascausedittobedescribedas nature’smoisturizer [4].Itgivestheskinits propertiesofresistanceandmaintenanceof theshape,andsupportsthepreservationof
thenaturaldegreeofhydrationoftheskincells. Itsconcentrationinthebodytendstodecrease withaging,andalackofitleadstoaskinweaknesspromotingtheformationofwrinkles [7]
Whileitisabundantinextracellularmatrices, HAalsocontributestotissuehydrodynamics, movementandproliferationofcells,andparticipatesinanumberofcellsurfacereceptorinteractions,notablythoseincludingitsprimary receptors,CD44andRHAMM.Upregulation ofCD44itselfiswidelyacceptedasamarker ofcellactivationinlymphocytes.HA’scontributiontotumorgrowthmaybeduetoitsinteractionwithCD44.ThereceptorCD44participates incelladhesioninteractionsrequiredbytumor cells.
Onthecellularlevel,HAishighlyhygroscopicandthispropertyisbelievedtobeimportantformodulatingtissuehydrationand osmoticbalance.Becauseofitshygroscopic properties,hyaluronansignificantlyinfluences hydrationandthephysicalpropertiesofthe extracellularmatrix.Hyaluronanisalsocapable ofinteractingwithseveralreceptors,resultingin theactivationofsignalingcascadesthatinfluencecellmigration,proliferation,andgene expression [21].
2.4Turnoverandeliminationpathways
TheconcentrationofHAinthehumanbody variesfromahighconcentrationof4g/kgin umbilicalcord,2–4g/Linsynovialfluid,0.2 g/kgindermis,about10mg/Linthoracic lymph,andthelowestof0.1–0.01mg/Linnormalserum [10].Dependingonthelocationof HAinthebody,mostofitiscatabolizedwithin days.Studiessuggestedthatthenormalhalf-life ofHAvariesfrom1–3weeksininerttissues suchascartilages,to1–2daysintheepidermis ofskin,to2–5mininbloodcirculation.ThepathwaysinvolvedinHAcatabolismincludeturnover(internalizationanddegradationwithin tissue)andreleasefromthetissuematrix,
1.Versatilehyaluronicacidnanoparticlesforimproveddrugdelivery
drainageintothevasculature,andclearancevia lymphnodes,liver,andkidneys.
Instructuraltissueslikeboneorcartilage withnoorlittlelymphaticdrainage,HAdegradationoccursinsituwithotherECMsuchascollagensandproteoglycans.Ontheotherhand,in skinandjoints,aminimalfraction(approximately20%–30%)ofHAdegradesinsitu.Since HAisrestrictedtothesmallintracellularspace ofskintissue,itshalf-lifeisslightlylongerfor daysandweeks [22].
3Preparationofhyaluronicacid nanoparticles
3.1Conjugateformation
Oneofthemostcommonlyusedtechniques forthepreparationofHAnanoparticlesisthe preparationofHA-drugnano-conjugatesby establishingacovalentbondbetweenthedrug ofinterestandHAwhichcouldimprovesolubility,pharmacokineticprofile,andinvivoplasma half-lifeoftheconjugateddrugs.Inmostcases, theconjugationisdesignedtobecleavedafter reachingthetargetsite,andincasesofcancer, theseconjugatesusuallyhavehigheraccumulationinthetumorsitesduetotheenhancedpermeationandretentioneffect(EPR) [23].Dueto thepresenceofmultiplefunctionalgroupson thebackboneofHAlikehydroxylandcarboxylic acidgroups,itwaspossibletogetconjugatedto variouscompoundsandmacromoleculessuchas paclitaxel [24,25],sodiumbutyrate [26,27],and ovalbumin [28].
TheuseofHAdrugconjugatesiskindofconvertingthedrugintoaprodrugderivativein whichthelinkbetweentheHAandthedrug moleculeshouldachievecertaincriteria.The mostimportantisthatthebondshouldbestable extracellularlytogivetherequiredinvivohalflife,andshouldbecleavedeasilyintracellularly toachievethedesiredeffect.Itisevidentthatthe releaseofintactdrugmoleculewithoutaffecting
thechemicalstructureisanotherrequiredproperty [29].Thetwomostcommonsitesforthe chemicalconjugationofdrugstoHAarethe hydroxylgroupsandthecarboxylicacidfunctionalitieswhiletheterminalaldehydegroup canonlybeusedtoprepareterminallymodified HA-ligandconjugatesaswellastograftHA oligomerstoanotherpolymercarryingamino groups.
