Enzyme-Sensitive Biomaterials for Drug Delivery

7/22/2019 Enzyme-Sensitive Biomaterials for Drug Delivery

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5.47 Enzyme-SensitiveBiomaterialsforDrugDeliveryYChauandJZhong,TheHongKongUniversityofScienceandTechnology,HongKong,China2011ElsevierB.V.Allrightsreserved.

5.47.1 Introduction 6055.47.2 Enzyme-SensitivePolymerDrugConjugate 6065.47.2.1 Overview 6065.47.2.2 PolymerDrugConjugatewithPeptideLinkers 6075.47.2.3 PolymerDrugConjugatewithNonpeptideLinkers 6095.47.2.4 TechnicalIssuesinDesignandCharacterization 6105.47.3 Enzyme-SensitiveHydrogel 6105.47.3.1 Overview 6105.47.3.2 HydrogelwithEnzymeSubstratesasPendantGroups 6115.47.3.3 HydrogelwithEnzymeSubstratesasCrossLinkers 6115.47.3.4 EnzymeSensitiveSelfAssemblingHydrogel 6145.47.3.5 TechnicalIssuesinDesignandCharacterization 6175.47.4 Enzyme-SensitiveParticulateCarriers 6185.47.4.1 Overview 6185.47.4.2 EnzymeSensitiveLiposomes 6185.47.4.3 EnzymeSensitivePolymericParticles 6195.47.4.4 EnzymeSensitiveSelfAssemblingPeptideandProteinParticles 6215.47.4.5 TechnicalIssuesinDesignandCharacterization 6225.47.5 Conclusions 622Acknowledgment 622References 622

Glossaryenhancedpermeationandretention(EPR)Aphenomenonofselective,enhancedaccumulation,andprolongedretentionofmacromoleculardrugsintumortissueduetotheleakyvasculatureandimpairedlymphaticsystemofsolidtumor.hydrogelAcrosslinkednetworkofhydrophilicpolymersthatareheldtogetherbychemicalorphysicalbonds.liposomes Nano ormicrosizedvesicleswithmembranesselfassembledbyabilayeroflipidmolecules.

polymerdrugconjugateApolymericdrugconsistingofdrugmoleculescovalentlyattachedtoapolymericcarriervialinkers.Thecleavageoflinkerallowsthereleaseofdrugmolecules.self-assemblyThespontaneousformationofsupramolecularstructuresbyrepeatingunitsorbuildingblocksviathermodynamicallyfavorablenoncovalentinteractions.

5.47.1 IntroductionEnzymesarethecatalystsofbiologicalsystemsandarethereforecentraltonumerousphysiologicalandpathologicalevents.Theycanselectivelyandefficientlyreactonawidespectrumofsubstrateswithvaryingchemistryundermildconditions.Thedesignandsynthesisofenzymesensitivebiomaterialsisaninterdisciplinaryfieldwherebiologicalsciencemeetsmaterialengineering.Withtemporalandspatialcontrolofdrugreleasebeingthemainpurposeindrugdeliverytechnology,animportantthemeistodevelopsmartsystemsthatreleasedrugsinresponsetostimuli.Toconstructasmartdrugdeliverysystem,astimulisensitivematerialisrequired.Muchefforthasalreadybeendevotedtodevelopmaterialsresponsivetophysicalstimuli,includingpH,ionicstrength,temperature, light,andmagnetic field.Withanincreasedunderstandingabout theprofile,expression,substratepreference,andlocationofenzymesand theirassociationwith diseases,aswellas theadvancement in chemicalsynthesis in tailoring materialproperties,onecurrenttrendistoexploitenzymesensitivebiomaterialsfordrugdelivery.

Undernormalconditions,enzymesaretightlyregulatedtocoordinatebiochemicalandbiophysicalactivitiesnecessarytosustainlife,suchasnutritionandenergymetabolism,selfreplication,andtissueremodeling.Enzymesaresoimportanttoorganismsthattheirmalfunctioncanbeanimmediatecauseofdiseases.Theenzymesanddiseasesarecloselyassociatedthereforetheexpressionandactivity of enzyme are good indicators of the progression and prognosis of diseases.This concept can be illustratedwith matrixmetalloproteinases(MMPs),afamilyofzincdependentproteolyticenzymesthatareassociatedwithtumors.TheexpressionofMMP2andMMP9iselevatedwithincreasingmalignantandangiogenicpotentialoftumors.ActiveMMP2evencouldnotbedetectedin

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606 Biopharmaceuticals,InVitroDrugTestingandDrugDelivery

benigntumors[1].Somemembraneanchoredmembersinthefamily,suchasMembraneType1(MT1)MMP,werelocalizedattheinvasivefrontoftumor[2].Thisuniquepropertyoftumorscanbeexploitedtoachieveselectivedrugreleaseonlyatthediseasedtissues(spatialcontrol),ortoreleasedrugsatafasterratewhenahigherdrugconcentrationisrequiredtocontrolthespreadoftumorcells(temporalcontrol).BuildingthistumortargeteddrugdeliverysystemrequiresbiomaterialsthatexhibitachangeofmaterialpropertiesupontheenzymaticreactionofMMPs.Duetotheremarkablesubstrateselectivityofenzymes,therateandextentofresponseofdrugdeliverybiomaterialscanbetailoredatthemolecularlevel.Aswillbediscussedinthenextparagraph,thereisalargevarietyofenzymesavailablefordrugdeliveryapplications.Theirsubstratescoverabroadrangeofbiomolecules,includingprotein/peptide,lipid,glucose,andnucleicacid.Thisbringsflexibilityinmaterialdesignandpreparation.

RecommendedbythenomenclaturecommitteeoftheInternationalUnionofBiochemistryandMolecularBiology(IUBMB),enzymesaredividedintosixclassesaccordingtothereactiontheycatalyze[3].Eachisdividedintosubclassesthatsharecertainspecificityofthereaction.Basedonthissystem,extendedandspecializeddatabasesweredeveloped[46].Uptodate,therehavebeenmorethan4000recordedenzymes.Thesedatabasesarepowerfultoolsforenzymestudyandexploitation.Inaddition,thegenomicandproteomicprojectsareadvancingtheknowledgeaboutenzymesatunprecedentedpace[7].EnzymesthathavebeenexploitedincontrollingdrugreleaseareshowninTable1andtherelatedexamplesarediscussedinthisarticle.Itisevidentthatonlyahandfulofenzymeshavebeenused.Thereisstillmuchroomforinvestigatingenzymesensitivebiomaterialsfordrugdelivery.

Enzymeresponsivenesshasbeenincorporatedinbiomaterialsofdifferentdosageforms,includingpolymerdrugconjugates,hydrogels,andcolloidalcarriers,allofthesetopicsarecoveredinthisarticle.Acommonthemeisdefinedbytheincorporationofenzymesubstrateswithinthebiomaterial.Theenzymaticreactioncausesthebiomaterialtoundergoachemicalorphysicalchangeresultingindrugrelease.Weprovideabriefoverviewaboutthestrategiestoincorporateenzymatictriggersandthedrugreleasemechanism in each type of dosage form.All these approaches involve the use of synthetic chemistry and synthetic biologytechniques, the advancement ofwhich paves theway forscientistsand engineers to invent newenzymesensitive biomaterials.The synthetic component is emphasized. Although some naturally occurring materials are inherently enzymesensitive(e.g.,collagenandfibrin),syntheticcomponentshavetheadvantagesofbeingmodifiabletoachievedesirableproperties.

Examplesfromliteratureareusedtoillustratethestrategiesindesigningenzymesensitivebiomaterialsandhighlightthemainresultsobtainedforeachdosageform.Applicationsdiscussedcoverawiderange,suchascancertargeting,colontargeting,woundhealing,andtherapeuticangiogenesis.Thedrugstobereleasedonenzymatictriggerincludelowmolecularweightchemicalsaswellashighmolecularweightbiomolecules.Wealsodetailthetechnicalissuestobeaddressedindesigningandcharacterizingenzymesensitivebiomaterials.

5.47.2 Enzyme-SensitivePolymerDrugConjugate5.47.2.1 OverviewA typical enzymesensitive polymerdrug conjugate is made up of a minimum of three parts: (1) a polymer carrier, (2) drugmolecules,and(3)enzymesensitivespacersbetweendrugsandthepolymer(Figure1).Althoughthepolymerusuallydominatestheoverallphysiochemicalfeatureandpharmacokineticperformance,inmostcasesitisthespacerthatdetermineswhenandhowthe drug molecules are released.A careful design of spacerswith proper length and structure allows them to act as preferredsubstratesofenzymesandenablesthereleaseofattacheddrugsviaspecificenzymaticreactions.Ideally,drugmoleculesshouldbestableduringdelivery,inactivebeforeenzymecleavage,andfullyfunctionalafter.

