ERROR LOADING HTML FROM SOURCE (http://ncf.sobek.ufl.edu//design/skins/UFDC/html/header_item.html)

FORMATION OF SMALL UNILAMELLAR VESICLES AND APPLICATIONS TO TARGETED DRUG DELIVERY

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004774/00001

Material Information

Title: FORMATION OF SMALL UNILAMELLAR VESICLES AND APPLICATIONS TO TARGETED DRUG DELIVERY
Physical Description: Book
Language: English
Creator: Gupta, Sonali
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Liposomes
Drug Delivery
Membranes
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Liposomes, or membranous vesicles, with a radius less than 100 nm are called Small Unilamellar Vesicles (SULVs). The formation of stable SULVs is problematic since tension in the membrane due to its high curvature destabilizes the particles. In this study the spontaneous formation of SULVs was observed through two separate pathways, both involving structural precursors. SULVs formed through the bicellar phase have disk-like precursors whose morphology is dictated by the molar ratio of long and short chain phospholipids in solution. Here DMPC and DHPC were used in a 3.2:1 ratio to create SULVs of 34.3 +/- 0.24 nm at a concentration of 0.75% lipid w/v and 35.6 +/- 0.45 nm at a concentration of 1.0% lipid w/v. Ellipsoidal precursors yielded liposomes ranging from approximately 58 to 66 nm in radius, at highly dilute concentrations (0.03 to 0.09 % lipid w/v) indicating that at low concentrations the molar ratio of phospholipids incorporated within the structures shifts from the ideal input ratio. Lamellar sheets were not shown to result in SULVs, but may have malleability at threshold concentrations where the lamellae are undergoing unbinding transitions. The dynamics of disk growth hints at a mechanism by which the metastable morphologies of SULVs with structural precursors are maintained for longer periods than SULVs created through high-energy methods such as sonication. These vesicles were created for the purpose of optimizing anti-tumor drugs to glioblastoma multiforme cells through the enhanced permeability and retention effect. SULVs were found to diffuse through uniform porous media at rates 50 times faster than those of LULVs (Large Unilamellar Vesicles), indicating that they may be able to penetrate tumors to a greater extent than LULVs, which are also hindered by lower circulation half-lives. A study of size dependent leakage rates of encapsulated agents remained inconclusive. Overall, stable SULVs suitable for use in targeted drug delivery were successfully created and show promise for biological applications that require penetration of semi-permeable membranes, tissues, or vasculatures.
Statement of Responsibility: by Sonali Gupta
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Colladay, Donald

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 G8
System ID: NCFE004774:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004774/00001

Material Information

Title: FORMATION OF SMALL UNILAMELLAR VESICLES AND APPLICATIONS TO TARGETED DRUG DELIVERY
Physical Description: Book
Language: English
Creator: Gupta, Sonali
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Liposomes
Drug Delivery
Membranes
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Liposomes, or membranous vesicles, with a radius less than 100 nm are called Small Unilamellar Vesicles (SULVs). The formation of stable SULVs is problematic since tension in the membrane due to its high curvature destabilizes the particles. In this study the spontaneous formation of SULVs was observed through two separate pathways, both involving structural precursors. SULVs formed through the bicellar phase have disk-like precursors whose morphology is dictated by the molar ratio of long and short chain phospholipids in solution. Here DMPC and DHPC were used in a 3.2:1 ratio to create SULVs of 34.3 +/- 0.24 nm at a concentration of 0.75% lipid w/v and 35.6 +/- 0.45 nm at a concentration of 1.0% lipid w/v. Ellipsoidal precursors yielded liposomes ranging from approximately 58 to 66 nm in radius, at highly dilute concentrations (0.03 to 0.09 % lipid w/v) indicating that at low concentrations the molar ratio of phospholipids incorporated within the structures shifts from the ideal input ratio. Lamellar sheets were not shown to result in SULVs, but may have malleability at threshold concentrations where the lamellae are undergoing unbinding transitions. The dynamics of disk growth hints at a mechanism by which the metastable morphologies of SULVs with structural precursors are maintained for longer periods than SULVs created through high-energy methods such as sonication. These vesicles were created for the purpose of optimizing anti-tumor drugs to glioblastoma multiforme cells through the enhanced permeability and retention effect. SULVs were found to diffuse through uniform porous media at rates 50 times faster than those of LULVs (Large Unilamellar Vesicles), indicating that they may be able to penetrate tumors to a greater extent than LULVs, which are also hindered by lower circulation half-lives. A study of size dependent leakage rates of encapsulated agents remained inconclusive. Overall, stable SULVs suitable for use in targeted drug delivery were successfully created and show promise for biological applications that require penetration of semi-permeable membranes, tissues, or vasculatures.
Statement of Responsibility: by Sonali Gupta
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Colladay, Donald

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 G8
System ID: NCFE004774:00001


This item is only available as the following downloads:


Full Text

PAGE 1

FORMATIONOFSMALLUNILAMELLAR VESICLESANDAPPLICATIONSTO TARGETEDDRUGDELIVERY BY SONALIGUPTA AThesis SubmittedtotheDivisionofNaturalSciences NewCollegeofFloridainpartialfulllmentoftherequirementsforthedegree BachelorofArts UnderthesponsorshipofDr.DonaldColladay,ProfessorofPhysics Sarasota,Florida May,2013

PAGE 2

Acknowledgements FirstlyIwouldliketothankmymotherforherunwaveringsupportand thefreedomshehasaordedmetoexploremyidentity,andpursuemy path.HerrstpriorityhasalwaysbeenmyeducationandforthatIam deeplygrateful.WithoutherIwouldnotbethepersonIamtoday.Sheis alsoaprofoundlyinteresting,caring,fun,andlovingperson,andaconstant sourceofinspirationtome.Iloveyou,mummy. Ialsowanttothankthemembersofmybaccalaureatecommittee: Dr.Colladay,forhisadvisingthroughouttheprocessofwritingthisthesis. WhenIlostdirectionandgotmiredindetails,youwereareorientingforce. Thankyouforyoureloquentlectures,andforyourpatiencewithme. Dr.Shipman,whohastaughtmethemeaningofhardworkandintegrity byexample.ThankyouforsomeofthebesttutorialsessionsIhavehad; yourlucidexplanationsofP.Chemconceptsleftmemoreinawethanyou know.Thankyouforbeingthekindofprofessorwhostaystill3a.m.ona Fridaybackingupthesisstudents'datawhenmasterkeysgomissing.After thelibraryclosedyourlabwasmyrefuge,withaconstantowofespresso. Mostofall,thankyouforrevealingtomethebeautyinP.Chemandfor inspiringmetowhollydedicatemylifetoscience. Dr.Clore,forbeingmyacademicadvisorthroughoutmostofmyundergraduatestudiesandservingonmybaccalaureatecommittee.Yourgreat lecturesincellbiologyandyourenthusiasmforthesubjectmovedmeto pursuebiophysics.Yourdisciplineandfocussetanexample,andIthank youforyourguidanceandyourpatiencetowardsme. Iamalsogratefulformyresearchmentor,Dr.Pai,forallhisguidance andinvaluablethesisadvice.Withouthimthisthesiswouldnotexist.I thankhimformakingmyrstresearchexperienceanincrediblypositive one,formotivatingme,andforteachingmethevalueofperserverancein doingresearch.

PAGE 3

IalsothankDr.BellamkondaforhiscounselandDr.AlbertoFernandezNievesforgenerouslyallowingmeuseoftheSoftCondensedMatterLabat GeorgiaTech. IespeciallywanttothankJohnS.HyattandAndreaScottifortheirinvaluablehelpwithDLSandfortakingtimeoutoftheirbusyschedulesto helpmerunexperiments.IalsowanttothankJohnHyattforwritingthe MATLABcodeIusedtoprocessmydata. FinallyIwouldliketothankmyfriendssimplyforbeingtheinteresting, awesomepeoplethattheyare.Thankyouforthewonderfultimewehave sharedtogether,forallthelaughs,andforallthelove.Ialsothankmy dog,Einstein,whohasbeenmyconstantcompanionandabundleofjoy.

PAGE 4

FORMATIONOFSMALLUNILAMELLARVESICLESAND APPLICATIONSTOTARGETEDDRUGDELIVERY SonaliGupta NewCollegeofFlorida,2013 Abstract Liposomes,ormembranousvesicles,witharadiuslessthan100nmarecalledSmall UnilamellarVesiclesSULVs.TheformationofstableSULVsisproblematicsincetensioninthemembraneduetoitshighcurvaturedestabilizestheparticles.Inthisstudy thespontaneousformationofSULVswasobservedthroughtwoseparatepathways, bothinvolvingstructuralprecursors.SULVsformedthroughthebicellarphasehave disk-likeprecursorswhosemorphologyisdictatedbythemolarratiooflongandshort chainphospholipidsinsolution.HereDMPCandDHPCwereusedina3.2:1ratioto createSULVsof34.3 0.24nmataconcentrationof0.75%lipidw/vand35.6 0.45 nmataconcentrationof1.0%lipidw/v.Ellipsoidalprecursorsyieldedliposomesrangingfromapproximately58to66nminradius,athighlydiluteconcentrations.03to 0.09%lipidw/vindicatingthatatlowconcentrationsthemolarratioofphospholipids incorporatedwithinthestructuresshiftsfromtheidealinputratio.Lamellarsheets werenotshowntoresultinSULVs,butmayhavemalleabilityatthresholdconcentrationswherethelamellaeareundergoingunbindingtransitions.Thedynamicsofdisk growthhintsatamechanismbywhichthemetastablemorphologiesofSULVswith structuralprecursorsaremaintainedforlongerperiodsthanSULVscreatedthrough high-energymethodssuchassonication.Thesevesicleswerecreatedforthepurpose ofoptimizinganti-tumordrugstoglioblastomamultiformecellsthroughtheenhanced permeabilityandretentioneect.SULVswerefoundtodiusethroughuniformporous mediaatrates50timesfasterthanthoseofLULVsLargeUnilamellarVesicles,indicatingthattheymaybeabletopenetratetumorstoagreaterextentthanLULVs, whicharealsohinderedbylowercirculationhalf-lives.Astudyofsizedependentleakageratesofencapsulatedagentsremainedinconclusive.Overall,stableSULVssuitable foruseintargeteddrugdeliveryweresuccessfullycreatedandshowpromiseforbiologicalapplicationsthatrequirepenetrationofsemi-permeablemembranes,tissues,or vasculatures. Dr.DonaldColladay,DepartmentofNaturalSciences iv