3.2Self-assembliesformation
AnothertypeofHAnanoparticlesarethe onespreparedbyHAassociationwithhydrophobicpolymerswiththeresultingconjugates abletoself-aggregateintheformofhydrophobic micelles.Examplesincludethehydrophobic associationofHAtopoly(lactic-co-glycolicacid) (PLGA) [30],poly(ethyleneglycol)-poly (ε-caprolactone)copolymers(PEG-PCL) [31], ortetradecylamine [32].Theseamphiphilic self-assemblingHAderivativeshavebeenpreparedbycouplingthecarboxylicgroupsofthe hydrophilicHAthroughcarbodiimidechemistrytodifferenthydrophobicmoietiessuchas PLGA [33],tetradecylamine [32],andPCL [31]. Thiskindofnanoparticlewouldcombinethe hydrophobiccoreneededfortheencapsulation ofmanytherapeuticagents,andthehydrophilic shellprovideslongercirculationtimesbyreducingunwantedproteinadsorption [23].
HydrophobicallymodifiedHA(HMH)was preparedbythecovalentconjugationtothe hydrophobictetradecylamine(TDA)using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC)andhydroxysulfosuccinimide(sulfoNHS).Thisreactionwasabletoproducenano self-aggregatesuponitsdissolutionandsonicationintoaqueousphosphatebuffersolution [32] Theself-associatedHMHnanoparticlesranged insizebetween197and285nmdependingon thedegreeofsubstitutionwithminorinfluence fromthepolymerconcentrationused.Thehigher thedegreeofsubstitution,thesmallertheparticles produced.AmphiphilicHA-5b-cholanicacid
conjugates(HA-CAconjugates)weresynthesized bychemicalconjugationofhydrophobicbileacid (5b-cholanicacid)tothehydrophilicHAbackbonethroughamideformationinthepresence ofEDCandNHS.VaryingthemolarratioofCA tothecarboxylicacidofHAvariedthedegreeof substitutionfrom2to10.Fortheproductionof HAnanoparticles,theproducedamphiphilic HA-CAconjugatesweredissolvedinaphosphate bufferedsalineandthesolutionwassonicated usingaprobe-typesonificationsystemfollowed byfiltrationstep.Theproducedparticlesranged insizefrom237to424nm [34].
3.3Ionicnanocomplexesformation
SinceHAisapolyanionicpolysaccharide,it canbeeasilyallowedtoreactwithcationiccompoundsandformsuccessfulioniccomplexes. ThisreactioncanbeinducedbydirectinteractionofHAwithpositivelychargedcargomoleculeslikeinthecaseofDNAandplasmids,orit canhappeninthepresenceofanotherpositively chargedpolymerlikechitosan [35].Examples includetheionicnanocomplexbetweenHA andTRAIL(TumornecrosisfactorRelatedApoptosisInducingLigand),whichwashighlystableandlongcirculatingcomparedtothe nativeTRAIL [36].Similarly,biopolymeric amphiphileswerepreparedfromtheEDC mediatedcouplingreactionbetweenHAand deoxcycholicacidproducingself-assembled nanocarriersinthesizerangeof100–600nm. ThestronginteractionbetweenHAandchitosan (CS)evenledtothefasterreleaseofthe entrappednegativelychargedcargomolecules, whosereleasecouldhavebeenhinderedincase ofinteractionwithchitosanalone.CS-HAplasmidnanoparticleswerepreparedbysimple mixingofbothsolutionsundermagneticstirring andDNAwasthenaddedtoformcomplexes. Themixturewasvortexedfor3–5sandthenleft atroomtemperatureforthecomplexestoform completely [37].Itwasfoundthatthesizeof thenanoparticlesincreasedandthezeta
potentialdecreaseduponincreasingtheratio ofHAtoCS.Similarly,HAcouldionicallyinteractwithpositivelychargedmetalliccompounds suchastheanticancerdrugcisplatin.CisplatinHAnanoparticleswerepreparedbysimple mixingofHAandthedrug,formingnanoparticlesinthesizerangeof80–160nm.Being largeinamount,thereleaseofthedrugfrom theparticlematrixwasaccompaniedbydisintegrationoftheparticles [38].