Thedevelopmentofenzymesensitivepolymerdrugconjugateanditsspacerdesignhasgonethroughseveralstages.ThefirstreportedwasanNvinylpyrolidine conjugateof mescalineviaadipeptide linker[26].At that time, effortswere focused on theconjugationchemistry.Thespacerswereeithernondegradableordegradablebynonspecifichydrolysis.ThepotentialofpolymericdrugsinbiologicalapplicationwasnotfullyrecognizeduntilitwasclearlydepictedbyRingsdorfinhismodelofpharmacologicallyactivepolymer[27].Thisideainspiredtheemergenceofavarietyofconjugateswithrationallydesignedspacers.Oneexamplewasthespacerssensitivetotheproteasesoracidityinendosomeandlysosomewiththeknowledgethatendocytosiswasthemajorrouteofcellular internalization ofpolymericdrugs.However, endocytosis isanormal event inmostcells, therefore the tumoritropicaccumulationofconjugateismainlyattributedtotheenhancedpermeationandretention(EPR)effect,whichwasfirstdescribedbyMaedain1980s[28].EPReffectisconsideredtoaccountforthepassivetargetingofmacromoleculardrugsinvasculatureimpaired

Table1 EnzymesexploitedformediatingdrugreleaseinenzymesensitivebiomaterialsClass Function Membersexploitedforenzyme-sensitivedrugreleaseinbiomaterialsOxidoreductase Catalyzeoxidation/

reductionreactions Azoreductase [8,9]TransferasesHydrolases TransferafunctionalgroupCatalyzethehydrolysisof

variousbondsTyrosinekinaseCathepsins[1013],alkalineandacidphosphatase[14],matrixmetalloproteinases[1517],

elastase[18,19],lipase[20],lactamase[21],thermolysin[19],plasmin[22,23],urokinaseplasminogenactivator[24],factorXa[25]


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Drug

Drug

Drug

Drug

Drug

Drug

Soluble polymer backbone

Cleavable spacer

Figure1 Schematicillustrationofthegeneraldesignofanenzymesensitivepolymerdrugconjugate.Drugsareinactiveuntiltheyareliberatedfromthepolymercarrierbytheenzymaticcleavageoflinkers.

tissuesandhasbecomeafoundationofthesubsequentdevelopmentofpolymerdrugconjugates[29].Morerecently,conjugatessensitivetodiseaseassociatedenzymesweredevelopedforactivetargeting.Incorporatingspacerslabiletointerestedenzymesisthemajorapproachtoachievethisgoal.5.47.2.2 PolymerDrugConjugatewithPeptideLinkersTheinvestigationof[N(2hydroxypropyl)methacrylamidecopolymerdoxorubicin](HPMAcopolymerDOX),whichwasnamedPK1later,isagoodexampletoillustratethedevelopmentofanenzymesensitiveconjugate.Encouragingresultswereobtainedatdifferentstagesfrominvitroassessmenttoclinicaltrials.DOXwascovalentlyboundtoHPMAcopolymerbyapeptidelinker.Itwasaimedtotargetthelysosomalthioldependentproteases(suchascathepsinB).AtetrapeptideGFLGwasidentifiedasalysosomalproteasedegradablelinkerbasedonascreeningstudy[30].Todeterminethestabilityofoligopeptidesincirculation,anHPMAcopolymercontainingpeptidesidechainsterminatinginchromophorewaspreparedandthenincubatedinratplasmaandserum[31].The release of chromophorewas monitored and the results proved that the sequence GGFLGPwas stable (less than 5%cleavage). Using the central tetrapeptide GFLG as spacer, a HPMADOX conjugatewas constructed and subject to in vitro andanimaltestwithalargepanelofmodeltumors[11].Theconjugatesweredegradedbyisolatedratlysosomalenzymesinvitroandinratliverfollowingintravenousadministration,toliberatefreeDOXinatimedependentfashion.DOXassociatedcardiotoxicitywas remarkably reduced and survival timewas significantly increased in all the cases. Pharmacokinetic study also showed anincreased area under concentrationtime curve (AUC) of total DOX up to 77fold compared to free DOX [32]. Based on thepreclinicaldata,thisconjugateenteredphaseIclinicaltrialasthefirstoneinthisnewclassofchemotherapeutics[33].Decreasedtoxicityandmaintainedanticancerefficiencywereshown.ThemaximumtolerateddoseofDOXof320mgm2wasfivefoldhigherthan free DOX. Itwas a proofofprinciple clinical study for polymerdrug conjugate. In a followup study, the importance ofenzymesensitive spacerwas demonstrated by HPMAplatinate conjugates containing degradable orundegradable peptide sidechain(GFLGorGG)[10].TheHPMAGFLGenPtsignificantlyincreasedthesurvivaltime(T/C=1.43),whileHPMAGGenPtwasalmostinactive(T/C=1.09)atsimilardosagebecausethelattercouldnotliberateanyactiveantitumordrug.

Althoughithasbeenshownthatlysosomalthioldependentproteasesareelevatedinhumantumors[34],theseenzymesmaynotprovidesatisfactoryspecificityduetotheirbroadsubstratespectrumandtheubiquitouspresenceinnormaltissue.Toincreasethedifferenceinenzymaticlevelbetweennormalandtumortissues,aconcepttermedpolymerdirectedenzymeprodrugtherapy(PDEPT)wasproposedbySatchiandDuncan[35].Itisatwostepantitumorapproachusingacombinationofanenzymesensitivepolymerdrugconjugateandpolymerenzymeconjugatetoreleasecytotoxicdrugsselectivelyatthetumorsite.Thecolocalizingofdrugs and enzymes at the tumor tissue is based on the EPR effect of polymers.This conceptwas proved by thework usingHPMAGFLGDOX(PK1)asthepolymerdrugconjugateandHPMAcathepsinBastheactivator[12].Althoughtheenzymaticactivity of cathepsin B decreased to2025% due to polymeric conjugation, the antitumor activity (T/C %)was raised by 16%


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comparedtoPK1alone.InanotherstudyofPDEPT,anonmammalianenzymelactamasewasusedtoactivateHPMAcopolymercephalosporinDOX.Cephalosporinisanlactamsantibioticandseveredasthespacercleavablebythelactamase[21].Majority(80%)ofenzymaticactivitywasretained.Whiletheattacheddrugsshowednoantitumoractivity,thePDEPTcombinationcausedasignificantdecreaseintumorgrowth(T/C=132%).Enzymesfromexogenoussourcearenotpresentinthehumanbodyandcanthereforeminimizenonspecificactivation.However,itincreasestheriskofimmunogenicity.Thisproblemcanbeaddressedbytheconjugationwithpolymerssuchaspolyethyleneglycol(PEG)andHPMA,whicharewellknownforreducingtheimmunogenicityofboundproteins.

Another approach to increase drugtargeting efficiency is to target enzymeswhose activity is minimal in normal tissues butelevated in diseased tissues.A familyofextracellular zincdependentproteases, MMPs,hasbeen shown critical ina number ofpathological events including tumor growth, migration, and metastasis. Because of their special location and correlationwithdisease progression, MMPs are considered promising targets for drug delivery [36]. Chau et al. [17] synthesized a dextranpeptidemethotrexate (MTX) conjugate and investigated important factors for optimal tumor targeting (Figure 2).The linkersequence ProValGlyLeuIleGlywas designed to allow high sensitivity toward MMP2 and MMP9,whichwas confirmedby the enzyme digestion assay. Serum incubation result also suggested satisfactory stability of conjugate in circulation.Antitumor study indicated the antitumor efficiency was significantly increased in vivo compared to free MTX [16]. Thebiodistribution data implied that the observed tumortargeting effectwas mainly attributed to the passive targeting and EPReffect. But the side effect in small intestine was significantly lowered in the conjugate with MMPsensitive linker whencompared to MMPinsensitive analog and an increased therapeutic indexwas thus obtained [15]. This work also revealedand investigated an important issue in enzymesensitive conjugates; that is, the remaining peptide fragmentattached toadrug

Figure2 Schematicillustrationofanexampleofapolymerdrugconjugatesensitivetoendogenousdiseaseassociatedenzyme.ThisconjugatereleasesanticancerchemotherapeuticagentsuponthecleavageofthepeptidelinkerbyMMP2andMMP9[16].Reprintedwithpermission fromChauY,TanFE,andLangerR(2004)SynthesisandcharacterizationofdextranpeptidemethotrexateconjugatesfortumortargetingviamediationbymatrixmetalloproteinaseIIandmatrixmetalloproteinaseIX.BioconjugateChemistry15(4):931941.Copyright2004AmericanChemicalSociety.


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molecule may affect its therapeutic effect.The potency of free MTX in vitrowas found to decreasewith increasing number ofattached amino acids.

Thespectrumofanticancerdrugshasextendedfromcytotoxicagentstoangiostaticandcytostaticagents.Antiangiogenesisandhormonetherapyareimportantsupplementstoconventionalchemotherapy.Thestrategyofenzymesensitiveconjugatewasalsoapplied to these emerging anticancer medicines.Antiangiogenesis inhibitorTNP470was conjugated to HPMA copolymerviapeptidelinker[37].ThelinkersequenceGFLGisselectedbecauselysosomalcysteineproteaseswerealsooverexpressedbytumorendothelial cells [38]. Selective accumulation of conjugate in tumorvessels suggested successful targeting. Neurotoxicity is acommonsideeffectaccompaniedwithTNP470athigherdosagenecessaryfor tumorregression. It isnoteworthy thatthissideeffectwascompletelydiminishedintheinvivostudybecausethebulkyconjugatewaspreventedfromcrossingthebloodbrainbarrier.

Similarly, the strategyof enzymespecific polymerconjugatewas exploited in hormone therapyand incombinationwithcytotoxicchemotherapy[13].Aminoglutethimide (AGM)isanaromataseinhibitorthatiseffectiveindecreasingestrogenleveland is commonly used in breast cancer treatment.AGMwas conjugated to HPMA copolymer by itself (HPMA copolymerAGM) or in combination with DOX (HPMA copolymerDOXAGM) via the tetrapeptide GFLG. Synergistic effect wasobserved in HPMA copolymerDOXAGM. Further investigation revealed that HPMA copolymerGFLGAGMwas able toinhibit themitogenic effect and aromataseactivity in breast cancer cells but failed to suppress the activityofaromatase inacellfreesystem.ThisresultconfirmedthattheintracellularenzymaticreleaseofAGMisacriticalstepfortheinhibitoryactivityofHPMAcopolymerAGMconjugate.Thisfeature,togetherwithEPReffect,mayfacilitatethelocalizedactivationofconjugateat tumorsite.

