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CellandTissueMorphogenesis

Cellsadoptdeﬁnedshapesthatareessentialfortheirspecializedfunctionsand thisofteninvolvesinteractionswithothercellstoformorganizedtissuesand organs.Disruptionofnormalcell cellinteractionsisakeystepleadingto theprocessofmetastasisthatisseeninlatestagesofcancer.

CellMigration

Oneofthemoststrikingfeaturesofnormalembryonicdevelopmentisthe large-scalemovementsandmigrationsofcellsastheyreorganizetoformthe differentbodycompartments.Outsideoftheimmunesystem,cellmigrations intheadultarenormallyrestrictedtolocalizedareaswithintissues.Afeature oflate-stagecancersismetastasis theabilityofcellstomigrateinappropriatelytootherareasofthebody andthisisresponsibleforthemajorityof cancerdeaths.

Signiﬁcanttechnicaladvancesinimaging,molecularbiology,andgenomics havefueledarevolutionincellbiology,inthatthemolecularandstructural processesofthecellarenowvisualizedandmeasuredroutinely.Drivingmuch ofthisrecentdevelopmenthasbeentheadventofcomputationaltoolsforthe acquisition,visualization,analysisanddisseminationofthesedatasets.These toolscollectivelymakeupanewsubﬁeldofcomputationalbiologycalled bioimageinformatics,whichisfacilitatedbyopensourceapproaches.We discusswhyopensourcetoolsforimageinformaticsincellbiologyareneeded, discusswhysomeofthekeygeneralattributesofwhatmakeanopensource imagingapplicationsuccessful,andpointtoopportunitiesforfurtheroperabilitythatshouldgreatlyacceleratefuturecellbiologydiscovery.

Bioimageinformaticsasadiscoverytoolincellbiologyimagingisusedas atoolfordiscoverythroughoutbasiclifescience,andbiomedicalandclinical research.Inthesedomains,advancesinlightandelectronmicroscopyhave transformedbiologicaldiscovery,enablingvisualizationofmechanismand dynamicsacrossscalesofnanometerstomillimetersandpicosecondstomany days.Fluorescentprotein-taggedfusionscanbeusedasreportersofbiomolecularinteractionsinculturedlivingcells[1],andthesamereportercanreveal thelocalizationandgrowthofatumorinalivinganimal[2,3].Inshort,thelast 20yearshaveprovideduswithawealthofsophisticatedbiologicalreporters andimagedataacquisitiontoolsforbiomedicalresearch.Manyoftheseimagingandinstrumentationdevelopmentshavebeendrivenbypartnerships betweenacademiclaboratoriesthatinventandprototypenewtechnologyand commercialentitiesthatdevelopandmarketthemascommercialproducts. Thisdevelopmentanddeliverypipelineofcommercialimaginginstrumentationandsoftwarehasbeenquitesuccessful,havingdeliveredthelaserscanningconfocal[4,5],spinningdiscconfocal[6,7],wide-ﬁelddeconvolution [8,9]andmultiphotonmicroscopes[10]thatareenginesofdiscoveryincell anddevelopmentalbiology.

Allofthesemethodologiesproducecomplex,multidimensionaldatasets thatmustbetransformedintoreducedrepresentationsthatscientistscan manipulate,analyze,sharewithcolleagues,andultimatelyunderstand.Despite thediversityofapplicationsofimaginginbiology,therearecommonunifying challengessuchasdisplayingamultigigabytetime-lapsemovieonalaptop screen,oridentifying,tracking,andmeasuringtheobjectsinthatmovieand presentingtheresultingmeasurementsinagraphthatrevealsthemechanisms thatdrivetheirmovements.Theserequirementshavespawnedthenewﬁeldof bioimageinformatics[11],whichaimstodelivertoolsfordatavisualization, management,storage,andanalysis.Whilestillarelativelyyoungﬁeld,bioimageinformaticshasalreadyhadamajorimpactincellbiologyparticularly intheareaofquantitativecellimagingwhereadvancedfeaturerecognition, segmentation,annotation,anddataminingapproachesareusedregularly [12 20].