3.4Nanogelsformation
HAnanogelscanbepreparedbyeitherphysicalorchemicalcrosslinkingofHAtoprovide colloidalstableparticlesinthemicroornano range.Thephysicalcrosslinkingwoulddepend onnoncovalentattractiveforcesbetweenthe polymerchainssuchashydrophobicinteractions,hydrogenbondingandionicinteractions. However,chemicalcrosslinkingwouldprovide particlesofhigherstabilityandsubsequently longerhalf-lives.Topreparechemicallycrosslinkednanogelparticles,itispreferableto spatiallylocalizetheHAmoleculesandcrosslinkersinverysmallvolumestoachievethe requiredreactioninthenanorange.Thiscould besuccessfullyachievedusingtheinverse w/omicroemulsiontechnique,whichis describedin Fig.1.2[29].Hyaluronanmicrosphereswerepreparedbythesurfactant-aided homogenizationofHAaqueoussolutionsand crosslinkerinmineraloil.Thisstepisfollowed bytheinitiationofcrosslinkingbyadding EDC [39].Otherexamplesofnanogelparticles formationbyamidationcrosslinkingwere describedintheliterature [40,41].However,this microemulsionchemicalcrosslinkingtechniquesdemandshighenergymechanicalstirringorultrasonicationandtheuseoforganic solvents,whicharenotconsideredfavorable conditionsforthelabilemoleculessuchasproteinsandnucleicacids.Physicalcrosslinkingis consideredtobemuchmilderintermsofaffectingthelabilestructuresofdrugmolecules [29].
FIG.1.2 InversephasewaterinoilmicroemulsiontechniqueforthepreparationofchemicallycrosslinkedHAnanogels. ReprintedwithpermissionfromOssipovDA.Nanostructuredhyaluronicacid-basedmaterialsforactivedeliverytocancer.ExpertOpin DrugDeliv2010;7:681–703. https://doi.org/10.1517/17425241003730399 andTaylorandFrancisLtd.
4.1Skinapplications
AlthoughHAispresentinmostofthebiologicalfluidsandtissuesandextracellular matrixofsoftconnectivetissues,skinisconsideredtobemostHA-abundanttissueinthe humanbody.Uponaging,especiallyafterthe ageof20,skincontentofHAcontinuously decreases [42]
TheuniquepropertiesofHA,includingits biocompatibility,biodegradability,viscoelasticity,andnonimmunogenicity,havemadeitan idealmaterialforcosmeticandbiomedical applications.Itexertsahydratingeffectonthe skin,whichmayhelptoenhancethepenetration ofdifferentdrugsthroughtheskin.However,its ownpenetrationisverylimitedduetothehigh molecularweightaswellastheenzymatic degradationrisk.ItwasreportedthatcrosslinkedHAprovedtopermeatethroughtheskin
deeperlayersmakingitsuitablecarrierfortransdermalapplications [42].HAitselfasmacromoleculeisnotabletopenetratetheskin beyondthesurfacelayersduetothestrongbarrierproperties [43].HAnanoparticlesprepared bytheanionicinteractionwiththecationicpolymerprotaminewereabletopenetratetheskin anddelivertheHAtothedermiswhilefree HApenetratednofurtherthestratumcorneum. Therefore,thesenanoparticleswereconsidered promisingfortheeffectiveskindeliveryofHA tocontributetobarrierrecoveryfollowingUV irradiation [44].Similarly,nanoparticlesofquaternizedcyclodextrin-graftedchitosanassociatedwithHAhavebeenalsoproven promisingforcosmeticsandskinhydration applications.Theirskinhydratingabilityaswell astheirsafetyonhumanskinfibroblastswere demonstratedinvitro [45].