Photodynamictherapy(PDT)isanothercancertreatmentthatbenefitsfromenzymemediatedactivation.ConventionalPDTuses combinations of chemical phtotosensitizers and light to produce cytotoxic singlet oxygen.The tumor targeting usuallycomes from local illumination. However, the poor specificity of photosensitizers causes phototoxicity to lightexposed tissuessuch as eyes and skin.A novel strategy termed proteasemediated PDT (PMPDT)was developed to achieve tumortargeteddeliveryofphotosensitizers[39].Multiplecopiesofphotosensitizerchlorinee6(Ce6)werecoupledtoapolyLlysinebackbonegraftedwith PEG to achieve selfquenching. Due to the conjugation to a polymeric carrier, improved pharmacokinetics andpassivetargetingofCe6totumorwereobserved.Thepolylysinebackbonewasdegradabletoseveraltumorassociatedproteasesincludingcathepsins andMMPs.Theselectiverecoveryof fluorescence and singletoxygengeneration ofCe6 in thepresence ofproteases and lightwere observed both in vitro and in vivo.Tumor growthwas suppressed by 50% in an animal study.Togeneralize this system to other enzymes associatedwith different diseases, a second generation of polymeric photosensitizerprodrugsinwhichpeptidelinkercanbetailormadewasreported[40].Toproveconcept,atrypsinsubstratesequenceGlyThePheArgSerAlaGlywas used as the linker between photosensitizer pheophorbide and the polymeric carrier polyLlysine.Selectiveactivationbytrypsinwasobserved invitro.5.47.2.3 PolymerDrugConjugatewithNonpeptideLinkersTheactivationcanbeachievedbyproteasesaswellasotherclassesofenzymes,byenzymesofhumancellsaswellasmicroorgan-isms.Asthelinkerservesastheenzymesubstrate,thelinkerchemistrymayvarydependingonthenatureofenzymaticreactions.Since microbial enzymes of azoreductase activity in colonwere found, sitespecific delivery can be achieved by polymerdrugconjugates containing aromatic azo linkers.An antiinflammatory drug 5ASAwas coupled to polyamidoamine dendrimerviaaromaticazobondforcolondiseasetreatment[8].Incubationtestintissuehomogenatesuggestedthatdrugreleasepredominantlytookplaceinlargeintestinebutnotintheupperintestine.DetailedbiodistributionandpharmacokineticstudywasconductedonanotheraromaticazolinkagecontainingconjugateHPMAcopolymer9ACforcoloncancertreatment[41].Itwasshownthattheconcentrationofreleased9ACfromconjugateexceededfree9ACincolontissueswhileconcentrationwaslowinplasma.Morerecently,anazoreductasesensitiveconjugatePEGmesalazinewastestedininducedcolitismodel[9].Encouragingresultsinbothtissuespecificityandtreatmenteffectivenesswereachieved.

Enzymesensitiveselfimmolativedendrimers[42]orcascadereleasedendrimers[43]consist ofanenzymesubstrateat thecore of the dendrimer and multiple drug molecules attached to the periphery of the tails (Figure 3).The linkages betweenthe drugs and the polymers are able to decompose through a chain of selfelimination reactions.The reactions are initiatedby the unmasking of an amine at the dendrimer core and completed by the subsequent shifting of conjugated electron pairs(1,8elimination[42]and1,4quinonemethiderearrangement[43])alongthedendrimerbackbone.Moreover,thesereactionscan take place in aqueous environment.These novel dendrimers are structurally designed to readily release all of its drugmoleculesanddissociateintofragmentsuponasingleenzymaticreaction.Thisisunlikethemechanisminpreviousdiscussionin which one enzymatic cleavage causes the release of one drug molecule.Another notable feature is that, for the drugsconjugatedvia its amine or hydroxyl groups, they can be released in their original formwithout any remaining fragmentfrom spacer.The application of this promising system in enzymesensitive drug deliverywas pioneered by Shabat. DOX andcamptothecin(CPT)wereconjugatedseparatelyorincombinationtoafirstgenerationselfimmolativedendrimertriggerablebyachemicalinduced antibody 38C2,whichwas designed to catalyze a sequence of retroaldol retroMichael cleavage reactions[44,45].Thedendrimersweremuchmoretoxicinthepresenceof38C2thanintactconjugatebytwoordersofmagnitude,andthe potencywas comparable to free drugs.When DOX and CPTwere loaded on the same dendron to form a heterodimericconjugate, a 1000fold potency difference before and after cleavagewas observed. In another conjugate, a single cleavage by


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Activation

Activation

(Overall)

Spontaneous

Spontaneous

Figure3 Schematicillustrationofacascadereleasedendrimer.Asingleactivationofasecondgenerationcascadereleasedendrimertriggersacascadeofselfeliminationsandinducesreleaseofallendgroups[43].ReproducedfromdeGrootFMH,etal.(2003)Cascadereleasedendrimersliberateallendgroupsuponasingletriggeringeventinthedendriticcore.AngewandteChemieInternationalEdition42(37):44904494.CopyrightWileyVCHVerlagGmbH&Co.KGaA.Reproduced withpermission.

penicillinGamidasewasabletoreleasefourCPTmolecules.Thereleaseprofileshowedefficientdrugliberationbyanenzymewhilefreedrugwasalmostnondetectableotherwise.Theactivatedconjugatewasmoretoxicthanitsintactformbythreeordersof magnitude in all cell lines tested [46].This dendrimerbased drugdelivery system is promising because it can amplify thesignalofenzymaticstimulusbyhigherdrugreleaseefficiency.Thisclassofselfimmolativedendrimersisespeciallysuitablefortumortargeting,becausetheintactbulkyconjugatecantakeadvantageoftheEPReffectandthefragmentsafterdecompositioncanbeeasilyexcreted.5.47.2.4 TechnicalIssuesinDesignandCharacterizationA number of tests are commonly performed to characterize an enzymesensitive polymerdrug conjugate. Drug loading is animportantparameterandmustbedeterminedtoallowthecomparisonoftheperformanceofaconjugatewiththecorrespondingfree drug at equivalent dosage. Spectroscopy (nuclear magnetic resonance, ultraviolet spectrum, infrared spectrum, and atomicabsorption),colorimetry(byintrinsicchromophoreorlabeledgroups),andhighperformanceliquidchromatography(HPLC)areconventionaltechniquesfordeterminingdrugconcentration.

Inmostcases,thedrughastobeliberatedfromtheconjugatetotakeeffect.Differentdesignsoflinkersallowawidevariationofcleavability. Drug release assay serves the purpose to (1) indicatewhether specific enzymatic reaction triggers drug release; (2)quantifythedrugreleasekinetics;and(3)measurethestabilityofpolymerdrugconjugatesduringcirculation.Conjugateswillbeincubated in a media mimicking environments that the conjugates encounter. For example, buffer at pH 7.4simulates thephysiologicalconditionofbodyfluidwhilelowpHbufferat5.5mimicstheconditionsinthelysosomalcompartment. Enzymeswillbeaddedtoprovidethetriggerfordrugreleaseandtheresultswithandwithoutenzymaticincubationarecompared.

Sometimes,thetherapeuticpotencyofaconjugateisgreatlyimpairedrelativetofreedrugeventhoughthereleaseisspecificandefficient.Amajorreasonisthatthereleaseddrugmaynotbeexactlyintheformoftheoriginalone.Dependingonthelengthandsiteoftheremainingfragmentofthespacer,andthetoleranceofparticulardrugreceptor,therecanbesevereornoinfluenceonthedrugpotency.Thestructureofreleasedtherapeuticcanbepredictedbasedontheunderstandingofcleavagereactionandcanbefurtherconfirmedbymassspectroscopy.

5.47.3 Enzyme-SensitiveHydrogel5.47.3.1 OverviewInthedesignofenzymesensitivehydrogel,thegoalistoprovidealocalizeddeliverydepotthatiscapableofreleasingdrugsorproteinsondemand,asdefinedbythelevelofspecificenzymes.Enzymesubstratesareincorporatedintothehydrogelaspendantgroupsorcrosslinkers.Inanemergingclassofmaterial,selfassemblinghydrogel,theenzymesubstrateisembeddedwithineach


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building block. Enzymatic reactions on these substrateswill lead to either breakage or change of property thatwill cause drugrelease,whichmayormaynotbeassociatedwiththedissolutionofthehydrogel.

5.47.3.2 HydrogelwithEnzymeSubstratesasPendantGroupsEnzymetriggeredrelease ispossiblewhenadrugorprotein isattached to thehydrogelmatrixviapendantpeptide linkers thatare cleavable by specific proteases.This idea has been implemented in a microbial infectionresponsive drugdelivery system[47,48].AsinfectionbyPseudomonasaeruginosaandStaphylococcusaureusleadstohigherthrombinlikeproteolyticactivity,shortpeptidelinkers,G(D)FPRGFPAGG,areconstructedaspendantgroupsintopolyvinylalcohol(PVA)basedhydrogelfor immobilizing gentamicin, an antibiotic. It was found that in the presence of the microbialinfected wound fluid,biologicallyactive gentamicin capableof killingbacteria cellswasreleased.When exposed tononinfected fluid, nogentamicinreleasewas detected.This hydrogel further demonstrated its usefulness aswound dressing by its in vivo bactericidal effect in aratmodel. Itwasproposed thatby targeting thereleaseviabacterialenzymemediation, theriskofemergenceofdrugresistantbacteria could be reduced. It should be noted that the proteolytic profile of the bacteriainfected wound was not fullycharacterized.The authors suggested that the thrombinlike activity cleaved the peptide between arginine and glycine, andleucine aminopeptidaselike activity was present to liberate the biologically active gentamicin from the remaining peptidefragment.