Almostallcommerciallyprovidedimageacquisitionsystemsinclude softwaretoolsthatprovidesophisticatedimagevisualizationandanalysis functionsfortheimagesrecordedbytheinstrumenttheycontrol.However,in recentyears,manynoncommercialprojectshaveappeared,almostalways basedinresearchlaboratoriesthatrequirefunctionalitynotavailablein commercialproducts.Here,wediscusstheapplicationofbioimageinformaticsincellbiologyandfocusspeciﬁcallyonthedevelopmentofopen sourcesolutionsforbioimageinformaticsthathaveemergedoverthelastfew years.

WHATARETHEINFORMATICSCHALLENGES INQUANTITATIVECELLBIOLOGYIMAGING?

Giventherapiddevelopmentinimageacquisitionsystemsinthelast20years, itisworthconsideringwhyacorrespondingrapiddevelopmentofinformatics toolshasoccurredonlyrecently.Certainly,oneofthebarrierstoproviding universaltoolsforbioimageinformaticsisthediversityofdatastructuresand experimentalapplicationsthatproduceimagingdata.Inopticalmicroscopy alone,thereareasubstantialnumberofdifferenttypesofimagingmodalities and,indeed,amethodlikefluorescencemicroscopyencapsulatesahugeand rapidlygrowingfieldofimageacquisitionapproaches[21].Informaticstools thatsupportthisrangeofmethodsmustbecapableofcapturingtherawdata (theindividualpixels)andthemetadataaroundtheacquisitionmethodology itself,includinginstrumentsettings,exposuredetails,etc.Thisdiversityof datastructuresmakesdeliveringcommoninformaticssolutionsdifficult,and thiscomplexityismultipliedbythelargenumberofcommercialimaging systemsthatuseindividuallyspecified,andoftenproprietary,fileformatsfor datastorage.Ourcurrentestimatesarethatthereareapproximately80proprietaryfileformatsforopticalmicroscopyalone(andnotincludingother commonimagingtechniques)thatmustbesupportedbyanybioimage

informaticstoolthataimstoprovideageneralizablesolution.Inshort,thelack ofstandardizedaccesstodatamakesthegenerationofinformaticstoolsquite difﬁcult.

Adeeperchallengeresidesineachindividuallaboratorythatusesimaging aspartofitsexperimentalrepertoire.Thesheersizeoftherawdatasetsand therateofproductionmeanthatindividualresearcherscaneasilygenerate manytensofgigabytesofdataperday.Thismeansthatlargelaboratoriesor departmentalimagingfacilitiesgeneratemanyhundredsofgigabytesto terabytesperweekandarenowenterprise-leveldataproductionfacilities. However,theexpertisefordevelopingenterprisesoftwaretoolsorevensimply runningthehardwarenecessaryforthisscaleofdatamanagementandanalysis rarelyexistsinindividuallaboratories.Inshort,thesophisticatedsystemsand developmentexpertisethatareusedtodelivergenomicsdatabasesand applicationsarerequiredinindividualimaginglaboratoriesandfacilities.The deliveryoftoolsthatprovideaccesstoabroadrangeofdatatypes,manage andanalyzelargesetsofdata,andhelprunthesystemsthatstoreandprocess thesedataisthechallengethatbioimageinformaticsseekstoaddress.

WHYAREOPENSOURCEAPPROACHESESSENTIAL?

Acriticaldevelopmentintheﬁeldofbioimageinformaticshasbeenthe introductionofmanyopensourceprojectsinthelastfewyears[11,22 30]. Theseprojectsrangefrombeingopensourcedistributionswherethecodeis availablebutnewdevelopmentisnotspeciﬁcallyencouraged,toopen developmentprojectsthatarecommunity-drivenprojectsthatactively encouragethehelpandparticipationofprojectsforthesupportandadditionof newfeatures.Therefore,beforeweproceed,itisworthconsideringwhat constitutesopensourceandopendevelopmenteffortsandwhytheyare valuableorevennecessaryforbioimageinformatics.