Besidestheuseofnanoparticlesforthedrug deliverypurposes,somecosmeticapplications
HA and drug Aqueous phase
Surfactant Organic phase
Nano gel
Crosslinks
Drug
Crosslinker or coupling agent
HA
havebeenproposed.Preparationsofslightly crosslinkedHAareusedasfillersforaugmentation,tofillfacialwrinklesanddepressedscars. SuchHAgelsaremoreeffectiveinmaintaining cosmeticcorrectionsthancollagen-basedproducts.Unlikecollagen-basedfillers,HAis extremelyelastic,providingtheelasticity requiredbyspacesinwhichitisinjectedand thehyaluronatepreparationsaremoresustained.ExamplesoftheuseofHANPsforcosmeticapplicationsaredescribedin Table1.1.
4.2Osteoarthritis
Theuseofintra-articularHAinjectionsasa viscosupplementtorestorethenormalviscosity ofsynovialfluidinosteoarthritispatientsisa well-establishedtherapeuticstrategy [11]. Cationicpolymericnanoparticleslinkedtohyaluronateprovedtobeeffectiveintheproduction ofionicallycrosslinkedhydrogelsinsituto increasetheretentiontimeofamodeldrugin thesynovialcavity [50].
4.3Tissueengineering
HAasoneofthemaincomponentsofbody tissueshasfrequentlybeeninvestigatedfor tissueengineeringapplications.HA-based sheetsserveasamatrixforsofttissue,cartilage, bone,andskingrowth,andasasubstratefor tissueregenerationandremodeling.ThreedimensionalscaffoldsofHA-basedmaterials canfacilitaterestructuringoftissuesandassist inregainingfunction.Thesematerialsareideal fortissuereconstruction,asthereisnohost immuneresponse,andareparticularlyuseful
Genetherapyhasbeenproposedasatreatmentmodalityfortargetingspecificpathological mechanismsandhencehelpingtotreatthe underlyingdiseaseorigin.HA-CS-plasmid nanoparticleswerepreparedasnovelnonviral genedeliveryvectorforthetreatmentofosteoarthritis.TheyutilizetheabilityofHAtobindtothe CD44tobeinternalizedbythetargetedcellsby theendocytosispathway.ThetransfectionefficiencyoftheseNPswasfoundtobesuperiorto theCS-plasmidNPS,suggestingthemasasafe andeffectivenonviralgenedeliveryvectorto chondrocytes [37].
TABLE1.1 CosmeticapplicationsofHAandHANPs.
HAPolyioncomplexformationwiththe cationicpolymerProtamine
HAPolyioncomplexformationwith quaternizedcyclodextrin-grafted chitosan
barrierpropertiesindamagedskin [44]
moisturizingproperties [45] HAHA/lysineNPsbyionicinteraction betweenHAandlysine
injectiontechnique
nanoparticlesbyionicgelation
[46]
forcellularskinlayersregeneration [47]
[48]
percutaneousabsorption [49]
forburnandtraumapatients.Stemcellsrequire anHA-richenvironmentformaintainingthe undifferentiatedstate.Vascularendothelialcells canbeselected,aswellasaorticsmoothmuscle cellsfortheconstructionofheartvalves,by seedingontoHAsheetsandmembranes.However,duetoHA’shighsolubilityandfastelimination,itsuseforscaffoldfabricationand structuralstabilityhasbeenchallenging.To overcometheselimitations,modificationand crosslinkingofHAhavebeenproposed.Various examplesontheuseofcrosslinkedHAnanoparticlesfordrugdeliveryarepresentedin Table1.2.Water-solublecarbodiimidecrosslinking,polyvalenthyadrazidecrosslinking,and othertechniqueshavebeenintroducedfortissue engineeringapplicationsofHA.ChemicalcrosslinkingisexpectedtoextendtheHAdegradationprocessinvivoandprovidelong-term stabilityforthevariousapplicationsinorthopedics,cardiovascularmedicine,anddermatology. Elsewhere,photocrosslinkedHAhydrogels havebeenalsointroducedfordifferentapplicationssuchascartilagetissueengineering,cardiac repair,moleculedelivery,valvularengineering, controlofstemcellbehavior,andmicrodevices [10].HAasanaturalpolymerhasbeenmixed withpoly(lactic-co-glycolicacid)nanoparticles todevelopaninsitucrosslinkablesystemwith drugdeliverypotential.Althoughsuchasystem hasshownfavorablemechanicalpropertiesfor tissueengineering,itsbiocompatibilityandtoxicityinvivoisstillamajorconcern [64].