AnotherhydrogelthatmaybeusedforwoundhealingisaPEGbasedhydrogelthatexhibitshumanneutrophilelastase(HNE)-responsivedelivery[18].Insteadoftargetingbacterialenzymes,thisgelrespondstoanendogenousproteaseexpressedbyactivatedneutrophilsduringinflammation.AnsethslabphotopolymerizedHNEsensitivepeptidesubstrates(e.g.,AAPVRGMG)intoaPEGdiacrylatehydrogelaspendantgroups.ThechainlengthofPEGbetweencrosslinkingpointswasselectedtobe10kDatoensurethatHNE(~29.5kDa)couldpenetrate into thegel toperform cleavage.Bychangingtheaminoacidsnext to thescissilebond,peptidesubstratesdisplayingvaryingdigestionkineticswereobserved,andtherateofpeptidecleavagewasfoundtocorrelatewiththereleaseofpeptidefragmentsfrom thehydrogel.Thus,altering thesubstratesensitivitytoward theenzymeprovidedadirectmethodtotunethedrugreleaserate.

Gemeinhart and coworkers [49] reported an MMP triggerable delivery system of cancer chemotherapeutics by complexingcisplatintopendantpeptidesinahydrogel.CysteineterminatedMMPsubstratesequences(CGLDD)weregraftedtoaPEGbasedhydrogelvia Michael addition reactionwith the acrylate groups on functionalized PEG (Figure 4). Itwas reported that theincubationwithMMP2andMMP9causedthereleaseofpeptidedrug,LDDcisplatin,giventhatthemeshsizeinthehydrogelallowedtheaccessofenzymes.Thus,thehydrogelwasenzymeresponsivewhenPEGchainlengthwas~4000butnotwhenthelengthwas~574.However,therewasasubstantialnonspecificreleaseoffreecisplatinduetotheweakchelationbondwithcarboxylgroups.AnotherconcernwasthatLDDcisplatinexhibitedadrugpotencyanorderofmagnitudelowerthanfreecisplatin.Theseproblemswereaddressed bycomplexingcisplatinwith theamine grouponMMPcleavablepeptide(KPAGLLGC).Therewasareductioninnonspecificreleasebuttheamountremainedsignificant(equalingapproximately1/3oftheMMPspecificrelease).ThedrugpotencywassimilarbetweenfreecisplatinandthereleasedproductcisplatinKPAG.Thehydrogelwaseffectiveinexertingcytotoxicityagainstbraincancercells(U87MG)invitro.

Proteins can be attached to the hydrogel network via proteolytically degradable linker to achieve triggered release.Recombinant proteins are genetically altered to make them amenable for covalent attachment to the polymer chains. Inone example, vascular endothelial growth factor (VEGF) was mutated to carry an additional cysteine residue at theCterminus to facilitate its grafting tovinyl sulfonefunctionalized PEGvia Michael addition [22].As a cleavage site forplasmin is present near the Cterminal ofVEGF, the immobilized proteinwas released by the incubationwith plasmin.Importantly,VEGF released retained thebiological activity of simulating the proliferation ofendothelial cells, aprerequisite for bloodvessel growth (angiogenesis). The celldemanded release is a desirable attribute to provide a spatial and temporalcontrol ofVEGF concentration to achieve therapeutic angiogenesis in ischemia andwound healing. Genetic engineering ofproteins offers newways to conjugate protein to a polymer chainwith different chemistries.Another example involved theadditionofashortsegmentofglutamineacceptorpeptidetoVEGF,allowingittocoupletoPEGcappedwithMMPsensitive lysine donor peptidesvia the transglutamase reaction of factorXIIIa [50].The release ofVEGF by MMP1 digestionwasdemonstrated.

5.47.3.3 HydrogelwithEnzymeSubstratesasCross-LinkersIn the second type of enzymesensitive hydrogel, peptides are incorporated as crosslinkerswithin otherwise nondegradablehydrogel. Macromers are either modifiedwith peptides at the chain ends for polymerization, or crosslinkedwith each otherusing peptide sequences (Table2). Drug molecules are mixedwith the macromers prior to the gelation reaction, and becomephysicallyentrappedwiththehydrogelnetwork.Duetotherelativelylargeporesizes,thesehydrogelmaterialsareusefulforthecontrolleddeliveryofmacromoleculartherapeuticssuchasproteinsbutnotlowmolecularweightcompounds.Theporesizewillaffecttherateofproteinleavingthehydrogelviadiffusion.Itwillalsoinfluencewhetherthehydrogelundergoesbulkdegradationandsurfacedegradation.Thedegradationprofileand,thus,thereleasekineticsofthehydrogelaredependentonthesensitivityofthepeptidelinkerstowardspecificenzymes.Thechoiceofpeptidelinkerthusbecomesanimportantdesignfactorforthistype


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Device

MMP

Cell

Cell death

Hydrogel backbone

MMP-sensitive peptide

Cleaved peptide

Chemotherapeutic agent

Figure4 Schematic illustration of an example of an enzymesensitive hydrogelcontaining enzyme substrates as pendant groups. This hydrogelexhibitsMMPtriggeredreleaseofchemotherapeuticagentsthatarecomplexedtothehydrogelviapeptide linkers[74].Reproduced withpermissionfrom Tauro JR and Gemeinhart RA (2005) Matrix metalloprotease triggered delivery of cancer chemotherapeutics from hydrogel matrixes.Bioconjugate Chemistry 16(5): 11331139. Copyright 2005 American Chemical Society.

ofmaterial.Thistypeofmaterialshaswidespreadapplicationsindrugdelivery.Thecellresponsivenessalsomakesthempromisingscaffoldingmaterialsforoftissueengineering.

West and Hubbell [23] pioneered a method for synthesizing proteasedegradable hydrogel by the photopolymerization ofpeptidecontainingblockcopolymers.TheblockcopolymerconsistsofPEGflankedwithshortpeptidesequencesonbothends,which are in turn cappedwith lightreactive acrylate groups. Itwas concluded that the peptide sequence did not interferewithpolymerizationandthepolymerdidnotinhibitproteolysis.Thishydrogelshowedbulkerosioninthepresenceofspecificenzymes,butnotunderconditionswithoutenzymesorwithotherenzymes.Intheproofofconceptexperiments,thehydrogelswerefoundsensitivetocollagenase(anothernameforMMP1)andplasmin.Theauthorspointedoutanddidshowlatertheirpotentialin


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Table2 ExamplesofhydrogelscontainingpeptidecrosslinkerscleavablebyspecificproteasesSequenceofpeptidecross- Mechanism ofdegradationand

Methodofhydrogelsynthesis linkers Targetedenzyme proteinrelease PotePhotopolymerizationofacrylatecappedpeptide APGL MMP1 Bulkdegradation Wou

PEGpeptideblockcopolymers GGLGPAGGK Plasmin meVRN ElastaseAAAAAAAAAK

RedoxpolymerizationofNIPAAM,AAcandpeptide QPQGLAK MMP13,also Bulkdegradation Bonediacrylate sensitiveto recollagenase os

Michaeladditionreactionofvinylsulfone GCRDGPQGIWGQDRCG MMPs(MMP1 Bulkdegradation;degradation- BonfunctionalizedmultiarmPEGsandbiscysteine GCRDGPQGIAGQDRCG andMMP2) controlledreleaseofprotein bocontainingpeptides precipitatedwithingel

Photopolymerizationofnorbornenefunctionalized CGAAPVRGGGGC HNE Surfacedegradation;degradation TreamultiarmPEGsandbiscysteinecontaining CGAAP(Nva)GGGGGC controlledzeroorderrelease prpeptides

PEG,poly(ethyleneglycol);NIPAAM,Nisopropylacrylamide;AAc,acrylicacid;MMP,matrixmetalloproteinase;HNE,humanneutrophilelastase;NVA,allpeptidesequencesarewritten inrepresentedbyNva.


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regenerativemedicine,inspiredbythefactthenaturalextracellularmatrixwasdegradedbyenzymeslocalizedoncellsurfaces[51].Althoughthedrugreleasekineticsfromthismaterialhasnotbeenreported,itshouldbeusefulforproteinreleasedirectedagainstinflammatorydiseases,whichoverexpresscollagenaseandplasmin,suchasvarioustypesofcancers.

UsingpeptidediacrylatetocrosslinkNisopropylacrylamide(NIPAAM)andacrylicacid(AA),KealyandHealy[53]preparedaproteolytically degradable, thermo (and pH)sensitive hydrogel.The peptide sequence, derived from type II collagen,was asubstratepreferredbyMMP13,anenzymeoverexpressedduringbonedevelopmentandseveralinflammatorydiseases(e.g.,breastcancerandosteoarthritis).Potentialapplicationsofthisgelincludebonetissueregenerationandsitespecificdrugdelivery.Becauseof the presence of NIPAAM, this hydrogel exhibits lower critical solution temperature (LCST) behavior, that is, an increase inmechanical rigidity above the critical temperature an ideal property for injectable material.To ensure that the gel is easilyinjectable,thepeptidecrosslinkingdensitymustbekeptatalowvalue,sothatthegelwouldflowbelowLCST.Asthetypesofdrugssuitableforreleasefromthehydrogel,thereleaserate,aswellasthedegradationofthegeldependsonthecrosslinkingdensity,thisconstraintwilllimittherangeofdrugdeliveryapplications.