PAGE 5

Contents ListofFiguresix ListofTablesxi ListofAbbreviationsxiii 1Introduction1 1.1LiposomesinTargetedDrugDelivery...................1 1.2ModelSystem:GlioblastomaMultiforme..................2 1.3Motivations..................................4 2FormationofSmallUnilamellarVesicles7 2.1MembraneComposition...........................7 2.1.1PhospholipidStructure.......................7 2.1.1.1AcylChainLength....................8 2.1.1.2Phosphatidylcholines...................9 2.1.2PackingParameter..........................9 2.1.2.1DerivationandApplicability...............9 2.1.2.2Calculating P forComponentPhospholipids......11 2.1.2.3PredictingtheBicellarPhase...............12 2.1.3TheMolarRatio...........................13 2.1.3.1MolarRatiosforanIdealBicellePhase.........14 2.1.3.2OverallSignicanceoftheMolarRatio.........15 2.2ConcentrationandTemperaturePhaseDiagram.............17 3Methods21 3.1FormationofSmallUnilamellarVesicles..................21 3.1.1LipidCake..............................21 3.1.2Path1:DilutionAboveCriticalTemperature...........22 3.1.3Path2:Disk-likePrecursorsBelowCriticalTemperature....22 3.1.4Path3:EllipsoidalPrecursorsBelowCriticalTemperature...22 v

PAGE 6

CONTENTS 3.1.5Extrusion...............................23 3.1.6DLSMeasurements..........................23 3.2DependenceofBaselineLeakageonSizeofLiposomes..........24 3.2.1QuantifyingRhodamineConcentrationsthroughFluorescenceIntensity.................................24 3.2.2RhodamineLoadedLULVs.....................24 3.2.3RhodamineLoadedSULVs.....................25 3.2.4MembraneDialysis..........................25 3.2.5FluorescenceImaging........................25 3.3DiusionThroughaUniformGel......................25 3.3.1SettingtheSephadex.........................26 3.3.2SynthesisofLULVswithFluorescentMembranes.........26 3.3.3SynthesisofSULVswithFluorescentMembranes.........26 3.3.4DLSofLiposomeswithFluorescentMembranes..........26 3.3.5InjectionandFluorescentImaging.................27 4DataandAnalysis29 4.1FormationofSmallUnilamellarVesicles..................29 4.1.1ProcessingDLSData........................29 4.1.2Path1:DilutionAboveCriticalTemperature...........33 4.1.3Path2:Disk-likePrecursorsBelowCriticalTemperature....36 4.1.3.1 =0.75..........................36 4.1.3.2 =1.0...........................38 4.1.3.3 =1.5...........................39 4.1.3.4ComprehensiveDiscussionofSULVsFormedviatheBicellarPhase........................41 4.1.4Path2:EllipsoidalPrecursorsBelowCriticalTemperature...43 4.1.4.1 =0.3...........................43 4.1.4.2 =0.6...........................43 4.1.4.3 =0.9...........................45 4.1.4.4ComprehensiveDiscussionofLiposomesFormedviaEllipsoidalPrecursors....................45 4.1.5DiscussionofPreferredPathwayforSubsequentExperiments..46 4.2DependenceofVesicleLeakageonSize...................48 4.2.1QuantifyingRhodamineConcentrationsthroughFluorescenceIntensity.................................48 4.2.2DependenceofLeakageRateonVesicleSize............50 vi

PAGE 7

CONTENTS 4.3DiusionthroughaUniformMedium....................53 5Conclusions59 6Appendix61 6.1CurvatureEnergy...............................61 6.1.1CurvatureUsingMongeParametrizationinOneDimension...62 References67 vii

PAGE 8

CONTENTS viii

PAGE 9

ListofFigures 1.1EectofIBintheContainmentofGBMCells...............4 1.2SurvivalRatesofMiceUnderVariousGBMTreatments.........5 2.1DHPCandDMPC..............................8 2.2DHPCPhases.................................12 2.3TheDisk-likeBicelle.............................13 2.4Cross-SectionofDisk-likeBicelle......................14 2.5DependenceoftheHydrodynamicRadiusonMolarRatio........16 2.6PhaseDiagramofPhospholipidStructuresintheDMPC/DHPCSystem17 2.7GrowthbyCoalescenceandClosureofDisk-likeBicelles.........19 4.1SamplePlotofAutocorrelatedIntensityvs.DecayRate.........30 4.2ScalabilityoftheScatteringVector.....................32 4.3RelationshipbetweenIntensityandtheAngleofDetection........35 4.4LiposomesFormedviaBicellarPhaseat0.75%w/v............37 4.5LiposomesFormedviaBicellarPhaseat1%w/v-Uncorrected.....39 4.6LiposomesFormedviaBicellarPhaseat1%w/v.............40 4.7LiposomesFormedviaBicellarPhaseat1%w/v.............41 4.8LiposomesFormedviaEllipsoidalPrecursorsPhaseat0.3%w/v....44 4.9LiposomesFormedviaEllipsoidalPrecursorsPhaseat0.6%w/v....45 4.10LiposomesFormedviaEllipsoidalPrecursorsPhaseat0.9%w/v....47 4.11FluorescenceIntensitywithVaryingRhodamine-BConcentrations...48 4.12DependenceoftheQuantumYieldofRhodamine-BonConcentration.49 4.13FluorescenceIntensitywithVaryingRhodamine-BConcentrations...50 4.14FluorescenceIntensitywithVaryingRhodamine-BConcentrations...51 4.15FluorescenceIntensitywithVaryingRhodamine-BConcentrations...52 4.16Sephadex50GelMedium[31]........................53 4.17Diusionof50nmLiposomesAcrossa0.4 mSpace...........54 4.18Diusionof100nmLiposomesAcrossa0.4 mSpace..........55 ix

PAGE 10

LISTOFFIGURES 4.19Diusionof200nmLiposomesAcrossa0.4 mSpace..........56 x

PAGE 11

ListofTables 2.1ExperimentallydeterminedmolecularparametersforDMPCandDHPC.11 3.1MolecularWeightsforLiposomeIngredients................21 3.2PhospholipidConcentrationsforallPath1Solutions...........22 3.3PhospholipidConcentrationsforallPath2Solutions...........22 3.4PhospholipidConcentrationsforallPath3Solutions...........23 4.1Temperature,ViscosityandDiusionCoecientfor =0.75......38 4.2Temperature,ViscosityandDiusionCoecientfor =1.0.......39 4.3Temperature,ViscosityandDiusionCoecientfor =1.5.......42 4.4AverageHydrodynamicRadiiforallLiposomeswithDisk-likePrecursors42 4.5Temperature,ViscosityandDiusionCoecientfor =0.3.......43 4.6Temperature,ViscosityandDiusionCoecientfor =0.6.......46 4.7Temperature,ViscosityandDiusionCoecientfor =0.9.......46 4.8AverageHydrodynamicRadiiforallLiposomeswithEllipsoidalPrecursors48 xi

PAGE 12

GLOSSARY xii

PAGE 13

ListofAbbreviations SULVs -smallunilamellarvesicles;liposomes lessthan100nminradius. LULVs -largeunilamellarvesicles;liposomes greaterthan100nminradius. GBM -glioblastomamultiforme;themostcommonandthemostdeadlyformofbraincancer. BBB -bloodbrainbarrier;ltersbloodowto thebrain. IB -imipramineblue;anagentthatstuntsthe radialspreadoftumorsbyinhibitingactinpolymerization. DXR -doxorubicin;acommonchemotherapy drug. VEG-F -vascularendothelialgrowthfactor;its upregulationcausesangiogenesisandneovascularizationneartumors. PEG -polyethyleneglycol;usedasaliposomal coattoincreasecirculationhalf-lifeandincrease thenanoparticle'sstealthtothebody'sltration mechanisms. ECM -extracellularmatrix. DMPC -dimyristoylphosphatidylcholine;a phospholipidwithacholineheadgroupandtwo 14carbonlongacylchains. DHPC -dihexanoylphosphatidylcholine;aphospholipidwithacholineheadgroupandtwo6carbonlongacylchains. DLS -dynamiclightscattering;atechniqueused toindirectlyobservethesizeofvesiclesinsolutionbasedonhowtheparticlesscatterlaserlight. NBDDHPC -1-hexanoyl-2-6-[-nitro-2-1,3benzoxadiazol-4-ylamino]hexanoyl-sn-glycero-3phosphocholine-afattyacidlabeleduorescent phospholipidwithaphosphatidylcholineheadgroupandtwo6carbonlongacylchains. xiii

PAGE 14

0.LISTOFABBREVIATIONS xiv

PAGE 15

1 Introduction 1.1LiposomesinTargetedDrugDelivery Drugsareoftenintendedtotargetspecicregions,typesofcells,orparticularbiochemicalpathwaysinthebody.Commonmethodsofdrugadministration,likeoral uptakeorinjections,exposemanybodilysystemstothedrugsbeforetheydiuseto theirtargetsites.Drugsarealsosubjecttothebody'sltrationsystem;somemore thanothers,dependingontheirmolecularproperties.Ingeneralhowever,thedosage administeredisgreaterthantheeectivedoseinordertoaccountforlossduetoltration.Themetabolismofdrugs,orsimplytheirpassagethroughvariousorgansand tissues,contributessignicantlytosideeects.Thisisespeciallytrueforoftencytotoxicanti-cancerdrugs,orimmunosuppressantswhichgreatlyincreasetheriskof cancersandinfectionsoverall,evenwhentheinammationthedrugsaretryingto combatislocalized.Thegoaloftargeteddrugdeliveryistotransportdrugstotheir targetsiteswhileminimizingexposuretotherestofthebody,therebyreducingside eectsandincreasingtheiroverallecacy. Nanocarriersarenano-sizedstructuresusedtotransportdrugsinvivothatcan beengineeredinavarietyofways,andwithspecicpropertiesdependingontheir intendeduse.Onetypeofnanocarrierwithgreatversatilityareliposomes,whichare hollowstructureswithaphospholipidbilayermembrane.Liposomeshavebeenformed inavarietyofshapessuchasspheres,ellipsoids,anddisks.Sphericalliposomesarethe mostcommon,withthosegreaterthan100nminradiuscalledlargeunilamellarvesicles LULVs,andthosebelowthissizerangecalledsmallunilamellarvesiclesSULVs.[1] Thetherapeuticagentisencapsulatedwithintheliposomeduringformationandseveral techniquescanbeusedtoreleasetheliposomeloadatthetargetsite. Thetechniquesemployedfordrugreleasearehighlydependentuponthenatureof thetherapy.Forinstance,nanocarrierswouldbelargelyineectiveindeliveringsiRNA 1