Opensourcesoftwareisawell-establishedmovementwithstrongparadigmsinmanyverysuccessfulprojectssuchasLinux(http://www. linuxfoundation.org/),Java(http://java.sun.com/),MySQL(http://www.mysql. com/products/database/),andApache(http://www.apache.org/).Afundamentaltenetofopensourcesoftwareprojectsisthatthecopyrightholder (usuallythesoftwaredeveloperorhis/heremployer)determinesthesoftware license,whichdeﬁneshowthesoftwareisdistributedandwhatendusersmay dowiththesoftware.Foropensourcesoftware,theoriginalsourcecodeis madeavailableunderthetermsofthislicense.Anopensourcelicenseusually allowsenduserstousethesoftwareforanypurpose,makechangestothe softwaresourcecode,orlinktheirownsoftwaretoitand,iftheydesire, distributethose“derivativeworks.”However,thesoftwarelicensealsodeﬁnes underwhattermsandlicensederivativeworksmaybedistributed.Forany usersordevelopers,thesedetailsareimportantandmustbeunderstoodgiven thegreatimplicationsfordevelopmentanddeployment.

agenciesshouldplaceonthecontinuedfundingofsoftwaredevelopmenttools foritsuse.Ifcontinuedfundingistobeconsidered,theapplicationand reviewingprocesseswillneedtobemodiﬁedtoproperlycaptureandassess thevalueoftheseprojects.Inouropinion,inexchangeforperiodicreviewand considerationforsustainedfunding,publiclyfundedscientiﬁcsoftwareprojectsshouldberequiredtofollowopendevelopmentmodels,whereengagementandsupportforthecommunityisrequired.Thiscanoccuronlyif fundingforsupportandcommunityengagementisavailable,andifcareer developmentandevaluationincludepublicationrecordanddeliveryofuseful toolstoandengagementwiththecommunity.

Incomparingopensourceandcommercialsoftwareproducts,oneofthe biggestdifferencesissupportforthesoftwareitself.Ingeneral,commercial softwarepackagesaresupportedwithinstructions,manuals,anddirectuser support,andthisisakeyadvantageofusingcommercialsoftware.Thecostof suchsupportiseitherincludedintheoriginalpurchasepriceorpaidforby purchaseofasoftwaremaintenanceagreement.Coveringthecostsofuser supportisdifﬁcultforopensourceprojectsbecausethereisnocorresponding feestructuretocoversuchsupportcostsand,often,theacademicgrantsthat fundopensourceprojectscoveronlytheinnovativeresearchcomponentsand donotsupportthepersonnelorinfrastructureneeded.Thisisgradually changingwithfundingagenciesandscientistsalikerealizingtheimportanceof producinginnovativeandfeature-richcodebutensuringthatitiswellsupportedandmaintained.Therearewell-establishedstandardsandtoolsinthe opensourcecommunityforsupport,mailinglists,userforums,screencast demos,andWiki-baseduserdocumentation,thatallcontributetomaking softwaresuccessful.WithinourownOpenMicroscopyEnvironment Consortium(http://openmicroscopy.org),weuseprojectmanagementtools suchasSubversion(http://subversion.tigris.org/)tomanageoursourcecode repository,Trac(http://trac.edgewall.org/)forallprojectmanagementand issueandrevisiontracking,Jabber(http://www.jabber.org)forreal-time communication,Hudson(https://hudson.dev.java.net/)forcontinuousintegration,Ploneformanagingourwebsite(http://plone.org/),andPHPBBfor runningouruserforums(http://www.phpbb.com/).Inadditiontothesetools, weholdannualusermeetingstoassessprogressanddeﬁneroadmapsfor futureworks.Weparticipateaspresentersorexhibitorsinlargemeetingsof thecommunityinordertocaptureasmuchfeedbackaspossible.Thesetools andactivitieshelpsupportandengageaverybroaduseranddeveloper communityandareanimportantpartofensuringcommunitywideadoption, butinstalling,running,andmaintainingthesetools,aswellasanswering queriesandmoderatingdiscussions,requiretimeandresources(bothpeople andmoney).Manysuccessfulopensourcepackageshaveshowntheimportanceoftransformingtheconventionaluserbaseintoanadditionalsupport mechanismwheretheusercommunityinteractswiththeoriginaldevelopers andwitheachotherforsupportandnewcodedevelopments.Usersand

optimumtherapeuticdrugconcentrationinthebloodwithminimumﬂuctuation,predictableandreproduciblereleaseratesforextendedperiodsoftime, enhancementofactivitydurationforshorthalf-lifedrugs,theeliminationof sideeffects,frequentdosing,wasteofdrug,optimizedtherapy,andbetter patientcompliance.11