The use of proteolytically degradable hydrogel for protein releasewas demonstrated by Hubbell and coworkers. Bonemorphogenicprotein(BMP2)andtransforminggrowthfactor(TGFb1)havebeenphysicallyentrappedinandreleasedfromanMMPdegradablePEGpeptidehydrogel[54,56].Intheformerexample,BMP2enhancedtheprocessofboneregenerationandwasfoundtoacceleratethehealingofcranialdefects.Inthelatter,TGFb1activatedMMP2andenhancedthehydrogeldegradationbyMMPsecretingendothelialcellsaprocessthatwouldpromotevascularhealing.ThehydrogelwassynthesizedbyamildandeffectiveMichaeladditionreactionbetweenvinylsulfonefunctionalized multiarmPEGsanddithiolcontaining peptide linkers.Enzymemediateddegradationofthehydrogel followedzeroorderkineticsindependentofthepeptidesubstrateconcentration.This observation supported that the enzymes were fully saturated with peptide substrates (i.e., substrate concentrationSoMichaelsMentenconstantKm).As4armPEGsofmolecularweightof20kDawereusedasthebuildingblocks,themolecular

weightbetweencrosslinkingpointsapproximatedto5000Da,yieldingalargemeshsizeinthehydrogelforboththediffusionofproteolyticenzymesandproteindrugs.IntheabsenceofMMPs,however,majorityoftheproteindrugsremainedentrapped.Therelease of proteinwas triggered by the proteolytic digestion of the hydrogel.The authors explained that the protein moleculesprecipitatedwithinthegelduetotheirpooraqueoussolubility,thattheywereliberatedonlyafterthegelwasdegraded.Becauseofthelargemeshsize,thebreakageofthepeptidecrosslinkersoccurredhomogeneouslythroughoutthegel.Bulkdegradationwasobservedinsteadofsurfacedegradation.

Surfacedegradationcanbeachievedintheproteolyticallydegradablehydrogelifthediffusionofenzymesismuchslowerthantheenzymaticcleavageofthepeptidelinkers.Forthistypeofhydrogel,thereleaserateoftheproteincanbebettercontrolledasitissolelydeterminedbythegeldegradationkinetics.Furthermore,aconstantreleaseratecanbeachievedprovided(1)thesurfaceareaofthehydrogeldeviceremainsconstant(suchasinthecaseofflatdisc)and(2)thegeldegradationfollowszeroorderkinetics(whenSoKm).AnsethsteamdemonstratedsuchanexampleofproteinreleasefromHNEdegradablePEGpeptidehydrogel.Thematerialwaspreparedusingthiolenephotopolymerization(Figure5).UnliketheworkfromHubbellgroup,4armPEGsof5kDawereused,andthusthemeshsizeoftheresultinghydrogelwassmaller.Thisrestrictedthemovementofboththeproteinentrappedandthepenetrationoftheproteolyticenzymesanecessaryconditionforproteinreleasedominatedbyenzymemediatedsurfacedegradation.Thehypothesiswasconfirmedbyshowingthatproteinsofdifferentsizeswerereleasedataratecorrelatingtothegeldegradationandthegelmaintainedthesamecrosslinkingdensitythroughoutthecourseofdegradation.5.47.3.4 Enzyme-SensitiveSelf-AssemblingHydrogelSelfassemblinghydrogelconsistsofrepeatunitsthatspontaneouslyorganizeintoasupramolecularstructurewiththecapabilitytoholdwater.Therepeatunitsarelowmolecularweighthydrogelators,whichinteractwitheachotherbynoncovalentforcestobuildnanoscale structures.Very often, nanofibers are formed by the ordered arrangement of the hydrogelators and these nanofibersentangletoformawaterencapsulatingnetwork.Toexploitnoncovalentinteractionseffectively,designthemesofselfassemblinghydrogelatorsareborrowedfromnature.Theseincludeamphiphilicityandselfassemblingpeptidemotifs.Asynergyofnoncova-lentforces,suchashydrogenbonding,vanderWaalsinteractions,andpipistacking,isrequiredtoconstructthenanostructures.Tomaketheselfassemblinghydrogelenzymeresponsive,atriggerthatisapreferredsubstrateofatargetenzymeisincorporatedintothehydrogelatororitsprecursortoregulatethenoncovalentinteraction.Upontheenzymaticreaction,theselfassemblingpropertyisturnedeitheronoroff.Designingenzymesensitivehydrogelasadrugdeliverydepotusingthisbottomupapproachrepresentsanewandemergingtrend.Triggerincludesawiderangeofenzymes:proteases,phosphatases,kinases,esterases,lipases,andbacterialenzymes.

Peptidesareamongthemostcommonlyusedbuildingblocksinselfassemblinghydrogel.Becausepeptidesarealsosubstratesof many enzymes, enzyme sensitivity can be readily incorporated by including a preferred amino acid sequence into the selfassemblingpeptide.Therearethreemajorclassesofselfassemblingpeptidesandtheexamplesofusingenzymestomediatedrugdeliveryineachgrouparediscussed.

Zhangetal. [57]pioneeredthedesignofionicselfcomplementarypeptides.Theselfassemblingsequenceconsistsofalternatingoppositelychargedaminoacidsspacedwithhydrophobicresidues.Zhangandcoworkersshowedthatahydrogelconstructedusingionicselfcomplementarypeptides((RADA)4)couldbeusedforthecontrolledreleaseofproteinscoveringarangeofmolecularweightsandisoelectricpointswhilepreservingtheirfunctions[58].Here,proteinsarephysicallyencapsulatedwithinthehydrogeland thereleasekinetics isdominated bydiffusion.Onecan envision thatenzymemediateddegradationof theselfassembling


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CH CH CH CH CH

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P1 P1' N C C C C C

n O

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CH3 CH3

NH2

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Figure5 Schematic illustration of an example of an enzymesensitive hydrogel containing enzyme substrates as crosslinkers. This hydrogel isformedby thiolenephotopolymerization andexhibits human neutrophilelastasetriggered release ofproteins [55]. Reproduced fromAimettiAA,Machen AJ, and Anseth KS (2009) Poly(ethylene glycol) hydrogels formed by thiolene photopolymerization for enzymeresponsive proteindelivery. Biomaterials 30(30): 60486054. Copyright (2009), with permission from Elsevier.hydrogelcouldbeusedasanothermechanismtocontroltherateofdrugrelease(achievablewhenthecharacteristicdiffusiontimeisrelativelylongcomparedtodegradationtime,e.g.,whenproteinmoleculesarelargeandtheselfassemblingpeptidedensityishigh).Toincorporateenzymesensitivityintoionicselfcomplementarysequence,weflankedasixaminoacidlongMMPsubstrate(PVGLIG)withthreerepeatsofRADAonboththeN andCterminals[59].Similartotheoriginal(RADA)4,thepeptidescouldselforganizeintobetasheetdominantsecondarystructureandformahydrogelinthepresenceofsalt.UponthedigestionbyMMP,thepeptideswerecleavedintoshorterfragmentsthatexhibitedmostlyrandomcoilsunderaqueousconditions.WhenthehydrogelwasexposedtoMMP,surfaceerosionwasobservedasevidencedbyadecreaseinsurfacehardness.However,proteinreleasestudyhasnotbeenperformedinthishydrogel.

Tung and coworkers [24] designed a urokinase plasminogen activator (uPA)sensitive ionic selfcomplementary peptidehydrogelanddemonstratedenhancedreleaseoflowmolecularweightdrugsinthepresenceofthetargetenzyme.ElevateduPAisassociatedwithanumberofmalignancies,andthereforethehydrogeldepotispotentiallyusefulfortumortargeteddrugrelease.Thepreferredsubstrate,SGRSANA,wasinsertedbetweenrepeatsofKLDL.Thepeptideswereable toformbetasheetsecondarystructures and assemble into a hydrogel under physiological conditions. Upon proteolysis, the cleaved peptides lost the selfassemblingpropertyandbecamedetachedfromthegelmatrix(Figure6).Tomakethehydrogelausefuldepotforthecontrolledreleaseoflowmolecularweightdrugs,thedrugmoleculeswereattachedcovalentlytotheselfassemblingblockstopreventtheirburstreleasefromthehydrogel.Inthisexample,fluorescenttagsasmodeldrugmoleculeswereattachedtotheaminoterminioftheselfassemblingpeptides.WhenthegelwasexposedtouPA,thefluorescentdrugs(stillappendedtothepeptides)werereleaseduponenzymaticdigestion.Toencapsulateamitochondrialdisruptionpeptide,whichisanapoptoticagentagainsttumorcells,thepeptidedrugwascovalentlyattachedtoKLDLrepeats.Similartothecaseoffluorescenttags,themodifiedapoptoticpeptideswerereleasedbythetriggerofuPAdigestioninanenzymeconcentrationdependentmanner.Therateofmatrixdegradationanddrugreleasedependedontheenzymesubstrateconcentrationwithinthegel.

Stuppandcoworkers[60]pioneeredthedesignofpeptideamphiphilemoleculescapableofselfassemblingintonanofibroushydrogel.ThemoleculeconsistsofapolarpeptidesequenceNacylatedwithanonpolarfattyacid.Selfassemblyintonanofibersisaffordedbyaproperbalanceofamphiphilicityandtriggeredbymildconditions(neutralpHandthepresenceofdivalentcations).Hartgerinkandcoworkers[61]incorporatedanMMP2cleavablesequence,GTAGLIGQ,betweenthepolarandnonpolarportionofthepeptideamphiphileanddemonstratedthatthehydrogelwasdegradablebytheproteolysisofMMP2.Macroscopically,theweight of the gel decreased significantlywhen incubatedwith the enzyme (but notwhen incubatedwith buffer containing no


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Fragments

Drug

5 mm

-sheetsegment

-sheetsegment

Targetenzyme

Solventexchange

Protease cleavagesite

Figure6 Schematicillustrationofanexampleofanenzymesensitiveselfassemblinghydrogel.Thebetasheetformingpeptidesselfassembleintoahydrogel.Drugmoleculesareattachedtothepeptideunitsandarereleasedsubsequenttotheproteolyticcleavagethatdisruptstheselfassemblingproperty[24].ReproducedwithpermissionfromLawB,WeisslederR,andTungCH(2006)Peptidebasedbiomaterialsforproteaseenhanceddrugdelivery.Biomacromolecules7(4):12611265.Copyright2006,AmericanChemicalSociety.

enzyme),indicatingthepeptideamphiphilemoleculesweredetachedfromthenanofibernetworkuponproteolysis.Interestingly,Transmission Electron Microscopy (TEM) images revealed that MMP2 caused a change in the nanofiber morphology, fromnanofiberswith nearly uniform diameters to a combination of eggshaped fibrillar aggregates and twisted ribbons ofvaryingwidths.Asthiskindofenzymesensitivehydrogelmimicshowtheextracellularmatrixisdegradedduringwoundhealingandtissuedevelopmentinnature,itisausefulmaterialforconstructingtissueengineeringscaffold.Theauthorsdemonstratedthatthegelcouldencapsulatecellsandallowtheirmigration.Potentially,thisgelwillalsobeusefulfordeliveringdrugsataratecontrolledbythelevelofdiseaseassociatedproteases.