4.4Cancertargeting
Self-assembledpolymericnanoparticleshave beeninvestigatedforcancertherapyduetotheir abilitytoencapsulatethechemotherapeutic agentsandreleasethemonasustainedmanner. Thisisevenenhancedbyrenderingtheirsurface hydrophilicwhichwouldenhancetheircirculationtimeleadingtohigheraccumulationinto thetumortissuewiththeknownEPReffectof nanoparticles [34,65].Inadditiontothispassive
targetingstrategy,activetargetingcanbealso achievedbybindingtheseNPstotargeting moietiestorecognizeandbindtothetumorcells andbeinginternalizedbyreceptor-mediated endocytosis.SinceHAisdistinguishedbyits abilitytobindtovariouscancercellsthat overexpressCD44,ithasbeenconjugatedto variousdrugloadednanoparticlesasatargeting moiety [66].CD44isoverexpressedinmanycancersofepithelialoriginandthereforetheuseof HAnanocarrierscouldincreasethetargetability andtheretentionintothecancertissue [66]. However,theuseofHAnanoparticlesasatargetingmoietyanddrugcarrierisanewtrend, andextensivestudiesareneededtounderstand thefactorsaffectingtheaffinitytoCD44andthe internalizationmechanismsofHAnanoparticles.ItwasfoundthattheslowCD44representation(24–48h)ledtolimitedavailabilityofHA internalizationreceptors.Therefore,ahigher affinitynanoparticleandahigherdegreeofclusteringwouldleadtoalowernumberofinternalizedparticles.Ontheotherhand,loweraffinity systemsmightleadtolessclusteringwithmore efficientHA-mediateddeliveryofdrugpayloads [67].AmphiphilicHA-CAconjugates nanoparticles,whichself-assembleintohydrophobiccorenanosizedparticles,surrounded byahydrophilicHAshellefficientlyaccumulate intothetumorsitecomparedtothepurewatersolubleHAaftersystemicadministration.Thein vivobiodistributionoftheseNPsintumorbearingmicewasinvestigatedusinganoninvasivenear-infraredopticalimagingtechnique, whichrevealedasignificantaccumulationof theHANPsinthetumorsite.Theaccumulation wasmuchstrongerthaninnormaltissuesand wassizedependent,whichembracestherole oftheEPRpassivetargetingpathway.However, whenanimalswerepreinjectedwithhighdoses ofHApolymerbeforetheinjectionofHANPs, theNPsaccumulationinthetumorsitewas remarkablyattenuated,suggestingthatthe interactionbetweentheHAfromtheNPsto theCD44receptorsonthecancercellsurfaceis
TABLE1.2 VariousapplicationsofHANPsindrugdelivery.