HISTORYOFCONTROLLEDDRUGDELIVERY

Ingeneral,thecontrolledreleasesystemmustbedevelopedsothatitissafe, reproducible,effective,andamenabletoscale-up.Theresearchofcontrolled drugdeliverysystems,firstcenteredonmicroencapsulation,beganin1949 withthepatentoftheWursterprocess.Thistechnique,whichutilizeda fluidizingbedanddryingdrumtoencapsulatefinesolidparticlessuspendedin midair,wasthefirstofmanymethodstobediscoveredwhichrevolutionized thefieldofdrugdelivery.Laterin1953,thecoacervationmethodwas developedwhichledtotheencapsulationofliquid.Between1956and1966, over50patentswerefiledformicroencapsulationtechniquesandthusbecame availableforpatientapplication.Otherimportantdeliverymethodsinclude implants,whichweredevelopedinthemid-1970sandtransdermalwerefirst introducedin1980.Muchoftherecentresearcheffortsoncontrolleddrug deliveryarefocusingonsite-directedsystemsasthenewtechnologyinthe industry.Manyscientistsbelievethattheuseofliposomesasdrugcarrierswill serveastheleadingresearchtopicintothetwenty-firstcentury.8,9

SUSTAINEDRELEASEDRUGDELIVERYSYSTEMS

Thehumanbodystrivesforbalance.Wehavedevelopedanetworkoffeedbackmechanismstoregulateeverythingfromhormonesecretiontoblood sugar.Theprincipleofhomeostasisgovernsallofourbodilyprocesses.The pharmaceuticalindustry,too,mustmaintaintheideaofbalance.Supplying theappropriateamountofmedicinetothebodyisessentialtothesuccessofa treatment.Thisconceptservesasthefoundationforsustainedreleasedelivery systems.Sustained,ortime,releasesystemsaremethodsofdrugdeliveryin whichonepreparationwillaccomplishthedesiredmedicinaleffectwithmore efficiencyandlongerdurationthanmultipledosagesofthesamedrug.The goalofthesesystemsistosupplytheoptimalconcentrationofadrugfora longertimethanconventionalsystemsallow.11 Undertraditionaltabletdosages,medicineisingestedatintervalsofspecifiedtime.Whenatabletis taken,drugconcentrationrisesrapidly,eventuallypeaks,andthenfallsuntil thenexttabletisconsumed.Afterthesecondtablet,theconcentrationof medicineinthebloodstreamagainrises,peaks,andfalls.Thecyclecontinues. Theproblemwiththisscenarioisthatoptimalconcentrationcannotbe maintainedandthepeaksmayoccurattoxiclevels.8,9 Also,humanerrormay causeadditionaldifficultiesifadosageisdelayedormissed.Sustainedrelease

bodybythemostefﬁcientmeanspossible.Thoughliposometargetingisfar frombeingperfected,itservesasavaluableresourceinthetargetingofdrugs. Liposomeshelptoreducethedangerofahighdrugconcentrationinthebody andwillcontinuetoaidthepharmaceuticalindustryinitsstruggletoﬁght disease.

NANOPARTICLESINDRUGDELIVERY

Nanoparticlesaresolid,colloidalparticlesconsistingofmacromolecular substancesthatvaryinsizefrom10to1000nm.14 Thedrugisdissolved, entrapped,adsorbed,attached,orencapsulatedinthemacromolecularmaterial(s).Nanoparticles,alsocallednanopelletsornanocapsules,canhavea shell-likewall,calledamicrosphere,orapolymerlattice.Itisoftendifﬁcultto determinewhethernanoparticleshaveashell-likewalloracontinuous matrix.10

AdvantagesandDisadvantagesofNanoparticles

Therearemanysimilaritiesbetweenliposomesandnanoparticles.Bothare similarinsizeandthusareoftenusedforsimilarpurposes,andbothhave manyadvantagesanddisadvantages.Liposomeshavetheadvantageofprimarilyconsistingoflecithinandcholesterol,whicharematerialsthatoccur naturallyinthehumanbody.Lecithinandcholesterolarealsopresentinthe bodyinlargeamounts,andthusdemandgoodbioacceptability.Nanoparticles havetheadvantageofbeingmorestable.Manytypesofdrugtargetingdepend ongoodstability.Abetterpenetrationoftheparticlesinsidethebody followingadministration,aswellaslongershelflife,isoneoftheseveral beneﬁtsofthegoodstabilityofnanoparticles.