Hydrophobic drugs can be encapsulated into the hydrophobic core of the nanofiber assembled by peptide amphiphiles.However,loadingefficiencywaslowbecauseofthesmallspaceandthelimitedpartitionofdrugsintothecore.UsingasimilarMMP2peptideamphiphileastheselfassemblingblock,Kimetal. [62]devisedanapproachtotunethereleaseofachemother-apeuticcompound,cisplatin,basedontheenzymeexpression.Insteadofusingcalciumions,cisplatinwasusedtotriggertheselfassemblybycomplexingwithcarboxylicgroupsofasparticacidlocatedneartheCterminalofthepeptideamphiphile.Inessence,cisplatinbecameanintegralpartofselfassembly,shieldingtherepulsionofnegativechargesandactingascrosslinkersbetweenpeptideamphiphile molecules and between nanofibers. High loading could be achieved.The gelwas mechanically strongercomparedtothatformedusingcalciumions.WhenincubatedwithMMP2,therewasnonoticeabledecreaseinthegelweight.However,TEMimagesrevealedshorteningofnanofibersandreleaseofcisplatinwasalsoaugmented.However,itshouldbenotedthatthisgelexhibitedasubstantialburstreleaseandahighpercentageofcisplatinwasreleasedevenwhenMMP2wasabsent.Thiswas probably because cisplatin moleculeswere only physically entrappedwithin the nanofiber networks. For those cisplatinmoleculesthatwerecomplexedwithpeptideamphiphiles,theywerereleaseduponenzymaticdigestionofpeptideamphiphiles.However,thereleaseddrugwasnotfreecisplatin,butcisplatincomplexedtoapeptidefragment.

Aromatic shortpeptidesareanother classofmolecules capable ofselfassembling intosupramolecularhydrogel.Theuse ofaromaticacidsforhierarchicalassemblyisathemeusedinnature,forexample,intheformationofamyloidfibers.Indesigningselfassemblingbuildingblocks,besidesadoptingaminoacidswitharomaticsidechains,thepeptidesmaybefurthermodifiedwitharomaticmoleculessuchasfluorenylmethyloxycarbonyl(Fmoc)andnaphthalene(Nap).Residuesthataresensitivetoenzymescanbeincorporatedintothebuildingblocktoregulateselfassembly.EnzymatichydrogelationhasbeendemonstratedbytheworkofXusandUlijnslabsusingarangeofenzymes(e.g.,tyrosinephosphatase,betalactamase,thermolysin,esterase,andMMP)[63].Forapplicationsindrugdelivery, itisdesirableto trigger thedrugreleasewithanenzymaticreactionwhentheenzymelevelisassociatedwiththediseaseprogression.Xuandcoworkers[64]designedashortaromaticpeptide,NapFFGEY,toachievethisgoal.Theincorporationoftyrosine(Y)inthesequenceenablestheuseofatyrosinekinase/phosphataseswitchtoalterthestatusofphosphorylation of this residue.When dephosphorylated, the sequence is capable of forming nanofiber networks.When thepeptideisphosphorylated,thenegativechargeofthephosphategroupincreasesthesolubilityofthesequenceandtherepulsionamongthepeptidesequences.Thenanofibersaredisassembledandthegelischangedintoasolution.Thisisapromisingmaterialforenzymetriggereddrugrelease.

Morerecently,theenzymesensitiveselfassemblingmotifiscovalentlylinkedtoadrugmoleculetoconstructabuildingblockfor formingaselfdelivery hydrogel. In addition tohigh loading efficiency andenzymemediated release, the advantage of thisapproachisthatthedegradationproductsofthehydrogelconsistofsimplydrugmoleculesandwelldefinedlowmolecularweightmolecules,asopposedtoacomplexmixturefromapolymerichydrogel.Achemotherapeuticdrug,taxol,wascovalentlyconjugatedtoanaromaticshortpeptidesequence,NapFFKYp[65].Uponincubationwithtyrosinephosphatase,ananofibroushydrogelwasformed.A steady drug releasewithout initial burstwas observed and the released taxol, albeit modifiedwith a short peptidesequence,retainedtheantitumorpotency.Duetothepresenceoftyrosineintheselfassemblingmotif,thedrugreleasecanpossiblybetriggeredbykinase.However,therehasnotbeenanyreportoftheexperimentalproofyet.


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Vermulaetal. [20]reportedaselfdeliveryhydrogel thatexhibits triggeredrelease inresponse to lipase.Acetaminophen,a common pain reliever, was covalently conjugated to a fatty acid to form selfassembling bolaamphiphiles. The selfassembly property required finetuning between hydrophilicity and hydrophobicity, and employed hydrogen bonding,pipi stacking, andvan derWaals forces to provide the enthalpic drive. Because the covalent linkage between acetamino-phen and fatty acidwas a lipaselabile ester bond, incubationwith lipase caused the gel to degrade and release the freedrug during the process. (The gel remained intactwithout any drug release in the absence of lipase.)The hydrogel couldalso provide enzymemediated release of a second drug.The authors demonstrated thiswith curcumin, an antiinflamma-torycompoundextractedfromcurry.Curcuminishydrophobicandcarriestwophenylgroups.Thisallowsthemoleculestobe trapped in the hydrophobic pockets of the selfassembled hydrogel and stabilizes the physical encapsulationvia pipistacking.Thus, in the presence of lipase, curcuminwas released from the gelwithout any initial burst release.The authorsprovided furthersupportthatthegeldegradationanddrugreleaseweretriggeredbylipasebyshowingthatselfassemblingbolaamphiphiles with the ester linkage replaced by a noncleavable bond remained intact and released no drug whenincubatedwith the enzyme.5.47.3.5 TechnicalIssuesinDesignandCharacterizationIndesigninghydrogelwithdrugsattachedaspendantgroupsviapeptidelinkers,thecompositionandlengthoflinkersaremostcrucial.Forlowmolecularweightdrugs,whichcanfreelydiffusethroughthehydrogel,theenzymatickineticsdeterminesthedrugreleaserate.Asenzymesmustfirstpenetrateintothehydrogelinordertoperformenzymaticreaction,themeshsizeofthehydrogelmustbelargeenoughandthisinturnrequiresconsiderationaboutthecrosslinkingdensityandthepolymerchainlengths.Whenenzymaticcleavagedoesnotreleasethefreedrugs,butratherdrugsattachedtopeptidefragments,potencymaybereducedanditisnotadesirabletraitforadrugdeliverysystem.However,thedrugpotencycanbepreservedwhenthechemicalmodificationdoesnotaffectthedrugbindingtothetarget.

Indesigningenzymesensitivehydrogelwithenzymesubstratesascrosslinkers,onecanvaryseveralparameterstocontroltheproteindrugreleasekinetics.Theseincludepeptidecomposition,crosslinkingdensity,andenzymeconcentration.First,therateofthe cleavageof crosslinkers by target enzymes depends on the composition of the peptide linkers. Changes in theaminoacidcomposition and sometimes the length of the sequence influence the digestion kinetics,which in turn affects the rate of geldegradationandtherateofproteinrelease.Experimentally,thecleavageofpeptideistrackedusingfluorescenceassaysinwhichtheincrease in free amine concentration correlateswith the intensity of anoptical signal.The data are conventionally fitted to theMichaelisMentenkineticsmodel.FromLineweaverBurkplots,theenzymeturnoverrate(kcat)andMichaelisMentenconstant(Km)areobtained.Thedigestionkineticsmeasuredforfreepeptidesusuallydifferfromthoseincorporatedwithinthehydrogel.However, the relative sensitivity of different free peptides is positively correlatedwith the relative rate of degradation of gelsincorporatingthesamepeptides.

Second,thecrosslinkingdensityaffectsboththemechanismofhydrogeldegradationandtherateofdegradation.Thecrosslinkingdensitycanbecontrolledbytheratioofthepeptidecrosslinkersandthepolymericchainsinsynthesizingthehydrogel.Hydrogelsundergoingbulkdegradationhavelargemeshsizeandthoseundergoingsurfacedegradationhavesmallmeshsizeforlimitingthediffusionofenzymes.Theswellingratioofthehydrogelduringthetimecourseofdegradationindicateswhichtypeofdegradation is dominant. In bulk degradation, the gelwill first swell due to a decrease in crosslinking density, followed bydisintegrationofthegelandthelossofgelmass.Insurfacedegradation,theswellingratioremainsconstantandtherateofthelossofgelmassisproportionaltothesurfacearea.Hydrogelswithlowerdensityofpeptidecrosslinkshavefasterdegradationrate,andthusfasterproteinreleaserate,becausefewerenzymaticcleavageeventsarerequiredtoliberatethepolymericchainsfromthegelnetwork.