PAGE 16

1.INTRODUCTION tocellsinordertosilencecertaingenesunlesstheyaredesignedtoensureintracellular delivery.Nanoparticleswithprotonspongeexteriorscomposedofcarboxylicacidand tertiaryaminegroupsatavariableratiohavebeenusedtotransportdrugsinside cells.[2]Byadjustingtheratiothenanocarrierscanbedesignedtocauseabuild-upof osmoticpressurewhileinanacidicendosomeenvironment,causingtheendosomesto burstduringtransportintothecellthroughtheendocytoticpathway.[2]Thisisjust oneexampleofhowmodicationstoananocarrierscanenabletargeteddelivery,but moreover,itisdemonstrativeofhowtheymustbeengineeredwithpropertiesspecic tothetherapybeingimplemented. Whenaspecictypeofcellistobetargetedsurfaceligandscapableofdistinguishingthecelltypeareoftengraftedtothenanoparticlesurface.Anothercommon techniqueisdecoratingdrugcarrierswithchemicalgroupsthatcaninducethecarrier toreleasethedruguponexcitationwhenexposedtoexternalradiation.Chainsofiron oxidesthatoscillateinaradiofrequencyeldhavebeenusedtocauseburstreleaseof liposomesthroughthismechanism.[3]Theseareonlyafewselectexamplesofliposome applicationsindrugdelivery,buttheyillustratehowliposomescanbecustomizedto meetimportantstipulationsofsometherapiessuchasintracellulardelivery,pHdependentrelease,cell-specicdeliveryandtimedrelease.Sinceliposomesareengineered tomeetthedemandsofadesireddrugdeliveryscheme,itisnecessarytoestablisha modelsystembeforetheirfunctionalitycanbeoptimized. 1.2ModelSystem:GlioblastomaMultiforme GlioblastomamultiformeGBMisthemostcommon,andalsothemostdeadlyform ofbraincancer.Ithasamortalityrateof96%after5years,andamediansurvival timeof15months.[4]Thehighmortalityrateisexpectedasthebrainisvitalin regulatingthebody'sfunctions.However,theincorrigiblenatureofGBMisalsodue toseveralcomplicatingfactorsinitstreatment,thebiggestdicultybeingthehighly metastaticnatureofthecancer.Becausebraintissueissosoft,thetumoriseasilyable toproliferateoutwards,makingtherateandextentofmetastasismuchhigherthan cancersinharder,densertissues.Thiscomplicatesneurosurgerysincegreateramounts ofbraintissuehavetobeexcised,whichmaycausefurtherdisability.Still,chances thatthecancerhasmetastasizeddeeperintothebrainremainhigh,andtargetingthese cellswithchemotherapyandradiationalsoaectshealthybraincells.Moreover,neural cellregenerationisslowandoccurstoalimitedextent.ThebloodbrainbarrierBBB isalsoanobstacletoecientdrugdeliverytoGBMcells.Forthesereasons,notonlyis GBMhighlymalignantbuttreatmentonlyminimallyprolongsthelifeofthepatient. 2

PAGE 17

1.2ModelSystem:GlioblastomaMultiforme GBMisanappropriatemodelsystemtotestliposome-assisteddrugdeliverybecause liposomesareabletopermeatetheBBB.Previousstudieshavedemonstratedtheir ecacyindeliveringdrugstothebrain,especiallydrugsthathavelowcirculation times,orthosethatarelargemoleculesunabletotthroughthetightjunctionsbetween endothelialcellscomposingtheBBB.[5]Onepromisingstudythatusedliposomesto deliverdrugstoGBMcellsusedanovelcompoundcalledimipramineblueIB,followed bydoxorubicinDXR,acommonchemotherapydrug.IBwasfoundtohaveantimetastaticpropertiessinceitinhibitsthepolymerizationofactin,thelamentsalong whichcancercellsspread.[6]ThespreadofhumanGBMneurospheresinacollagen matrixwassignicantlymorecontainedupontreatmentwithIB,asshowninFigure1.1. Eventhoughtheirgrowthrateislargelyunaected,theircontainmentallowsGBMcells tobebettertargetedbythefollow-uptreatmentofliposomaldoxorubicinL-DXR. Anotherreasonliposomesareespeciallysuitedforuseinthisdrug-deliveryschemeis becausefreeIBhasaverylowcirculationtime,withahalf-lifeofonly11minutes. Thisfact,alongwithitslargemolecularsize,makesitvirtuallyimpossibleforIBto crosstheBBB.WhenencapsulatedwithinliposomesL-IB,thecirculatoryhalf-life increasesto18hours,andtheabilitytocrosstheBBBisgreatlyenhanced.[6] ThemostexpedientpartofthisschemeisthattheliposomesonlytargetGBM cells,sparinghealthybraincellsfromcytotoxiceects.Thisisduetotheenhanced permeabilityandretentioneectseeninthevasculaturesurroundingcancercells,especiallyduringmetastasis.Tumorcellsmaintaintheirnutritionalneedsbyreleasing certainangiogenicfactors,likevascularendothelialgrowthfactorVEGF,inexcessto createnewbloodvessels,therebyincreasingtheirsurfaceareacontacttovasculature. Duringmetastasis,connectionsareformedbetweenthenewandexistingvasculature, encouragingbothtumorgrowthandspread.[7]Vasculargrowthinhealthycellsdepends onatightbalanceandafeedbackbetweenpositiveandnegativeangiogenicfactors.A disruptioninthisbalancecausesthehaphazardgrowthofbloodvesselsincontrast tohealthyvasculaturewhichisuniformlyaligned.Thisrapid,disorderlygrowthis responsiblefortheincreasedporosityofthisneovasculature,causingporesrangingbetween100-300nminthebraintumorselsewhereinthebodyhaveneovasculatures rangingfrom200-780nm.[8,9]Sincehealthyvasculaturehasporesoflessthan10 nmonaverage,[10]liposomesgreaterthan25nminsizewillonlyexitthecirculatory systemwhentheyowthroughcancerousregions.Thispassivecollectionofliposomes inandaroundtumorcells,overtheirlongcirculatoryhalf-lives,minimizestheeect ofthedrugsontherestofthebrain.Thiswastestedinthesamestudy,onmice withGBMusingL-IBandL-DXRwithanaverageradiusof160nm,andcoatedwith polyethyleneglycolPEGwhichfurtherextendscirculationtime.[6]Asisevidentin 3

PAGE 18

1.INTRODUCTION Figure1.1: EectofIBintheContainmentofGBMCells PicturedaretwohumanGBMneurospheresinacollagenmatrix,treatedwithoutand with10 MIB,stainedwithrhodamineanduorescentlyimaged.[6]Thescalebar represents100 m. Figure1.2,micetreatedwithL-IBfollowedbyL-DOXhada100%survivalthroughout the200daylongstudy,whereasnoneoftheuntreatedmicesurvivedpast35days. 1.3Motivations OnewaytooptimizetheL-IB/L-DXRtreatmentforGBMistomaketheliposomes smaller.Withanaverageporediameteraround260nm,the160nmliposomesonly exitthecirculationthroughporesonthehighestendofthesizerange.Foraparticle tofreelydiusethroughthevascularjunctions,itsdiametershouldbe 1 3 to 1 5 the diameterofthepore.[11]Thelargestporesizesareduetoprolongedupregulationof VEGF,andreectthepathophysiologyoflatestagecancer.Toadministertargeted earlytreatment,withalesserextentofneovascularization,thedrugwillhavetobe incorporatedintoliposomeslessthan50nminradius. UponenteringtheendothelialjunctionsinthevasculaturetheLULVsusedinthe GBMstudyarelikelytoremainclosetovascularwalls,leakingsomeoftheircargo 4

PAGE 19

1.3Motivations Figure1.2: SurvivalRatesofMiceUnderVariousGBMTreatments Graphsshowingthesurvivalratetopandtumorvolumebottomofuntreatedmice andthosetreatedwithonlyL-IB,onlyL-DXR,andbothL-IBandL-DXR.[6] backintocirculation.SULVs,duetotheirsmallsize,arebetterabletodiusethrough theextracellularmatrixECMtotheperipheryofthetumor.Liposometreatments relyontheaccumulationofdrug-carryingliposomesintothevasculatureporeswith everypassthroughthecirculatorysystem.UsingSULVswouldincreasetheclearance aroundthepores,allowingmoreliposomestoaccumulatewitheachcirculationcycle. TheliposomesusedintheGBMstudyslowlyreleasetheirencapsulateddrugsthrough passivediusionacrossthemembrane.Afasterandmoreconcentratedpresenceof SULVsatthetumorsitewouldalsoensuremostoftheliposomalloadisreleasedat thetargetsite. Previousexamplesdemonstratehowliposomesweretailoredforspecicavenues oftreatment,buttheliposomesusedheredidnotneedanyornamentationbeyonda standardPEGcoating,especiallysinceasmooth,sphericalparticleisoptimalforprolongingcirculationtime.Surfaceligands,suchashumaninterleukin-13,couldincrease theliposomes'anitytoGBMcellsbutwouldalsolowerthepassiveaccumulationof liposomesattumorsduetostericdestabilizationandltrationbythekidneys.[12]Iron oxidechains,likeintheexampleabove,couldallowliposomestoreleasetheircargoat specictimesbuttheradiationcancausenerveablationinhealthytissue,anddamage 5