Artiﬁcialornaturalpolymersaretheprimaryconstituentsofnanoparticles. Polymersareusuallyrestrictedbytheirbioacceptability.Thebioacceptability isaffectedbythepolymerandthesupplementarycomponents,aswellasby particlesize.19 Areductionintheparticlesizeofthepolymericparticleshas manyadvantages,whicharelistedbelow.

1. Intravenousinjectioncanbeallowedifthereisadecreaseintheparticle size.

2. Intramuscularandsubcutaneousdistributionsrequiresmallparticlesize.

3. Usingsmallparticlesizeminimizesirritantreactionsattheinjectionsite.

4. Carcinogeniceffectsdependonparticlesize.

Choosingtheappropriatepolymer,particlesize,andmethodofproduction woulddependonthreemajoraspects:bioacceptabilityofthepolymer,physicochemicalpropertiesofthedrug,andthetypeoftherapythedrugshould have.7 ClinicalUtility/RelevanceofCellBiologyTechniques

DrugLoadingProcedures

Drugsareloadedintothenanoparticlesbyseveralmethods.Onemethodisby producingthenanoparticleinthepresenceofthedrug.Drugsmayalsobe loadedintoemptyparticlespreparedaheadoftime.Thedrug,thepolymer,and themeansofpreparationallinﬂuencethetypeofinteractionwiththepolymer, theattachmentofthedrugtothenanoparticles,andtherateoftheir interaction.

ApplicationsofNanoparticles

Someimportantpracticesforwhichnanoparticlesareusedincludethe adsorbingandcoatingoforgansandtissues,peroraladministrationofdrugs, vaccinations,thedeliveryofanti-inﬂammatorydrugs,andthedeliveryof drugsfordiseasesandtumors.

Nanoparticlescanbetargetedtospecificorgansortissuesbycoatingtheir surfacewithdifferentmaterials.10 Nanoparticlesareusefulinthisapplication duetotheirstability.Liposomes,ontheotherhand,oftendisintegratedueto thesurfactantsthatarepresentinthismethod.Nanoparticlesarealsooften usedforperoraladministration.Anexampleofthiscanbeappliedtothe digestivesystem.Inordertoinfluencethemodeofinteractionoftheintestinal fluidswiththeintestine,thepropertiesofthesefluidsaremonitoredin differenttissuesinthegut.Thesaltspresentintheseintestinalfluidsoften depleteliposomes,whereasnanoparticlesarestableinthepresenceofthese substances.Invaccinationmethods,nanoparticleshaveanadvantagedueto theirslowdegradation.Thisslowprocessallowsforalongerinteractionofthe antigenwiththeimmunocompetentcellsofthebody.

Nanoparticles,aswellasmanyothercolloidalcarriers,areenablingnew methodsfortherapyindrugdelivery.Althoughmanyapplicationshavenot beeninvestigatedyet,thecurrentdevelopmentsarebecomingincreasingly moreusefulintheﬁeldofmedicine.

NANOSPHERES

Nanospheresconsistofadensepolymericmatrix,inwhichthedrugcanbe dispersed,whereasnanocapsulespresentaliquidcoresurroundedbyapolymericshell.Twomainapproacheshavebeenproposedforthepreparationof nanoparticlesofsyntheticpolymers.Theﬁrstoneisbasedontheemulsiﬁcationofthewaterimmiscibleorganicsolutionofthepolymerbyanaqueous phasecontainingthesurfactant,followedbysolventevaporation.Thesecond approachisbasedontheprecipitationofapolymeradditionofanonsolventof thepolymer.Concerningnanoparticlesformedofnaturalmacromolecules, nanoparticlescanbeobtainedbythermaldenaturationofproteins(suchas

ClinicalUtility/RelevanceofCellBiologyTechniques Chapter j 1 17

isamplydemonstratedbytheexistenceoflivingorganismswhoseform, function,andevolutionaregovernedbytheinteractionsofnanometer-scale structures.