Hydrogelscontainingpeptidecrosslinksarehypothesizedtobedegradablebyproteases.Toconfirmthishypothesis,thegeldegradationandtheproteinreleasemustbecomparedinthepresenceandabsenceoftheprotease.Thegeldegradationrateshouldincreasewithincreasingenzymeconcentration.InthecasethatthegeldegradationfollowsazeroorderkineticsatarateequalingkcatEo(whenSoKm),thedegradationrateisproportionaltotheenzymeconcentration.Lastly,toensurethatthereleasetriggerisspecific,itisnecessarytodemonstratethatthehydrogeldoesnotdegradeorreleaseproteindrugswhenincubatedwithanotherproteaseofdifferentsubstratespecificity.

In characterizing enzymesensitive selfassembling hydrogels,TEM is frequently used to observe the morphology of thenanofibers.Inmanycases,theenzymaticreactionwouldresultinachangeofthenanofibermorphology,whichthenleadsto gel degradation.The gel formation and gel degradation could be tracked by rheological measurements using dynamicmechanicalanalysis,whichprovidedvalues ofstoragemodulus(G)andlossmodulus(G).GelationisindicatedwhenGexceeds G, and G is also a measure of the mechanical strength of the gel. As enzymatic reaction switches off theselfassembling property of the building blocks, they may become detached from the matrix,whichwill cause the gel toloseweight.Toconfirmthatenzymaticincubationresultsinachemicalmodificationoftheselfassemblingbuildingblock,mass spectroscopy and HPLC can be used to show the changes.Toverify that enzymes are the trigger for drug release,control experiments need to be performed. The release profile is compared between incubation with and withoutenzyme (orwith an enzyme plus its inhibitor). Furthermore, hydrogels selfassembledwith building blockswithout theenzyme trigger but otherwise similar structures are tested for the degradation and drug release in the presence of theenzyme.


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5.47.4 Enzyme-SensitiveParticulateCarriers5.47.4.1 OverviewExamplesofenzymesensitiveparticulatecarriersfordrugdeliveryarefoundinliposomes,polymeric/hydrogelparticles,andthreedimensional(3D)containersselfassembledbysyntheticpeptidesandgeneticallyengineeredproteins.Particulatecarriers,espe-ciallynanocarriers,areparticularlysuitedfortargeteddrugdeliverysincetheyaresmallenoughtocirculateinthebody.Increasedaccumulation at the target tissues can be achieved by surface modification. Enzymes as biochemical signals specific to thepathophysiologyofdiseasesareusefulforeithertriggeringthereleasefromtheparticulatecarriers,orenhancingthebindingofthecarrierstothetargetcellsforincreasedinternalization.5.47.4.2 Enzyme-SensitiveLiposomesLiposomesarewidelyusedasdrugcarriersbecauseoftheirbiocompatibilityandtheabilitytoencapsulatehydrophiliccompounds.Theyarevesiclesformedbytheselfassemblyoflipidsandrangeindiameterfrom~30to500nmdependingonthepreparationproceduresand lipidcomposition.Enzymetriggeris incorporatedintoliposomesbyconjugatinglipidorcholesterolmoleculeswithenzymesubstrates.Thehybridmoleculesareformulatedwithconventionallipidstoformliposomes,allowingtheenzymesensitivity tobetunedby thepercentageofenzymesensitivecomponents.Onestrategy is tounmask thefusogenicpropertyofliposomeswithcellularmembranesupontrigger,enabling thecargoestobetransferredintothecells.Anotherstrategyinvolvesusingenzymestodestabilizethemembranestructureoftheliposomeinordertoreleasetheencapsulatedcontents.

Meerslabconstructedatriggerablefusionliposomesbydesigninganenzymecleavablelipopeptide[66].Adipeptidesequence(AA)waslinkedviaanamidelinkagetodioleoylphosphatidylethanolamine(DOPE)tomaskthelipidsfusogenicproperty.Theshort peptidewas cleavable by elastase, an enzyme associatedwith inflammatory and cancerous conditions.The lipopeptideconjugate,NAcAADOPE,wasmixedwithotherlipidmolecules(dioleoyltrimethylammoniumpropane(DOTAP)andPE)toformliposomes.Itwasdemonstratedthatthefusogenicpropertiesoftheseliposomewereswitchedonbythedigestionofelastase,andtheextentwasdependentontheenzymeconcentration.TheenzymaticcleavageyieldedDOPE,whichitselfisazwitterionicfusogenic lipid; it also caused a charge reversal of the liposomes from net negative to net positive, favoring their electrostaticinteractionwiththenegativelychargedcellmembrane.Liposomefusionwasevidentbyaugmentedlipidmixingandsuccessfultransferoffluorescentdextranprobesbytheelastasetreatedliposomesintotheredbloodcellghosts.

AvariationofthisapproachwastouseanenzymecleavablePEGderivativeasamaskinggroup toregulatecellularbinding(Figure7).ThismethodcarriesanadditionaladvantagebecausePEGpreventsopsoninadsorptionandreducesclearanceduetouptakebymacrophages.PEGylationminimizesaggregationofliposomesandimprovestheirstabilityincirculation.ThePEGcoatstericallyshieldsthetargetingligandsonthesurfaceofliposomesfrominteractingwiththecellularreceptors.UponremovalofPEGby enzymatic reaction, the ligands are exposed and receptormediated endocytosis is triggered to enhance cellular uptake.The

= Receptor

Cellular uptake byreceptor-mediated

endocytosis

= Drug

= Pegylated MMP-2 substrate

= Ligand

MMP-

2

Figure7 Schematicillustrationofanexampleofanenzymesensitiveliposome.UponMMP2cleavage,thePEGcoatingisremovedandthetargetingligandsareexposedtoenhanceuptakeofliposomesintothecells[75].ReproducedwithpermissionfromLawBandTungCH(2009)Proteolysis:Abiologicalprocessadaptedindrugdelivery,therapy,andimaging.BioconjugateChemistry20(9):16831695.Copyright2009,AmericanChemical.


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concept was demonstrated using an MMP2 cleavable PEGpeptideDOPE incorporated in galactosylated liposomes [67].Galactosylatedliposomesaremoreefficientlyinternalizedbylivercellsduetotheexpressionofasialoglycoproteinreceptorsonthecellsurface.Totargettheuptakebylivercancercells,whichoverexpressMMP2,MMP2cleavablemaskinggroups(consistingofthe peptide GPLGIAGQ conjugated toPEG on oneend andDOPEon the other end)were incorporated into the galatosylatedliposomes.ThedegreeofgalatosemaskingandthesensitivityoftheseliposomestowardMMP2weretunedbyvaryingtheamountofPEGpeptideDOPE.At0.5mol.%,theuptakebycellsoverexpressingasialoglycoproteinreceptorswasinhibited,eveninthepresenceofalowlevelofMMP2(1g ml1).TheuptakewasincreasedbyoverfivefoldwhenMMP2concentrationwasincreasedto10g ml1.ThissubtletuningisimportantfordesigningdrugtargetingvehiclesthatpreservethePEGmaskingfunctionduringcirculation in the blood streamwhere aresidual level of MMP2 is present. Enzymemediated delivery against cancer cellswasdemonstratedusingthisliposome:achemotherapeuticcompoundwasencapsulatedandthecytotoxicpotencyagainstcancercellswashigherinthepresenceoftheMMP2.

Enzymescanbeusedtoregulatethedrugreleaseratherthanthecellularuptakeofliposomes.Inoneexample,DavisandSzoka[14]attachedaphosphatemonoesterheadgrouptoacholesterolmoleculeviaaphosphatasesensitivelinker.Alkalinephosphataseisanextracellularenzymethatisassociatedwithcancers,whileacidphosphataseisanintracellularenzymefoundinendosomesandlysosomes.LiposomeswerepreparedbymixingthecholesterolphosphatederivativeswithDOPE.Thedoublynegativechargeonthecholesterolphosphatederivativeiscrucialinstabilizingtheliposomestructure,asDOPEalonecannotformvesicles.ItwasfoundthatphosphatasetreatmentcouldleadtotheremovalofthechargedphosphategroupsaccordingtoHPLCanalysis.Bothalkaline and acid phosphatasewere able to induce sufficient structural change in the liposome to cause the release of theencapsulatedfluorescentprobes.Therateofreleaseincreasedwhilethelagtimeofreleasedecreasedwithincreasingphosphataseconcentration.As heatinactivated phosphatase failed to trigger drug release, the authors rebuked the possibility that enzymepartitionintotheliposomeratherthanenzymaticcleavageaccountedforthedrugreleasemechanism.Supportingthisargumentwasthelackofenzymetriggeredreleasewhentheliposomeswereformulatedwithnaturallyoccurringcholesterolphosphatethatwasinsensitivetophosphatase.