PAGE 20

1.INTRODUCTION bloodvessels.Liposomesareusedfortheirabilitytotargettherapyatacellularlevel, andexogenoustreatmentslikeradiationnegatethatbenet.Finally,iftherateof leakageofthedrugfromtheliposomecanbeapproximated,dosagecanbecontrolled toacheivesustainedreleaseratherthanburstrelease. Becausethistreatmentworkswellwithstandardliposomes,itcanbeusedtotest whetherintrinsicpropertiesofliposomessuchassizeandphospholipidcomposition canpredictthestabilityanddiusionofliposomes.SULVsshouldtheoreticallydiuse easilythroughboththeneovasculatureandtheECM,buthaveinherentinstabilitydue tohighmembranetension.SULVscreatedusingtraditionalmethodslikesonication havehighsurfacecurvature,oftencollapsingintolargeraggregates.[1]Thisstudyuses protocolsforthecreationofthermodynamicallystable,orkineticallytrappedSULVs fromstructuralprecursors.Testingwhetherthealteredsurfaceareatovolumeratioin SULVsaectsratesofpassivediusionofthedrugthroughtheliposomalmembraneis importantingauginghowthesizeofvesiclesinuencedosage.Leakageofthedrugfrom theliposomemightbeinuencedbythegreatertensionwithinSULVmembranes.The goalofthisstudyistoformstableSULVsusingstructuralprecursorsfordrugdelivery tobraintumorsandtesttheirleakageanddiusionagainsttheLULVsusedinthe GBMstudy. 6

PAGE 21

2 FormationofSmallUnilamellar Vesicles WhilevariousmethodsforSULVformationexist,liposomalsolutionsandtheconditionsofformationvarywidelybetweenprocedures.Thestability,permeability,and encapsulationeciencyoftheliposomesarehighlydependentontheirpathwayofformation.Thischapterisadiscussionofhowmembranecomposition,temperature,and concentrationdetermineliposomeproperties.IntheGBMdrugdeliveryscheme,stabilityandencapsulationeciencyarekeytothefunctionalityoftheSULVs.Hence thesecharacteristicswillbeprioritizedwhendeterminingtheoptimalprocessofSULV formation. 2.1MembraneComposition Therststepincreatinganyliposomeistocombinethecomponentsofitsmembrane intoalipidcake,ahomogenousmixtureoftheconstituentphospholipidsintheirrespectivemolarratios.Thestructureofeachphospholipid,itschargedistribution,and itsmolarratiohavealreadypreordainedmembranedynamicsthroughouttheprocess ofliposomeformation.Thissectiondetailstheconsiderationsthatgointomembrane composition,withanemphasisonfactorsthatfacilitateSULVformation. 2.1.1PhospholipidStructure Phospholipidsareamphiphilicmoleculeswithaphosphateheadandahydrophobicacyl chain.Inanaqueoussolutionthishydrophobictailrestrictsthenumberofpossible hydrogenbondsofthesurroundingwatermolecules,reducingthelocalentropy.The arrangementofphospholipidsintobilayerswiththeacylchainspointedinwardsis thereforeentropicallyfavoredsinceitminimizesthesurfaceareaofthehydrophobic 7

PAGE 22

2.FORMATIONOFSMALLUNILAMELLARVESICLES region,andincreaseshydrogenbondingbetweenwatermolecules.Thiseectalso explainswhybilayerstendtowraparoundattheedges,formingclosedstructuresand furthershieldinghydrophobicareas.Thepolarphosphategroupallowsthemembrane atlargetofreelytravelthroughaqueousmedia. 2.1.1.1AcylChainLength Thelengthofthephospholipids'acylchainsinuencesmembraneelasticityinseveral ways.Aphospholipidisconsideredtohavealongacylchainifithasmorethan14 carbons.Longerunsaturatedacylchainshavethegreatestamountofmolecularexibility,andthuscontributetooverallmembraneelasticity.However,iftheacylchain issaturated,itslengthonlycontributestoitstendencytoaggregatewithotherphospholipidswhichcanbeproblematicinmakingSULVswhichalreadyhaveatendency tofusetogether.[13]Whilelongeracylchainsareabletobendeasily,theyalsooccupy agreatervolumewithinthebilayer.Largerstructureswithlowercurvaturecanaord longacylchainstheirspace,butinSULVstheinnerbilayerbecomescramped,pushing theentirestructureapart. Inordertoretainexibilitybutminimizestrain,long-chainphospholipidscanbe attenuatedwithshort-chain,rigidphospholipids.Thesemoleculestypicallyhavefewer than8carbonsineachchain,andallowtheouterleaet'sheadgroupstoremainassociatedwhilestillallowingthelongacylchainsroomtouctuate.HenceforSULV synthesis,dimyristoylphosphatidylcholineDMPC,commonlyusedinliposomemembranes,willbethelong-chainphospholipidwithtwoacylchainof14carbonseach. TheshortchainphospholipidwillbedihexanoylphosphatidylcholineDHPC,with twoacylchainsof6carbons. Figure2.1: DHPCandDMPC 8

PAGE 23

2.1MembraneComposition 2.1.1.2Phosphatidylcholines DMPCandDHPCwerechosenbecausetheyarebothphosphatidylcholinesPCs,the mostnaturallyabundantclassofphospholipidsinthebody,comprisingapproximately 80%ofbiologicalmembranes.Theyarealsoeasilyextractedforlaboratorypurposes fromeggyolksorsoybeans,andareoneofthemostcommonlyusedphospholipids inliposomestudies.PCshaveanoverallneutralheadasthecationiccholinegroup neutralizesthenegativechargeonthephosphategroup.Otherclassesofphospholipids includephosphatidylglycerolPGandphosphatidylserinePS,bothwithnegatively chargedheadswhichcompose10-20%ofphospholipidsfoundthebody.Thechargeon aphospholipidcanaecttheliposome'sabilitytopenetratecertaintissuesandcells, andhencechargeisoneaspectofoptimizingdrugdelivery.Theinteractionsofcharged phospholipidsarecomplicatedandcanaectoverallmembranestability.Byonlyusing phospholipidswithneutralheadgroups,theseinteractionscanbeconsiderednegligibleandmembranestabilitycanbestudiedasdependentononlythephospholipids' structureandtheirrespectiveconcentrationswithinthemembrane.Theheadgroup's dimensionsalsovarywidelybetweenclassesofphospholipidssousingonlyoneclass, suchasPCs,minimizesthevariationinsurfaceareaandvolumeperphospholipid,and simpliescalculations. 2.1.2PackingParameter Theheadgroupsurfaceareaandvolumeofphospholipidscanbevaluableinpredicting thestructureoftheiraggregatesthroughthehighlysimpliedgeometricalconstruction ofapackingparameter, P .IntroducedbyIsraelachviliin1979toexplaingeometricalconstraintsinselfassembly,thepackingparameterisbasedontheideathatthe shapeofanaggregateplaceslimitationsonthestructuralparametersofitscomponent phospholipids.[14] 2.1.2.1DerivationandApplicability Considertheaggregationof N phospholipidsintoaspheremadeupofasinglelayered phospholipidmembrane,calledasphericalmicelle,andassumethatmostofitsvolume, V ,isoccupiedbythephospholipidtails.Thesurfaceareaandvolumeoftheaggregate willbethesumofthesurfaceareasandvolumesofthe N phospholipidscomposing theaggregate.Thisisexpressedinthefollowingequationsforthecaseofaspherical aggregate, 4 R 2 = N a .1 9

PAGE 24

2.FORMATIONOFSMALLUNILAMELLARVESICLES 4 3 R 3 = N v .2 where a isthesurfaceareaoftheheadgroupofonephospholipidand v isthevolume theentiremoleculeoccupies.Solvingbothequationsfor N andequatingthemyields, 1 3 = v Ra .3 Thus,usingapurelygeometricalargument,theaggregateofaphospholipidwill besphericalifthephospholipid'sdimensionssatisfytheaboverelation.Theradiusof theaggregate, R ,isdeterminedbythemaximumtaillength, l ,ofthephospholipid. Longertailscanalsocompressandfoldtoaccomodateasmallerradius,sothepacking parameterforaspherecanberewrittenintermsof l as, P v la 1 3 .4 Similarargumentsforpackingparametersofcylindricalaggregatesleadstothe condition, 1 3 P 1 2 .5 andforbilayersheets[15], 1 2 P 2.6 Above P =2aggregatesstarttoexhibitreversephasemorphologieswiththeir tailsorientedoutwards.[15]Itmustbenotedthatspanning P isnotrepresentativeof allpossibleaggregatestructuresforagivenphospholipid.However,whenaggregates areknowntoformundercertainconcentrationandtemperaturerangesinahomogenoussolutionofphospholipids,thepackingparametercanbeusefulinapproximating thestructure.Forinstance,atverylowconcentrationsDHPCandDMPCarefree moleculesinsolution,andatsucientlyhighconcentrationsallphospholipidswillarrangeintolamellarsheets.[16]Neitherofthesescenariosaredictatedbythepacking parametersincetheeectsofconcentrationandintermolecularandmolecule-solvent interactionsaredominant.However,bothphospholipidshaveaconcentrationrange wheremoleculesinsolutionareabletondandinteractwitheachother,andarrange intoenergeticallyfavorablestructuresdictated,inpart,bythemolecule'sdimensions. ThelowerboundofthisrangeiscalledthecriticalmicellarconcentrationCMCof whichDMPC'sis6nMandDHPC'sis15mM.[17]InspiteofbelongingtothesamePC 10