Drug HAnanoparticles Particle size (nm)Application Reference
PlasmidDNAPolyioncomplexofHAandchitosan andplasmidDNA 100–300Nonviralvectorforgene deliveryforchondrocytes [37]
siRNA Inversew/oemulsion(nanogel)200–500SelectivetargetingofHCT-116cells [51]
PlasmidDNADihydrazidemediatedcrosslinking5–20 μmControlledrateDNAdelivery [39]
DoxorubicinHA-PEG-PLGApolymeric nanoparticlesbynanoprecipitation 93–186Selectivetumortargeting [33]
HA
Self-assembliesofhydrophobic 5β-cholanicacid-HA
HA Self-assembliesofhydrophobic 5β-cholanicacid-HA
DoxorubicinBioreduciblecore-crosslinked polymericmicellebasedon hyaluronicacid
Cy5.5and doxorubicin
Self-assembliesofamphiphilic iodinatedhyaluronicacid
–400Tumortargeting [52]
–424Passiveandactivetumortargeting [34]
[53]
200Theranosticsystemforcancer [54]
Paclitaxel Hyaluronate-cholanicacidmicelles258TargetingCD44overexpressionin cancercells [55]
Cy5.5 (PEG)-conjugatedself-assembledHA nanoparticles 217–269Cancertherapyanddiagnosis [56]
Cy5.5 Self-assembliesofhydrophobic 5β-cholanicacid-HA
Cy7or 89ZrEDCandsulfo-NHScrosslinkedHA andcholanicacid
237–424Targetingstabilin-2andCD44 receptorsoverexpressedin atherosclerosis [57]
90Theranosticapplicationin atherosclerosis [58]
pEGFPorpβ-gal asmodelplasmid Hyaluronicacid-chitosanionotropic gelation 100–235Genetransferandtargetingtoocular cells [59]
DexamethasoneHA-chitosannanoparticles – Enhancedocularbioavailability [60]
InsulinReverse-emulsion-freeze-drying182Oralinsulindelivery [61]
PerfluoropentaneOil-in-water(O/W)emulsification350Ultra-long-acting,liver-specific, Ultrasoundcontrastagent [62]
CuSandCy5.5EDCandsulfo-NHScrosslinkedHA andcholanicacid
227Image-guidedphotothermal therapyofcancer [63]
alsoresponsibleforthehighaccumulationon thetumorsite [34,52].TheuseofcorecrosslinkedHAmicellespreparedwithasimple methodofdisulfidebondformationloadedwith doxorubicinhasshownanenhancedtherapeutic efficiencyandtumoraccumulationaswellas improvedstabilityinvivocomparedtothe uncrosslinkedmicellesandthefreedrug.The superioractivitywascorrelatedtotheability ofthesecarrierstounloadthedruginsidethe tumorcellonlywiththemicellarstructuredissociatedinresponsetotheglutathioneattheintracellularlevel.2,3,5-Triiodobenzoicacid(TIBA) wasconjugatedtoanHAoligomerasa computedtomography(CT)imagingmodality andahydrophobicresidueandself-woven nano-assemblieswereproducedforthetumortargeteddeliveryofdoxorubicinmwhich presentedapromisingtheranosticsystemfor cancerdiagnosisandtherapyoftumorsthat expressCD44receptors [54].PolymericnanoparticulatemicellesofHA-CApaclitaxelwere foundtobespecificandefficientchemotherapeutictreatmentforCD44overexpressing tumorsandcancercells [55].However,amajor drawbackofHA-basedconjugatesornanoparticlesforcancertargetingistheirpreferential accumulationintheliveraftersystemicadministration.PEGylatedHA-NPsformedselfassemblednanoparticlesinthesizerangeof 217–269nmofimprovedcanceraccumulation andtargetabilitycomparedtoHA-NPswhen testedintumor-bearingmice [56].
4.5Atherosclerosis
TheuseofHA-NPshasbeenproposedasa potentialtoolforbothdiagnosticandtherapeuticapplicationsinatherosclerosis.Amajor observationinthepathogenicprocessofatherosclerosisistheoverexpressionofreceptorsof HAsuchasstabilin-2andCD44.SelectivestrongeraccumulationofHA-NPsinatherosclerotic lesionswasobserved,whichwasprobably explainedbyanactivetargetingmechanism
aftersystemicadministration [57].Aminefunctionalizedoligometrichyaluronanconjugated withcholanicesterandlabeledwithfluorescent orradioactivenanoparticleswastestedfor targetingatherosclerosisassociatedinflammation.The90nmparticlesaccumulationwas 30%higherinatheroscleroticaortasthanwild typecontrols.TheplaquestreatedwiththeHA nanoparticlescontained30%fewermacrophagescomparedtocontrolandfreeHAtreated groups.Therefore,thesenanoparticleswere proposedforPETimagingofatherosclerosisassociatedinflammationduetotheirfavorable targetingpotential [58].