More recently, Srivastava and coworkers [68, 69] described a new approach to use MMP9 to trigger release fromliposomes.Thedesign involved thesynthesisofa lipopeptidewithanMMP9substrateconjugatedtoa lipidmolecule (stearicacid, oleic acid, and palmitic acid).The peptide sequence, GPQGIAGQR(GPO)4GG, contains a cleavage site (GI) for MMP9and the motif for triple helix formation (repeats of GPO). Lipopeptides incorporated in liposomeswere capable of formingtriple helices, as revealed by circular dichroism spectroscopy. For an enzyme to cleave the peptide, it must be able to firstunwind the triple helix and this additional requirement enables targeting to be more specific toward MMP9. IncubationwithMMP9 led to a decrease in the triple helix content and the release of the encapsulated fluorescent dyes in an enzymeconcentrationdependent manner.When incubatedwith trypsin or noncollagenase MMPs that are incapable of unwinding thetriple helix, much lower releasewas observed.The authors proposed that the lipopeptideswere demixed in the lipid bilayer,which upon enzymatic cleavage, therewas a charge reversal (from positive to negative) and a reduction in the polar headgroup size in the lipopeptiderich domains.These changeswould destabilize the membrane structure and domain reorganiza-tionwould happen.The defects resulted from membrane perturbation then caused the release of the encapsulated contents.Thisproposedmechanismwassupportedby theobservation thatboth therateand extentofrelease increasedwhen therewasa larger mismatch between the acyl chains of the lipopeptide and other phospholipids in the liposomes. The structuralmismatch favored the demixing of lipopeptides and the forming of larger lipopeptiderich domains in the liposome. Theenzymatic cleavage should cause a more severe defect such that annealing by lipid organizationwas less effective, resulting inmore leakage of the encapsulated contents.5.47.4.3 Enzyme-SensitivePolymericParticlesAninterestingapproachusingenzymesensitivepeptideactuatorstotriggerdrugreleasefrompolymerichydrogelparticleshasbeendescribedbyUlijnsteam[19] (Figure8).Poly(ethyleneglycolacrylamide)(PEGA)microparticles(~250um)werefunctionalizedwithenzymecleavablepeptidesflankedbyoppositelychargedaminoacids.Inoneexample,thesequenceDAARwasevaluatedasapeptideactuatorrespondingtoelastaseandthermolysin.Theprincipleofdrugloadingandreleasewasbasedontheswellingofthehydrogelparticleduetoelectrostaticrepulsionofthelikechargegroupsattachedonthepolymernetwork.Therewasnonetchargeon the peptide actuator at physiological pH.The mesh size at this stage had a cutoff of 40kDa, making it accessible for smallenzymesincludingthermolysinandelastase,butinaccessibleforlargefluorescentdextransof77kDa.Toentrapthemacromole-cules,anacidicpHwasusedtoturntheparticleintoapositivelychargedparticlewithenlargedmeshsize,followedbythereturnto physiological pH to close the hydrogel network. Upon enzymatic cleavage, FmocDAARPEGA particleswere converted toARPEGAparticles,makingthempositivelychargedagain.Theresultingincrease in themeshsizeof thehydrogelnetworkwasevident by an increase in the particle size and the concomitant release of the encapsulated dextran probes.Therewere severallimitationsinthisdesignandsomewereaddressedinlaterstudies.

First,whennegativelychargedproteinswereencapsulated,theywereattracted tothepositivechargeofthehydrogelparticleuponenzymaticcleavage.Evenwithincreasedmeshsize,negativelychargedproteincouldnotbereleasedfromtheparticle.Thisproblemcouldbesolvedbydesigningthepeptideactuatorsinsuchawaythatthechargepropertyofthehydrogelparticlesafterenzymaticcleavagematchedwiththatoftheproteins[70].Thus,whileFmocDAARPEGAthatwasenzymaticallyconvertedtoARPEGAwassuitableforcationicproteins,FmocRRAADDPEGAthatwasenzymaticallyconvertedtoADDPEGAwassuitable


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Figure8 Schematicillustrationofanexampleofanenzymesensitivepolymeric/hydrogelparticle.Uponenzymaticcleavage,netchargewaspresentonthepeptideactuatorsattachedtothepolymericchains.Theelectrostaticrepulsioncausedanincreaseinthemeshsizeandfacilitatedthereleaseoftheencapsulateddrugmolecules[19].(a)Themolecularstructureofpolymerandanenvironmentalscanningelectronmicroscopy(SEM)imageofhydrogelparticles(scalebaris100lm).(b)Chemicalstructuresofanenzymecleavablelinkerandconsequentcleavageproducts.(c)Pendantaminegroupsmaybereadilyfunctionalizedwithzwitterionicpeptidelinkersthatconfernooverallchargeonthehydrogel.Enzymecatalyzedhydrolysisrevealsdoublychargedpeptidefragmentscausingthehydrogelparticletoswell.(d)Hydrogelparticlesmaybeloadedwithamacromolecular payload(representedbytriangles)byloweringandsubsequentrestoringthepH.FromThorntonPD,MartRJ,andUlijnRV(2007)Enzymeresponsivepolymerhydrogelparticlesforcontrolled

release.

Advanced

Materials

19(9):

1252

1256.

Copyright

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Verlag

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KGaA.

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permission.

foranionicproteins.Thisconceptwasprovenwith twosimilarsizedbutoppositelychargedproteins, theanionicalbuminandcationicavidin.

Second,asthereleasedependedonelectrostaticrepulsionafterenzymaticcleavage,theresponsewassuppressedbycounterionspresentinthemediumatphysiologicalionicstrength.Thisproblemwaselegantlysolvedbydesigningabranchedpeptideactuatorstoenhancethechargedensityanddecreasethedistancebetweenneighboringcharges[71].Comparedtotheoriginaldesignusinglinearpeptideactuators,therewasanaugmentedincreaseinthemeshsize.Moreimportantly,triggeredreleasebyspecificenzymeswasuncompromisedbyincreasingionicstrengthuptoaround0.3M.


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Theapproachwasdemonstratedforrelativelylargemicroparticles.However,itshouldbeapplicabletosmallersizedhydrogelparticlesofsimilarchemistry.Thiswouldfacilitatethedrugdeliveryapplicationsincludingthoserequiringinjectionandcirculationinthebody.Practicalproblemsthatstillneedtobeaddressedaretherelativelylowproteinloading(


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Proteinrepeatunitsareusedbynaturetoconstruct3Dcontainers.Forexample,viralcoatsandtheclathrincoatsofendocytoticvesicles are assembled by thisway.Another example is provided by small heatshock protein (sHSP) from hyperthermophilicarcheon,whichcanselfassembleintoan~12nmsphericalnanocapsulecontaining24proteinunits.Thecavityofthisnanocapsulecan be used to carry drugs.To trigger release from the nanocapsule by a specific enzyme, Murata and coworkers used geneticengineering tocreate mutantsof sHSP that carrypeptide sequences cleavableby factorXa(IEGR) [25].Among the 10mutantscreated,itwasfoundthatonewouldpreservetheselfassemblingpropertyoftheprotein,allowtheenzymetoaccessthesubstratepeptide, and decrease the thermal stability of the nanocapsule upon enzymatic cleavage.Thus, a nanocapsule of the samedimensionasthenativeproteinwasobserved.Cleavageofthepeptidefragmentswasconfirmedbymassspectrometry.Althoughthe nanocapsules from native sHSP couldwithstand high temperature, those formed by the mutants collapsed at 90 oC afterenzymaticcleavage.Thephysicalmechanismastowhythecleavagedecreasesthermalinstabilityremainsunknown.Inprinciple,thisapproachcanbegeneralizedtootherproteasesthataremorediseaserelevant.Amajorproblemtobeaddressedbeforepracticalapplicationsistolowerthetemperaturethresholdrequiredtodisassemblethenanocapsules.5.47.4.5 TechnicalIssuesinDesignandCharacterizationActivetargetingbyparticulatecarrierscanbeachievedbyenhancedbindingtotargetcellsand/orincreasedreleaseofencapsulateddrugsatthetargetsite.Buildingcomponentsofthemicro ornanocarriersareradiolabeledortaggedwithfluorescencetoallowthedetection of their associationwith cells. Fluorescent probes or fluorescencelabeled drugs are often encapsulatedwithin theparticular carriers to enable ready measurement of the drug release kinetics. In enzymesensitive particulate carriers, enzymesubstrates are incorporated as the trigger.The degree of cellular binding and the rate and extent of drug release can be tunedbytheenzymesubstratecomposition(e.g.,theaminoacidsequenceofacleavablepeptide)aswellasthesubstrateconcentration(e.g.,thenumberofpeptidespresentwithineachparticle).

To demonstrate that a specific enzymatic reaction triggers the drug release, proper control experiments must be performed.Resultsmustbecomparedbetweencaseswithandwithoutthetargetenzymesintheactiveform.Todistinguishfromthepossibilitythatthepartitionoftheenzymeintotheparticlescausingtherelease,negativecontrolcanbeconductedusinginactivatedenzymes.Alternatively,techniquessuchasHPLCandmassspectroscopyareusedtoconfirmthepresenceoftheproductsfromenzymaticreactions.

Dynamiclightscatteringisroutinelyusedtocharacterizetheparticlesizeofparticulatecarriers.Thelossofthestructuralintegrityofparticulatecarriersduetoanenzymaticreactioncanbetrackedbyachangeinsizedistribution.Forlargeparticles,thechangecanbevisualizedbylightorfluorescentmicroscopy.TEMandSEMareusefulinrevealinghowthemorphologyofparticleschanges.Thedetailedmoleculararrangementinselfassemblingparticlescanberevealedbycirculardichroismandfluorescencespectroscopy.

Drugmoleculesthatarephysicallyentrappedwithinparticularcarriersmaynotexhibit100%releaseeveninthepresenceofanenzymatictriggerandafteraprolongedperiod.Bindingbetweendrugmoleculesandthebuildingcomponentsofthecarriermayexist,blockingtheirescapefromthecarrier.Anotherpossibilityalsoexistsinselfassemblingsystems(includingliposomes).Thebuildingblocksmayreorganizeaftertheenzymaticreaction,annealingthestructuraldefectsandpreventingfurtherreleaseofdrugmolecules.

5.47.5 ConclusionsEnzymesensitivebiomaterialsarenovelmaterialscapabletoachievedesiredtransitionincontrollablemannerbyrespondingtoenzymes.Theapplicationindrugdeliveryhasbeenextensivelyexploredandremarkableadvancehasbeenachievedinrecentyears.Avarietyofdosageformsincludingpolymerdrugconjugates,hydrogels,andparticlesweredevelopedtoservefordifferentpurposeindifferentdiseases.Enzymesaresuchcentralandversatilebiomoleculesthattheuseofenzymeinmaterialengineeringopensupgreatfuturepossibilitiesofsmartdrugdeliverysystem.

AcknowledgmentWeacknowledgethefundingsourceofthiswork(RGCHKUST6407/06M).

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