PAGE 25

2.1MembraneComposition class,andbeingneutralmoleculeswithminimalelectrostaticinteractions,thethresholdconcentrationatwhichmicellesforminDHPCis6ordersofmagnitudegreater thanDMPC,indicatingthatdimensionalityhasalargeeectonaggregatestructure. Thus,withinamedianconcentrationrangethepackingparametercaninformaggregatestructureforeachphospholipid.Dimensionalitycanalsobeovershadowedbyhigh temperatureswherethekineticenergyofeachmoleculeistoogreatforittobeconstrainedwithinanaggregate.Thepackingparameterlosesmeaningforsolutionsheld abovethechain-meltingtemperatureofthephospholipid,whichisapproximately23 CforDMPCand45 CforDHPC.[1,18] 2.1.2.2Calculating P forComponentPhospholipids Quantity DMPC DHPC v A 3 1480 1126 a A 2 58.5 100 d A 25.3 23.9 P 1.182 0.471 Table2.1: ExperimentallydeterminedmolecularparametersforDMPCandDHPC. Table2.1displaysexperimentallydeterminedmolecularparametersofDMPCand DHPCalongwiththecalculatedvaluefor P .DMPCwitha P of1.18ispredicted tohaveplanarbilayeraggregates.Thisisconsistentwiththepreferenceforlower curvature,andhencelargervesiclesize,ofpureDMPCliposomesascomparedwith thosethatincludeDHPC.Asubtledistinctionistobemadeherebetweenthestacked bilayersoflamellarsheetsandthebilayeraggregatepredictedby P sinceconating thetwoimpliesthatconcentrationhasnoeectonDMPC'sfavoredstructure.In lamellarsheets,foundathighconcentrationsandminimalhydration,themoleculesare interdigitatedandhavegreaterintermolecularinteractions.[19]Thebilayersfoundat moredilutedconcentrationsareplanaruidmembranefragmentsthatariseinsolutions offreeDMPC,andarenotnecessarilyderivativeofthesheets.[1] The P valueforDHPCof0.471impliesformationofcylindricalmicelles.Sincethe DMPCexperimentaldatarepresentedafullyhydratedstate[20],theDHPCparametersforfullhydrationwerealsoselected.Figure2.2showsvariousphasesofDHPC andtheireectonmoleculardimensions.NotethatsinceDHPC'suidphaseistiedto atemperaturegreaterthanitschain-meltingthreshold,applyingitsuidphasedimensionstothepackingparameterwouldbeinappropriateforreasonsexplainedabove. SincetheDMPCdataaccountedfortheswellingofmembranewithwater,themaximummembranethicknessofahydratedDHPCmembranewashalvedinordertond 11

PAGE 26

2.FORMATIONOFSMALLUNILAMELLARVESICLES Figure2.2: DHPCPhases gel,interdigitatedfullyhydratedanduidfullyhydrated,T > 44 C.[18] thespaceoccupiedbythetail.Thisvalueisincloseagreementwiththe0.2nmlength approximatedby[21],whosedataforDHPCwillbeusedinalatersectiontorenea modelpredictingliposomesize.ThereisaninconsistencybetweentheDHPCsurface areafoundby[21]ofapproximately1nm 2 ,andthevalueof77.2A 2 givenin[18]. Theformervalueisemployedherenotonlybecauseofconsistencywithlatermodels, butbecause[18]displayshydratedDHPCasinterdigitated,whichistypicallynotthe caseinDHPCmicellesespeciallywhentheirheadgroupsareorientedoutwards,allowingspaceforwatermoleculesinbetween.Becauseswellingeectswereconsideredin membranethickness,theircontributiontotheheadgroupareawasalsoaccountedfor inusingtheheadgroupareadeterminedby[21]. 2.1.2.3PredictingtheBicellarPhase Thepurposeofusing P todeterminethepreferredstructuresofaggregatesinhomogenoussolutionswastwofold;itdemonstratedhoweventwoneutralphospholipidswith thesameheadgroupcanformdierentstructuresinthesamesolventandunderthe sameconditions,andwillnowbeusedtopredictstructuresinmixedphospholipidsolutions.ThisispossibleduetotheexistenceofabicellephaseinmixturesofDMPCand DHPCatparticulartemperaturesandconcentrations.Thedebateovertheconditions underwhichthebicellephaseemergeswillbeaddressedshortly,butNMRstudiesstate thatthebicellephaseischaracterizedbyloweredintermolecularinteractionsbetween DMPCandDHPCindicatinglimitedphysicalcontactbetweenthephospholipids.[22] Atthesametime,thebicellephaseemergesfrompreviousphaseswherethetwophospholipidsweremixedwithinaggregates,whetherthisbelamellarsheetswithDHPC linedpores,ormixedmicellesathighlydiluteconcentrations.[1]Thereforeinthebicel12

PAGE 27

2.1MembraneComposition larphasethespontaneousseparationofthetwoPCsoccurswithinthesamestructure allowingtheuseofthepackingparametertopredicttheindependentaggregationbehaviorofthePCsuponsegregation. P predictsthatDMPCwillformplanarbilayers andDHPCwilltendtowardscylindricalmicelles.Infact,thisispreciselythedisk-like structureassociatedwiththebicellarphase,withtheDMPCbilayersmakingupthe planarregionsofthediskandDHPCmoleculeslocalizingtothecurvedmicellarrimof thedisk.[1,22,23]ThecylindricalDHPCmicellepredictedby P canbeenvisionedas cutinhalfalongitslengthandwrappedaroundtheDMPCbilayer. Figure2.3: TheDisk-likeBicelle [24] Thepackingparameterisnotapplicablefortheentirespectrumofstructurescreated bymixturesofDMPCandDHPCsince,evenmorethantheabsenceofsegregation, thestructuresarehighlydependentontemperature,concentration,andmolarratiosof thePCs.Theireectswillbediscussedinturn,butsuccessfullyemployingthepacking parametertoexplainthedisk-likemorphologyofthebicellarphasehasdemonstrated howmoleculardimensionscandictateaggregatestructure. 2.1.3TheMolarRatio Theprevalenceofthedisk-likebicelle 1 inDMPCandDHPCmixtureshavenowbeen establishedbutyettobeaddressedistheirrelevancetotheformationofSULVs.When thetemperatureofthesolutioninbicellarphaseisraised,thediskscoalesceandundergo astructuralchangeintoSULVs.[1]Thismechanismwillbefurtherdiscussedinthe sectionofthischapterthatdiscussestemperatureeects.Themorphologyofthedisks willbeexploredfurtherherebecauseitisillustrativeoftheeectthemolarratiosof thephospholipidscanhaveonstructuresinsolution. 1 Theterm'bicelle'isamixtureoftheterms'micelle'and'bilayer'sincethedisk'splanesare attenedmicellebutitsend-statestructureresemblesabilayer. 13

PAGE 28

2.FORMATIONOFSMALLUNILAMELLARVESICLES 2.1.3.1MolarRatiosforanIdealBicellePhase Figure2.4: Cross-SectionofDisk-likeBicelle [25] Theplanarradiusofadisk, R ,tiesthesurfaceareaofDMPCbilayerstothedisk's perimeter,madeupofmicellarDHPCwithacross-sectionalradiusof r ,asshownin Figure2.4.HencelimitationsareplacedonthesizeofthediskbytheratioofDMPC toDHPC.IftheratioofDMPCtoDHPCistoohigh,disksinthebicellarphasewill havealoweraverageradiuscorrespondingtothesizeofdisksthatcanbefullyenclosed bythegivenconcentrationofDHPC.IftheDMPC:DHPCratioistoolowthesolution willcontainanexcessofDHPCmicellesandtheradiuswillbelimitedbythesizeofthe planarDMPCbilayers.Adistinctionthereforeneedstobemadebetweenthemolar ratio,denotedby q ,andtheeectivemolarratioofphospholipidsincorporatedwithin thedisk,calledq eff .Inanidealbicellesolutionq=q eff .[26]developedamodelfor themolarratioofdisksinanidealbicellesolutionbynotingthatifq=q eff theratio ofDMPCtoDHPCisgivenbytheratioofthesurfaceareasofthedisk'splanesA P tothesurfaceareaoftheircurvedrimsA C A P =2 R 2 .7 A C =2 r R +2 r .8 q = [ DMPC ] [ DHPC ] = A P A C = R 2 r R +2 r .9 Rearrangedintoaquadratic, R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(qrR )]TJ/F15 10.9091 Tf 10.909 0 Td [(2 r 2 q .10 14

PAGE 29

2.1MembraneComposition Usingthequadraticformulatosolvefor R intermsof q givestheidealbicelle equation, R = rq 2 [ + p 2 + =q ].11 Gloveret.al.renedthismodeltoaccountforthefactthattheDHPCheadgroup occupiesagreatersurfaceareapermoleculeinthecurvedrimthanDMPCoccupies inthebilayerplane.[25]ThiscorrectionwasaccountedforinTable2.1fromwhich k theratioofheadgroupsurfaceareaofDHPCtoDMPCisdeterminedtobe1.71.This parameterisincorporatedintothemodelbyGloverresultingintherelation, R = krq 2 [ + p 2 + k=q ].12 Thediameterofthediscs R canbeadjustedfrom5nmto40nmbyvarying q .[25]ThehydrodynamicradiioftheSULVsareconnectedtothecombinedradii, r 0 oftheplanarandthemicellarregionbythefollowingrelation.[25] r 0 = R + r .13 R H = 3 2 r 0 0 @ s 1+ t 2 r 0 2 + 2 r 0 t ln 2 4 t 2 r 0 + s 1+ t 2 r 0 2 3 5 )]TJ/F24 10.9091 Tf 16.872 7.38 Td [(t 2 r 0 1 A )]TJ/F22 7.9701 Tf 6.587 0 Td [(1 .14 Figure2.5showsthataftera q ofapproximately3,thereexistsalinearrelationship between q and R H .Hence,themolarratioof3.2usedintheprotocolbyNiehet.al. increatingdisk-likebicellesasstructuralprecursorstoSULVswillbepreservedinthis study. 2.1.3.2OverallSignicanceoftheMolarRatio InthisstudySULVswillbecreatedfromdisk-likebicelleprecursorsandtheiraverage hydrodynamicradiuswillbecomparedtothevaluepredictedbyEquation2.14.The eectofthemolarratioonthesizeofthedisksisnotaseasilylegislatedforother shapes,butgeneralrulescanbestatedforspheresandellipsoids,theothertwomain structuresrelevanttothisstudy.Forspheresahigherq eff correspondstolargerradii andanincreaseinvesiclesizebecauseloweramountsofDHPCarepresenttostabilize themembrane'scurvature.Thedierencebetweenqandq eff forspheresdependson themethodofsynthesis;ifdisk-likebicellesformedunderidealmolarratiosareused asprecursorsthisdierencewillbenegligibleintheresultantSULVs. 15