4.6Oculardrugdelivery
Duetothestrongdefensivemechanismsofthe eye,thetransportofdrugsviatopicalinstillation intheeyesislimited,causingrestrictedbioavailability.Theuseofhyaluronan-coatedchitosannanoparticleswasinvestigatedforthe enhancedoculardeliveryofdexamethasone. TheHA-coatednanoparticleshaveshown 2.14-foldhigherAUC0-24h comparedtodexamethasonesolution,whichwasexplainedby theprolongedprecornealretentioncausedby thehighlymucoadhesiveHA [60].HA-chitosan nanoparticleswithsizesbetween100and235 nmwerealsotestedforoculargenedelivery andwereabletoachievehightransfection efficiencywithoutaffectingcellviability [59]. HAisalsoparticularlyusefulasaspace-filling matrixintheeyetomaintaintheshapeof theanteriorchamberduringsurgeries.HAsolutionsalsoserveasaviscosity-enhancingcomponentofeyedropsandasanadjuvanttoeyetissue repair.
4.7Insulinsensitivityanddiabetes
Recently,ithasbeendemonstratedthatCD44 receptorsinpro-inflammatorycellsinobese adiposetissuesareinvolvedinthedevelopment ofadiposetissueinflammationandinsulin
resistanceintype2diabetespatients.Therefore, emptyHAnanoparticleswereusedasatherapeutictoolforadiposetissueinflammationand insulinresistancebyselectivelyaccumulating andclusteringtheCD44receptorsininhibiting theinteractionofthelowmolecularweightHA withthesereceptors,leadingtoimprovedinsulin sensitivityandglycemiccontrol.TheHAnanoparticlesarehenceproposedasatherapeutic agentinthetreatmentoftype2diabetespatients [68].HAnanoparticleshavealsobeenproposed fortheoraldeliveryofinsulinasanalternative forinsulininjectionsforthetreatmentofdiabetes.Insulin-loadedHAnanoparticleswerepreparedbyareverseemulsionfreeze-drying methodinsizesofapproximately180nm.The particlesproducedhadveryhighentrapment efficiencyupto95%.ThesepH-sensitivenanoparticlesprovidedtherequiredprotectionfor insulinfromtheacidicenvironmentofthestomachandatthesametimehadnoeffectonthejunctionintegrityofepithelialcells,whichisan importantparametertoconsiderincaseof chronicuseofmedicaments,whichistheusual caseofinsulin.ThepresenceofHAenhanced thetranscellulareffluxofinsulintransported throughCaco-2cellmonolayersviathetranscellularpathways.Theresultsofpermeability througharatsmallintestineshowedthatinsulin transportthroughtheduodenumandileumwas enhanced,andthetherapeuticefficiencyofthe producednanoparticleswasalsoprovedinadiabeticratsmodel [61].
4.8Theranosticandimaging
Forhigherdiagnosticprocedures’efficiency fordifferentdiseases,thereisagreatneedfor instantreal-timeimagingtechniquesthatcould favorablytargetcertainorgansortissuetypes. Oneofthemostcommonlyusedimagingtechniquesinmedicalfieldsisultrasoundimaging. NewechogenicHAnanoparticleshavebeen developedandpresentedasanultra-longacting,liver-specificultrasoundcontrastagent.