“Dry”nanotechnologyderivesfromsurfacescienceandphysicalchemistry,focusesonfabricationofstructuresincarbon(e.g.,fullerenesandnanotubes),silicon,andotherinorganicmaterials.Unlikethe“wet”technology, “dry”techniquesadmituseofmetalsandsemiconductors.Theactiveconductionelectronsofthesematerialsmakethemtooreactivetooperateina “wet”environment,butthesesameelectronsprovidethephysicalproperties thatmake“dry”nanostructurespromisingaselectronic,magnetic,andoptical devices.Anotherobjectiveistodevelop“dry”structuresthatpossesssomeof thesameattributesoftheself-assemblythatthewetonesexhibit.

Computationalnanotechnologypermitsthemodelingandsimulationof complexnanometer-scalestructures.Thepredictiveandanalyticalpowerof computationiscriticaltosuccessinnanotechnology:naturerequiredseveral hundredmillionyearstoevolveafunctional“wet”nanotechnology;theinsight providedbycomputationshouldallowustoreducethedevelopmenttimeofa working“dry”nanotechnologytoafewdecades,anditwillhaveamajor impactonthe“wet”sideaswell.”

Itistooearlytotellwhetheranyoralloftheseapproacheswillbesuccessfulortowhatdegree.Therearemanyinthefieldwhothinkthatthemost likelyscenarioisthatsuccesswillcomefromacombinationofthese approaches.Forexample,BruceSmith formerlyofWolframResearchand nowestablishinghisownnanotechnologyfirm isprogrammingaDNA sequence(wetnanotechnology)toforcemoleculesintoveryspecificareas, allowingcovalentbonds(drynanotechnology)tooccuronlyinveryspecific ways.Theresultingshapescouldbeengineeredtoallowpositionalcontroland fabricationofnanostructures.NedSeemanatNYUisworkingonahybridthat usesDNAtomakescaffoldingforthestructures.22(p.997)

Biologicalsystemshavethecapabilityofcreatinghugevolumesofvery complexmaterialsinveryshorttimes(forexample,trees).

“Ageneisamoleculardevicethatdirectsthesynthesisofproteins.The abilitytoaddorremoveagenefromachromosomeinvolvesmanipulationon ananometerscale.Anenzymeisawholechemicalfactoryonananometer scale.Inthisview,bothdryandwetformsofnanotechnologycanbeintentionallyengineered,andtheycanevenbeusedincombination”.17(p.1 5)

Fewpractitionersintheﬁeldarecompletelycomfortablewiththecalculus ofpurelymechanicalmethodsandmostinsistthatsuccesswillbedependent uponacross-disciplinaryapproachbecausenosingledisciplinehaswithinit alloftheneededtools.23

Howeveritisachieved,theseauthorsagreethatnanotechnologywillinvolve humansmanufacturingmolecule-sizeddevicesthatcanreplicatethemselves andmakeotherthingsinquantity.Itisintendedthatthese“assemblers”willbe

bioabsorbablepolymershavebeenusedasaculture-likefoundationforthe adhesionofcell.Braidsuturesbearingchondrocytesproducedsigniﬁcant amountsofcartilageinthesubcutaneouslayerinmice.Pericardialtissuein sheephasbeenregeneratedalsothroughthinlyslicedplatesofbiopolymers. Onemustwonderifthetissuethatis“regenerated”isnotjustscartissue formingfrommechanicalstressandtoxicreactions.

POLY(ORTHOESTERS)

Intheprocessofcreatinganewpolymer,fourattributesweresoughtafter. Theyare:

l Thepolymershoulddegradebyawell-deﬁnedreactiontosmallwater solublemoleculesthatmustbetoxicologicallybenign.

l Hydrolysisrateofthepolymershouldbeadjustablewithinwidelimitsby simplelimitsbysimplemanipulationsofpolymerstructureoruseof excipients.

l Thepolymershouldbecapableofundergoingsurfaceerosion.

l Mechanicalpropertiesshouldbevariablebysimplechangesinpolymer structure.

Dealingwithpropertynumbermaybeofthemostimportant,especially whendealingwithdrugdeliveryandfractureﬁxation.Whenthepolymerisin aphysiologicalenvironment,controlofthedeliveryanddegradationshouldbe plannedbeforetheoperationandimplantation.Thiscallsforapolymerthatis highlyhydrophobicwherethesurfaceerosionismuchhigherthantheinner bulkerosionrate,whileatthesametimeallowingforthediffusionofthe encapsulateddrug.Hydrolysisdeterminestheselimitingfactorsandisthe majorcontributortomanybiodegradationproblems.Thepolymermustnot onlybehighlyhydrophobictolimitwaterpenetrationintothebulkmaterial, butitmustalsocontainlinkagescapableofrapidhydrolysis.