PAGE 30

2.FORMATIONOFSMALLUNILAMELLARVESICLES Figure2.5: DependenceoftheHydrodynamicRadiusonMolarRatio Here r sph = R H .[26] Theeectsof q onellipsoidalvesiclesisverysimilartodisksexceptthatDMPC, undergoingachangefromauidbilayertogelphaseatdiluteconcentrations,becomes morerigidandorientsitselfsimilartothegelphasepicturedinFigure2.2.Thisshear eectintheplanarregioncauseswhatwouldbeadisktobecomeoblate.Thiseect isfurtherpronouncedwhenthedilutelipidconcentrationsfallbelowthehighCMC ofDHPC,causingittoloseitsmicelleshapeandinsteadbridgetheplanarDMPC gellayerswithfreemoleculesinstead.[1]Thusellipsoidalprecursorsfacesimilarsize limitationsdueto q becauseabalancingbetweenplanarsurfaceareaandthestructure's perimeterisstillatplay.Thedissolutionofthemicellarregionandphasechanges inDMPCmakeitdiculttorelate q tothesizesofSULVsresultingfromheated ellipsoids.Thelossofmicelleswithinthestructurehintstowardsalowerincorporation ofDHPCwithinthestructure,indicatingthatforellipsoidsq eff isgreaterthan q .The implicationsofthisonSULVradiiwillbeconsideredwhenanalyzingSULVdatafrom ellipsoidalprecursors. Thefactthatavarietyofstructurescanbeformedwiththesamephospholipids atthesamemolarratiosunderscoresanimportantprinciplethathasbeenalluded toseveraltimesthusfar;noonefactorlikethepackingparameter,themolarratio, theconcentration,nortemperaturecancompletelyaccountfortheemergenceofall structuresinsolution.Rather,atvariousphasesintheformationofSULVssomefactors 16

PAGE 31

2.2ConcentrationandTemperaturePhaseDiagram haveamoredominantroleindeterminingstructurethanothers.Ellipsoidsonlyemerge inanarrowrangeofverydilutelipidsolutionsasopposedtodiskswhichexistinvarious alignedornon-alignedstatesoveramuchbroaderrangeofconcentrations.[1,22,25] TheconversiontoSULVsfromprecursorsontheotherhandisentirelymediatedby temperaturechanges.Inthisspirit,thedatagleanedfromthisstudywillbeanalyzed mainlythroughthelensoftemperatureandconcentration,whicharethechiefvariables thatdictateconversionsbetweenstructuresinsolutionandinfactformanenergy surfacethattheparticlesnavigatetogettothermodynamicallystablestates.Other factors,suchasthemolarratioandsolvent,willbeheldconstantthroughoutallSULV solutionsandincludedintheanalysisonlyforthosestages,likeduringthebicellar phase,whentheybecomeprimaryinuencesonstructureorsize. 2.2ConcentrationandTemperaturePhaseDiagram Figure2.6: PhaseDiagramofPhospholipidStructuresintheDMPC/DHPCSystem [1] Becausethephospholipidsinsolutionandtheirmolarratiosarexed,concentrationandtemperaturebecomegoverningforcesbehindmorphologicaltransformations.Theconcentration, ,isdenedasthelipidweighttovolumepercent.Forthe DMPC/DHPCsystem,theemergentstructuresareoutlinedinFigure2.6.Thisdia17

PAGE 32

2.FORMATIONOFSMALLUNILAMELLARVESICLES gram,basedonthephasebehaviorofthephospholipidsandexperimentalNMRand SANSdata,isthebasisfortheprotocolfollowedinthisstudyforcreatingSULVs. Athighconcentrationsthemixturewillformporouslamellarsheets.Thepores arelinedwithDHPCmoleculesbecauseDHPC'sshorteracylchaincontributesto itsincreasedsolubility.[1]Figure2.6indicatesthatbicelledisksemergeatthesame concentrationsaslamellarsheets.ThisisrefutedbysomeNMRstudieswhichmeasured thepresenceofthebicellesbythealignmentoflipidbilayersinsolution,andfound magneticalignmentonlywithinanarrowrangeofafewdegreesabovechain-melting temperature.[22]Yetarguably,therapidtumblingofthebicellardisksprecludes theirmagneticalignment,andnumerousstudiesreportevidenceofdisk-likestructures acrossawiderangeoftemperaturesandconcentrations.[1,23,25].Infact,some studiesreportthatmagneticalignmentactuallyrepresentstheporouslamellarphase. [24,27]Thisstudyisbasedonthewidelyacceptedideathatbicellespopulateawide rangeofconcentrationsbelowthechain-meltingtemperature T c ,andissupportedby thepredictionofindividualphospholipidmorphologiesbythepackingparameter. Figure2.6showsthatpolydisperseULVcanforminsolutionregardlessoftemperature.When T>T c polydisperseULVemergeasproductsoftheunbindingtransition ofthelamellarphasewhichoccurswhen1.25 << 2.5.[1]PolydisperseULVarea kinetictrapthatindicateanon-idealmixingofphospholipidswithinthestructure,and requireahighenergyinput,suchassonication,tobreakupthevesicles.Evenifthe concentrationishighenoughthatcoolingyieldsbicellardisks,therewillstillexistin solutionexcludedDHPCmicelles,perturbingtheideal q eff .Thisstudydidnotemploy serialdilutionsinordertoreachadilutelipidconcentrationtoavoidfallingintothe trapofpolydisperseULVs.Insteadallsolutionswillbecreatedthroughaone-step dilutionfollowingbyrigorousvortexingandequilibrating. TheconversionofdiskstoSULVsismediatedentirelybytheincreaseintemperature beyondthechain-meltingtemperatureofthephospholipids.Raisingthetemperature increasesthekineticenergyofthedisks,whichatatrstseemstohintthatagreater diusioncoecientisresponsibleforthecoalescenceofdisksintoSULVs.Anyincrease indiusionwasshowntohaveanegligibleeectoncoalescenceinastudybyLeng et.al.whofoundthatexperimentallyobservedtime-scalesfordiskgrowthwere10 4 to10 6 timesslowerthanthetheoreticaltimescalculatedforcoalescencebydiusion. [23]Thisindicatesthatcoalescencedoesn'toccurimmediatelyuponcontact,andthat temperaturemediatestheconversiontoSULVsthroughanothermorepotentmechanism,specicallybyincreasingthemiscibilityofthetwophospholipids.[28]The DHPConthecurvatureoftherimmigrateintotheplanarbilayer,causingthedisk linetension,whichisthetensioncontainedintherimofthediskinholdingthedisk 18

PAGE 33

2.2ConcentrationandTemperaturePhaseDiagram Figure2.7: GrowthbyCoalescenceandClosureofDisk-likeBicelles [23] together,toincrease.Inordertostabilizetherim,thediskwillcoalescewithother disks.Thisoccursataratemuchslowerthanpredictedbycontactthroughdiusion becauseforthecoalescencetobeenergeticallyfavored,thedisksmustbealignedneck tonecksuchthattheDHPCrimisthecontactpointoffusion,aspicturedinFigure 2.7.[23]Plane-to-planecontactandplane-to-rimcontactbetweendisksareassociated withhigheractivationbarriersbecausetheywouldnecessitateanincreaseinareabeforestabilizationbyaddingDHPCtotherim.Theentirediskgrowsuntilthedisk-line tensionreachesitsthresholdatthediskradiusgivenby, R d = + G .15 where isthedisklinetension,and and G arethebendingandGaussianmoduli ofthebilayer.[1]TheULVcontainsbothanextrinsicandintrinsiccurvatureandboth areaccountedforintheHelfrichHamiltonian,whichgivesthetotalenergydueto curvature. R d istheradiusofthediskatthethresholdwheretheenergycontainedin therimtokeepthediskfromfallingapartisequaltotheenergyduetocurvatureof theULV.[29] Aderivationoftheintrinsicandextrinsiccurvaturesofasphericalvesicleusing MongeparameterizationisgivenintheAppendix.Notably,thecurvaturesarefound tobedependentonlyontheradiusoftheULV,indicatingthatallsphericalvesicles ofagivensizehavethesamecurvatureenergyregardlessoftheirformationpathway. WhythenwouldsonicatedSULVsbeinherentlymoreunstablethanthosecreated throughthebicellarorellipsoidalpathways?Thisquestionisnotonlyinterestingfrom abiophysicsperspective,butisrelevanttodrugdeliveryapplicationsofliposomes 19

PAGE 34

2.FORMATIONOFSMALLUNILAMELLARVESICLES sincetheirstabilitydictatesthecirculatoryhalf-lifeinsidethebody,drugstorage,and administration.ThisstudyaimstoidentifyuniquepropertiesofSULVsthatcontribute togreaterstabilitybyanalyzingdynamiclightscatteringdataofSULVsformedthrough themechanismsdetailedinthischapter. 20

PAGE 35

3 Methods 3.1FormationofSmallUnilamellarVesicles ThreepathwaysforSULVformationwillbetestedusingDMPCandDHPCatthesame molarratio,q,butatdierenttemperaturesaboveandbelowthecriticaltemperature, T c andatconcentrationsbelow2%. T c is25 C,thechainmeltingtemperatureofthe mixturesinceDMPChasachainmeltingtemperatureof23 CandDHPChasasmall positiveeectontheoverall T c ofthesolution.[19] 3.1.1LipidCake Thephospholipidsarerstcombinedwiththemolarratioofphospholipids3.2DMPC :1.0DHPC:0.043Tm 3+ inalipidcakealongwithsolventattheirmaximumhydratedconcentrationatabout25g/mLor25%w/vandhomogenizedthroughabrief vortexing. Molecule Mol.Wt.g/mol DHPC 453.507 DMPC 677.933 DPPC 734.039 Cholesterol 386.654 Fluor.DHPC 631.612 TmCl 204.3874 Table3.1: MolecularWeightsforLiposomeIngredients 25mgDHPCwasdissolvedin250 L1XPBSand12.1 Lofthissolutionwas combinedwith5.46mgDMPC.16.72mgTmCl H 2 Owasdissolvedin500 L1XPBS. 1.24 Lofthiswasaddedtothephospholipidmixture.Anadditional6.6 Lof1X 21