Theparticleswerepreparedwiththeoilina wateremulsificationmethodandcontainedperfluoropentaneasanultrasoundgasprecursor. Theparticlesweremorestablecomparedtothe conventionalmicrobubblesusedforultrasound (US)imaging.Theirlongcirculatingproperties allowedforseveralsystemiccirculationsfollowedbyintenseaccumulationattheliver, whichembracedtheirpotentialasatargeting imagingsystemfordiagnosisofliverdiseases. Thetargetabilitytotheliverwasrelatedtoboth thesmallhydrodynamicsizeoftheparticlesas wellasthehighaffinityofHAtotheliver.More interestingly,thepreparedparticleswereableto discriminatebetweenthenormallivertissues andlivercancerinalivertumor-bearingmice model.Thecanceroustissuewasfoundtobe morecompactthanthenormaltissue,andhence lowerUSsignalswereobserved [62]
Ironoxide-basedmagneticnanoparticles bearingHAonthesurfacehavebeendeveloped totargetactivatedmacrophagesforimagingand therapeuticapplicationsininflammatorydiseases.Theparticleswerepreparedby co-precipitationproceduresfollowedbypostsyntheticfunctionalizationwithbothHAand fluorescein.Particlesexpressedasignificantbiocompatibilityandstabilityinserum.Significant uptakebyactivatedmacrophageswasobserved, whichwasHAdependent.Inaddition,themagneticcoreoftheparticleswasfoundtobeonly transientlypresentinthecells,whichindicates lowerrisksoftoxicity.Fluoresceinwasfound tobesuccessfullydeliveredtothecellularnuclei, whichshowedthepotentialofusingthesenanocarriersalsoasatargeteddrugdeliveryand molecularimaging [69].HAnanoparticleswere synthesizedandlabeledwiththenearinfrared dyeCys5.5forimagingpurposes.Forusingthe sameparticlesbutforphotothermalpurposes, anadditionalstepofloadingtheparticleswith CuSwasperformed.Theobtainedparticleshad asizerangeofaround200nm.Thefluorescent signalofCys5.5wasquenchedbythepresence ofCuS.Whentheenzymehyaluronidasewas
addedtotheparticles,thefluorescencesignal wasrestoredinatime-dependentmanner,which suggestedthattheparticlescouldbeusedasa nanoprobetobeactivatedbythehighlyoverexpressedhyaluronidaseinthecanceroustumor areas.Therefore,theCuS-loadedHAnanoparticleswereusedforphotoacousticimaging, utilizingthestrongabsorbanceoftheCuS.After intravenousadministration,theparticlesaccumulatedinthetumorareaovertime,andwhen irradiatedwithalaser,agoodtumorinhibition rateof89.74%wasobservedonday5,indicating thegreatpotentialofthesenanoparticlesfor theranosticapplications [63].
5Clinicalstatus
Asalreadydiscussed,HAseemstobeavery promisingmoleculeforutilizationasavehicleor asanactiveingredientformanydrugdelivery purposes.TheuseofHAasadermalfillerand forintra-articulardeliveryisalreadywellestablished,andseveralproductshavebeenproved safeandapprovedbytheFDAforclinicaluse [70].However,mostofthescientificresearch conductedontheuseofHAandHANPsfor clinicalapplicationsinotherareasisstillinthe phaseoflaboratoryresearchandpreclinical evaluation.Thismaybeduetotheconsideration ofHAasanewchemicalentityeverytimeitis linkedorconjugatedtoadrugoranymolecules forchangingtheirproperties.Duetothecomplexprocessesinvolvedinsuchreactionsand thevariousfactorstobestudied,HAandits derivativesarealsodifficulttoindustrialize [1] However,severalproductshavereachedphase IIandphaseIIIclinicaltrials.AlchemicaoncologyinAustraliahaveproducedseveral HA-basedsystemsforthemanagementof cancersuchasHA-irinotecan,HA-DOX,and HA-5FU.AphaseIclinicaltrialwasconducted on12patients,andHA-Irinotecanhasproved tobesafeandwelltoleratedwhilepreserving
theanticanceractivityofirinotecan [23].Another phaseIItrialwasdoneon41patients,and showedtheadvantagesoftheHAnanoformulationsintermsofprogression-freesurvivaland safety [71].Unfortunately,phaseIIIclinicaltrials didnotachievetheexpectedresultsandthe reasonforthisisnotyetclear [1].Therefore, theindustrializationandwidespreadclinical utilizationofHAandHANPsasdrugcarriers arestillrichareasthatrequiremoreextensive researchandhavealongwaytogo.
6Conclusion
HAisoneofthemostimportantnaturalcomponentsofhumantissues,makingitabiocompatible,biodegradable,andpromisingcarrier forvariousdrugdeliveryapplications.Theuse ofnanotechnologyhasenhancedtoagreat extentthebenefitsachievedfromthisbiomaterial.Thiscouldbebyusingsystemsthatcan activelytargetcertaintissuesorachieve enhancedpenetrationorpermeationthrough certainbodybarriers.Futurestudiesareneeded toenhancethefabricationtechniquesand ensuretherequiredefficacyandsafetyofthe producednanosystems.
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