Twosuchlinkagesareanhydridesandorthoesters(1).Therateoferosion fororthoesterscouldbecontrolledbytheacidicorbasicsubstituents. Poly(orthoesters)atthistimewereexploredingreaterdetail.Fourfamilies weredevelopedtotestthedifferentproperties.

Poly(orthoester)IAphysicalproblemwithpoly(orthoesters)istheirlow glasstransitiontemperaturewhichhappenstobeonly3 Cabovenormal humanbodytemperature.Thisisduetothehighlyﬂexiblecarbon oxygen bondthatallowsthepolymertorotateandbend.Asolidstateimplantmadeof thismaterialwouldbealmostimpossible.Understandardroomtemperature,it appearstobeanointment-likegelundergoingrapidhydrolysis.

Thec101cis/transpolymerhasbeentestedasabioerodiblecontraceptive inlaboratoryanimalswhereinﬂammatoryandallergenicresponsesoccurred. Thesameresponseoccurredwhenthesameexperimentalconditionswere carriedoutinhumans.

ClinicalUtility/RelevanceofCellBiologyTechniques

Thec111polymertestedforitsbioerodibleointmentpropertiesinthe treatmentof Pseudomonasaeruginosa inratburns.Thec111polymer-treated burnshadahighersurvivalratethantheSulfamyloncreamusedcommercially.

Poly(orthoester)IIasstatedbefore,poly(orthoesters)areveryhydrophobiccompoundsandarestableenoughtobestoredinthepresenceof moisture.Onlywhenanhydridesareaddedtothepolymeritbecomessensitive tomoistureorheat.Thismixtureaccelerateshydrolysisandhydrophilicity;to reversetheprocess,abaseexcipientisadded.Hydrolysisoccursmorerapidly whenanacidisinvolvedduetothelowerpHthatspeedsupthehydrolysisof theorthoesterbonds.Whenthehydratingfrontislargerthantheerosionfront inthepresenceofanacid,theorthoesterlinkageswillhydrolyzeandbulk erosionwilltakeplace.Whenthehydratingfrontandtheerosionfrontis equal,onlysurfaceerosionwilltakeplace.

TheaboveprocesswasveryimportantinastudythatwastestingLevonorgestrel,asteroid,forbirthcontrolinhumans.Abaseexcipientisaddedto allowforthehydratingfronttobeequaltotheerosionfront,sothaterosion onlytakesplaceatthesurface.

Inthepast,metallicprostheseswereusedforfractureﬁxationanditwas quitesuccessful.But,itrequiredinsomecasesasecondsurgerytoremovethe implementsanditwasobservedthatthebonealmostneverrecoveredits originalstrength.Useofabioabsorbablepolymerwouldallowforeffective healingandtheretentionofthenaturalloadstrength,andsurgicalremoval wouldbeunnecessary.

POLY(ORTHOESTER)III

Thisfamilyshowedonlyointment-likepropertieswhichhadlimitedusefulnessasanenzyme-releasedeliverysystem.

POLY(ORTHOESTER)IV

Whenthisfamilyofpolymershadanadditionalmethylgroupattachedtoone ofitsbranches,itprecipitatedoutofthemixtureinorganicsolvents.The additionofanethylgroupmakesforasolublecompoundinorganicsolvents.

POLYANHYDRIDESASCARRIERSOFDRUGS

Althoughinthepastdecadevastresearchandmoneyhavebeendedicatedto theproductionandadvancementoflacticacidandglycolicacids,polyanhydridesposeasanewensembleofbiopolymers.Lacticandglycolicacids alwaysprovedtobeaproblemindrugdeliveryduetotheirreleasekinetics. Thesepolymersallowfordiffusionofwaterintotheencapsulateddrugarea, whichcauseseitherquickorunpredictabledegradation.Itwassoughttoﬁnda compoundwherecontrolofthepolymerhydrolysiswaspossibleandsurface erodibilityconcurredwithtime-releasetherapy.