PAGE 36

3.METHODS PBSwasaddedtobringthesolutiontoavolumeof19.96 Lwitha25%lipidw/v ratio.Thissolutionwasmixedthoroughlybypipettingandvortexing. 3.1.2Path1:DilutionAboveCriticalTemperature Thispathwayattemptstotakethephospholipidmixturefromitsmaximumconcentration,withlamellarsheetsinsolution,toverydiluteconcentrationsofSULVsinasfew dilutionsaspossibleatT > T c .Thephospholipidandsolventamountsfortherangeof concentrationstestedarelistedinTable3.2.Thelipidcakeand1XPBSwereheated to45 C,combined,andimmediatelyvortexedonthehighestsettingfor5minutes. Immediatelyfollowingvortexingthesolutionswereheatedtoandmaintainedat45 C forextrusion. Concentration 2.5%1.25%0.5%0.25%0.1% 25%PC L 52.52.52.51.25 1XPBS L 4547.5122.5247.5311.25 Table3.2: PhospholipidConcentrationsforallPath1Solutions 3.1.3Path2:Disk-likePrecursorsBelowCriticalTemperature Whenlamellarsheetsarecooledbelowtheircriticaltemperaturedisk-likemicellesform insolutionwhich,whenreheated,coalesceintoSULVs.The25%w/vphospholipid solutionwascooledto4 Calongwithaaskof1XPBS.Thesewerecombinedin threeseparatesolutionsaccordingtotheamountsinTable3.3.Thesolutionswere vortexedfor5minutesimmediatelyaftermixing,placedina45 Cheatblock,and subsequentlyextruded. Concentration 0.75%1.0%1.5% 25%PC L 13.51827 1XPBS L 436.5432423 Table3.3: PhospholipidConcentrationsforallPath2Solutions 3.1.4Path3:EllipsoidalPrecursorsBelowCriticalTemperature BelowT c atverydiluteconcentrations,oblateellipsoidsemergeinsolutionandbecome SULVsuponheating.Table3.4belowshowstheamountsof25%w/vphospholipid solutionand1XPBSthatwerecombinedtoproduceliposomalsolutionsatthese concentrations.ThephospholipidsandPBSwereonceagaincooledto4 Cbefore combining,vortexedfor5minutesandallowedtoequilibrateat45 Cforextrusion. 22

PAGE 37

3.1FormationofSmallUnilamellarVesicles Concentration 0.03%0.06%0.09% 25%PC L 0.541.081.62 1XPBS L 449.46448.92448.38 Table3.4: PhospholipidConcentrationsforallPath3Solutions 3.1.5Extrusion Aminisyringeextruderwasusedtoltertheliposomalsolutionstoparticlesizesbelow 50nm.Theextrusionchamberwasrstloadedwitha200nmnanoporemembrane. Eachliposomesolutionat45 Cwasforcedfromoneextrudingsyringe,intothechamber,andoutintothesecondsyringe.10suchpasseswereperformedforeachsolution afterwhichthesolutionwasreturnedtothe45 Cheatblock.Theentireapparatus wasthoroughlycleanedandloadedwitha50nmnanoporemembrane.Theliposome solutionspreviouslypassedthroughthe200nmmembraneweresimilarlyextruded throughthe50nmmembrane. 3.1.6DLSMeasurements AllextrudedsolutionsweretransferredtoanNMRtubeandallowedtoequilibrateina 37.5 CbathbeforeDLSmeasurements.100nmpolystyrenespheresinasolutionwith lowopacitywereusedasacontrolbeforeeachDLSruntoensurethesetupwasworking correctly.EachNMRtubecontainingasolutionwasimmersedina37.5 Cdecylene baththroughwhichlaserlighttravelledduringthecourseofDLSmeasurements.The decyleneensurestherefractiveindexoftheglassisthesameasthetube'ssurroundings. Forsomesolutionstherewassignicantvolumelossduringextrusion,so1XPBSat 37.5 CwasaddedtotheNMRtubeimmediatelyprecedingmeasurementstobringthe solutionlevelapproximately2cmfromthebottomofthetubesothatthelasercrosssectionwassafelybelowtheminiscusofthesolution.WhileDLScanbedonewith onlyonelaser,usingtwoinacross-sectionreducesmultiple-scatteringeects.Before eachmeasurement,3DSpec,theprogramusedtocontroltheDLS,wassettoread cross-sectionallaserdata,andtheintensityofthelaserwassettoapproximately3500 Hzat20 .Quickreadingswerecollectedat20 and140 todetermineifthesignal tonoiseratiowastoolowattheseextremeanglesduetoscatteringeects.Weak signalsattheseanglesweretypicallyfoundindensersolutionsandthosewithhigher polydispersity;hereangleswereadjustedin5 incrementsuntiltheygaveconsistent readings.Thenewanglesthendenedtherangeofthedatacollectionset-up.The programwassettorecordtheintensityateachdetectorover2minutesforeachangle, 23

PAGE 38

3.METHODS progressingwithastepsizeof5 ,andrepeatingthemeasurementfortwodatapoints foreachangle. 3.2DependenceofBaselineLeakageonSizeofLiposomes Inordertotestwhetherahighsurfacecurvaturecontributestotheleakinessofthe liposome,thebaselineleakageofvarioussizedliposomesloadedwithrhodamine-B,a uorescentmolecule,wasmeasuredinthisexperimentusingmembranedialysis.The largerliposomeswerecreatedusingprotocolsfromearlierGBMstudies.TheseformulationsdonotincludeDHPCandinsteadincorporatecholesterolandDPPC.The SULVswererecreatedusingthemostsuccessfulpathwayfromtheprevioussection. 3.2.1QuantifyingRhodamineConcentrationsthroughFluorescence Intensity Measuringliposomeleakageinthisexperimentreliesontranslatingtheuorescenceof thesamplestotheconcentrationofrhodamine.Todothistheuorescenceofknown concentrationsofrhodaminewillbemeasuredandplottedagainstthepercentsaturation.Themaximumsaturationofrhodamineinwateris50mg/mL.Therefore50mg rhodamine-Bwasaddedto1mL1XPBSandvortexeduntilitwasdissolved.200 L ofthiswasdepositedintoonewellofa96wellplate.Theremainingvolumeof800 L wasthendoubledwith800 L1XPBS,resultingina50%saturatedsolution.After abriefvortexing200 Lofthiswascollectedanddepositedintoasecondwell.These serialdilutionswererepeated,halvingthepercentsaturationeachtime,untilapercent saturationof0.0061 3.2.2RhodamineLoadedLULVs TomaketheLULVs201.8mgDPPCand77.4mgcholesterolwerecombinedin1 mLof100%ethanolandheatedto65 Cuntilthesolutionwasclear.Arhodamine concentrationofapproximately12.5%itssaturationinwaterwasused,at6.25mg/mL ofrhodamine.Hence56.25mgofrhodaminewasdissolvedin9mLwaterandheated to65 C.Thephospholipidsolutionwascombinedwiththerhodamineandstirred at65 Conalowspeedfor1hr.Thesolutionwascentrifugedat15000rcffor20 minutes,afterwhichthepelletwasresuspendedin10mL1XPBS.Theliposomes weresonicatedonhighfor1hr.Halfofthesolutionwasextrudedthrougha400nm nanoporemembrane,andtheotherhalfthrougha200nmnanoporemembrane. 24

PAGE 39

3.3DiusionThroughaUniformGel 3.2.3RhodamineLoadedSULVs A25%w/vlipidcakewascreatedusingtheprocedureintheprevioussectionand cooledto4 C.Then6.88mgofrhodaminewasaddedto1067 L1XPBSandcooled to4C.33 Lofthe25%w/vsolutionwasaddedtotherhodamine,resultingina solutionof0.75%lipidw/v.Thiswasimmediatelyvortexedfor5minutesandheated to45 C.Theliposomeswereextrudedthrougha200nmmembrane,andthenthrough a50nmmembrane.1XPBSwasheatedto45 Candaddedtothesolutiontobring thevolumeupto3mLbeforeDLSandmembranedialysis. 3.2.4MembraneDialysis 3Thermo-Scienticmembranedialysiscasettesof10000MCWwithacapacityofup to3mLswereimmersedinabeakerof1XPBSfor2minutes.Usinga20gauge syringeonevalveofeachcasettewaspuncturedandinjectedwithliposomesofeach size,removingasmuchexcessairfromthechamberaspossible.Thesecasetteswere onceagainimmersedin1XPBSfor2hours,afterwhichthebuerwasonceagain exchangedfor20mLoffresh1XPBSat37.5 C.Thecasettesweremaintainedatthis temperatureoveraperiodof36hours,with200 Lsamplescollectedfromthesolvent every3hours,andreplacedwith200 Loffresh1XPBS. 3.2.5FluorescenceImaging TherhodamineloadedliposomeswerenotmeasuredwithDLSduetotimeconstraints. FluorescenceimagingwasdoneusingaBiotekSynergy4Reader.Allsampleswere collectedandimagedina96wellblackplatewithaclearbottom.Theuorometer wassettoanexcitationwavelengthof544nm,andanemissionwavelengthof627nm. Twolterswereused,onewithagainsettingof50andtheotherwithagainof100 themaximumgainbeing200. 3.3DiusionThroughaUniformGel Forthisexperiment,liposomesofthesamesizerangeastheprevioussectionwere injectedintoauniformgelcalledSephadexG-50whichconsistsofcross-linkeddextran withepichlorohydrinresultinginparticlesizesof40to160 mwhichformalattice oncethegelisprepared.Iftheliposomes'abilitytopenetratethisgelmediumissize dependent,itstandstoreasonthatthepassivediusionofliposomesoutoftumorous vasculatureisalsosizedependent. 25

PAGE 40

3.METHODS 3.3.1SettingtheSephadex Tosetthegel1.5gofSephadexG-50wascombinedwith17.5mL1XPBSinasmall cellculturedish.Threesuchdisheswerepreparedandlefttosetforthreehours.Excess waterwasdecantedandallowedtoevaporateresultinginagelwithavolumebetween 13.5mLto16.5mL. 3.3.2SynthesisofLULVswithFluorescentMembranes LULVsweremadebycombining50.45mgDPPCwith17.98mgcholesteroland1.37 mgBodipyCholesterolin250 Lof100%ethanolandheatingto65C.Next2.25mLof 1XPBSwasalsoheatedto65C,combinedwiththephospholipidsolution,andstirred onlowspeedfor1hour.Thesolutionwascentrifugedat15000rcffor20minutes,after whichthepelletwasresuspendedin2.5mL1XPBS.Theliposomesweresonicatedon highfor1hr.Halfofthesolutionwasextrudedthrougha400nmnanoporemembrane, andtheotherhalfthrougha200nmnanoporemembrane.Theliposomeswerestored at4 C. 3.3.3SynthesisofSULVswithFluorescentMembranes 43.02 LoftheDHPCsolutionmadeinsection4.1.1wascombinedwith21.84mg DMPC.2mgNBDDHPCuorescentlylabeledDHPCwasdissolvedin100 L1X PBSand27.1 Lofthissolutionwasaddedtothephospholipidmixture,alongwith 4.96 LoftheTmClsolutionalsofrom4.1.1.Anadditional4.76 Lof1XPBSwas addedtobringthesolutiontoavolumeof79.84 Lwitha25%lipidw/vratio.This solutionwasmixedthoroughlybypipettingandvortexing,resultinginahomogenized lipidcake.SULVswerethencreatedusingPath2,andextrudedto50nmasdetailed insection4.1.5.Theliposomeswerestoredat4 C. 3.3.4DLSofLiposomeswithFluorescentMembranes TheexcitationwavelengthofNBDDHPCis464nm,sothescatteringoftheDLS laserlightat632.8nmwillnotbeaectedbyabsorptionandemissionoflightby theuorescentliposomes.300 LoftheextrudedLULVsolutionsweretransferred toanNMRtubeandallowedtoequilibrateto37.5 C.Afterensuringthe100nm polystyrenespheresgaveappropriatereadings,theNMRtubeswereimmersedinthe 37.5 Cdecylenebath.Theintensityofthelaserwassettoapproximately3500Hzat 20 and3DSpecwassettorecordtheintensityateachdetectorover2minutesfor eachangle,progressingwithastepsizeof5 26

PAGE 41

3.3DiusionThroughaUniformGel 3.3.5InjectionandFluorescentImaging Approximately8weeksaftertheliposomeswerecreated,thegelswererehydratedand lefttositagainfor3hours.Theliposomes,whichhadbeenstoredat4 Cwereheated to37.5 C.0.5mLwereinjectedintothecenterofthegelplateusinga20gauge syringe.Theliposomeswereallowedtodiusefor2minutes,afterwhichtheplatewas placedundera4Xobjectivelensofauorescencelightmicroscope.Themicroscope wassetatagaintotheFITClter,whichexcitesat495nmanddetectsemittanceat 519nm,andthesoftwarewassettorecordimagesatagainof1000withamaximum gainof2000.Theplacementofthegelplatewasadjustedusingthestand'scontrol knobsuntiltheuorescentparticleswerebarelyvisibleontheborderofthescreen. Thisimagewasrecordedandtheexcitationlightwasturnedotoavoidquenchingthe liposomemembranes.After30secondsthelightwasturnedonagaintogaugetherate atwhichtheliposomeswerediusingthroughthegel.At30secondstheSULVshad diusednoticeablysosubsequentimageswererecordedat30secondintervalswithout movingthegelplateuntiltheliposomeshaddiusedacrossthescreen.Signicant diusionwasnoticedforthe100nmliposomesafterthree30secondintervals,setting theintervalforthefollowingrecordedimagesto90seconds.The200nmliposomesdid notshowsignicantdiusionevenafter3minutes.Fortheseliposomesimageswere recordedevery5minutesuntiltheliposomesreachedtheendofthescreen. 27

PAGE 42

3.METHODS 28

PAGE 43

4 DataandAnalysis Theoveralloutlineofthisstudywastorstdetermineareliablepathwaytocreate stableSULVs,andthentestthemagainststandardLULVs,likethoseusedinthe GBMstudy,fordiusivityandpermeability.ThischapterbeginsbypresentingDLS dataforSULVscreatedusingthethreepathwaysoutlinedinChapter2.Themost reliablepathwayisthenusedtocreateSULVswhicharecomparedwithLULVsused intheGBMstudiesforleakageratesanddiusionthroughuniformmedia. 4.1FormationofSmallUnilamellarVesicles ThethreeSULVformationpathwaysstudiedweremadeusingformulationswithidenticalmembranecompositions,phospholipidratios,andsolvents.Theonlyvariables thatdierentiateonepathwayfromanotherarethelipidweighttovolumepercentage andthetemperatureatwhichthesolutionswereallowedtoequilibrateafter vortexing.DLSmeasurementsofeachsolution,alsoperformedunderidenticalconditions,measuredtheintensityoflaserlightateachdetectorovertime.Thissection rstdemonstrateshowtheaverageradiusofparticlesinsolutionisgleanedfromthis rawintensitydataandsubsequentsectionspresenttheprocessedDLSdataforvarious solutions.Fromthisdataboththesizeandstructureofvesiclesinsolutionwillbe discussedandexplainedintermsofconcentrationandtemperaturemodulation. 4.1.1ProcessingDLSData OneinterestingaspectofDLSisthatthewavelengthoflaserlightusedis632.8nm,yet theradiiofparticlesinsolutioncanbedeterminedtoaresolutionthatislessthana tenthofthewavelength.Atrstglancethisseemstoviolatetheuncertaintyprinciple, accordingtowhichthewavelengthoflightusedtoobservetheparticlesdetermines theprecisionofthemeasurement.DLShoweverevadestheproblemposedbythe 29

PAGE 44

4.DATAANDANALYSIS uncertaintyprinciplebecauseratherthanmeasuringanindividualparticle,itmeasures theintensitydistributionoveraspaceasafunctionoftherelativeorientationofall particlesinthespace.Aparticulardistributionofparticlesatagivenmomenthasan eectonincidentlightsimilartoaseriesofslits;thelightisscatteredandaninterference patternofspecklesisdetected.Thisinterferencepatternshiftsastheparticlesdiuse, makingtheintensityofonepointickerwithtime.Ratherthanadirectmeasurement, cumulantanalysisoftheickeringintensityoflightautocorrelatedintimeprovidesthe decayratefromwhichthediusionconstantoftheparticlesinsolutioniscalculated. Therateofdiusionisthenrelatedtothesizeoftheparticlesinsolutionbythe Einstein-Stokesrelation. Figure4.1: SamplePlotofAutocorrelatedIntensityvs.DecayRate Intensityuctuationsareautocorrelatedintimeastheyaremeasuredforeachangle,therebygivingameasureoftheextenttowhichthescatteringintensityatonetime pointisrelatedtothatatanothertimepoint.TheintensitywasautomaticallycorrelatedbytheDLSsoftwareasitwasmeasuredresultinginacharacteristicexponential decayplotseeninFigure4.1.Thedecayrate, denesthetimeittakesforscattered lightdatatakentaparttolosecorrelation.AMATLABcodethendeterminesthe electriceldcorrelationfunctionusingtherelation, g 1 = Z 1 0 e i )]TJ/F25 7.9701 Tf 5.289 0 Td [( P\051d)]TJ0 g 0 G [-11526(.1 whereP\051istheintensity-weightedsumofeachparticlesize,dependentuponthe decayrateoftheirindividualintensities.Therelativeorientationoftheparticleswill 30

PAGE 45

4.1FormationofSmallUnilamellarVesicles maketheelectriceldinterfereconstructivelyatsomepointsanddestructivelyatothers.Theelectriceldcorrelationfunction,givenby g 1 takestheseinterferenceeects intoaccount.Thus g 1 describescorrelatedparticlemovementasgleanedfromthe uctuatingintensity.Theelectriceldcorrelationfunctionandtheintensitycorrelation functionarerelatedtoeachotherthroughtheSiegertrelation,givenby g 2 =1+ g 1 2 .2 whereg 2 istheintensitycorrelationfunction.TheMATLABcodeusestheintensity datatottheSiegertrelation,nding values,andperformsacumulantexpansion ong 1 tondthedecayconstantofthesumofalltheparticles,.Cumulantanalysisis beyondthescopeofthisstudy,andhereitwillsucetosaythatthedecayconstantis obtainedfromtheintensitydataforeveryangleatwhichameasurementisperformed. Astheangleofthedetectorschanges,dierentscatteringvectorsarepickedup. Thescatteringvector, q ,isafunctionoftheangleofthedetector, ,thewavelength oflaserlight, ,andtherefractiveindexofthesample, ,thelattertwoofwhichare constants. q = 4 sin 2 .3 Itisevidentthat q hasdimensionsofm )]TJ/F22 7.9701 Tf 6.587 0 Td [(1 .Thereforethequantity l = 2 q .4 hasdimensionsofmanddeterminesthesizeofparticlespickedupinthesolution. Thisquantitymustbeonthescaleoftheparticlesbeingmeasured;hencethewavelengthoflightandtheangleofdetectionarewhatdeterminetheresolutionoftheDLS measurement.Since l isdependentupon ,changingtheangleallowsthedetection ofparticleswithradiispanningdierentlengthscalesasdepictedinFigure4.2.As theangleofthedetectorincreases,radiionsmallerlengthscalesarebetterdetected. Maximizingtherangeof isoptimalbecauseitallowsdetectionofawidersizerange ofparticles.Thisalsomeansthattheanglescanbeadjustedtoonlypickupcertain speciesofparticlesinsolution,asinthecaseofbimodalsizedistributions.Thevariationinsizewith q canalsogiveasenseoftheextentofpolydispersityinthesolution, andwillbediscussedforeachSULVpathwayinlatersections. Thedecayconstant,,isrelatedtothediusioncoecient, D ,by )-278(= )]TJ/F24 10.9091 Tf 8.485 0 Td [(D q 2 .5 31

PAGE 46

4.DATAANDANALYSIS Figure4.2: ScalabilityoftheScatteringVector Thedimensionallinesrepresentthevaryingscaleof l .Justasonewouldn'tusea metersticktomeasurethesizeofacellorthelengthofaskyscraper,thesizeofthe particlesbeingmeasuredmustbeonthesamescaleas l Plottingthe)-232(valuesinthedata'sMATLABcodeoutputagainst q 2 yieldsastraight line,theslopeofwhichgivesthediusioncoecient.Iftheliposomesareassumedto besphericalparticles,theEinstein-Stokesequationgivestheaveragehydrodynamic radius, R H oftheliposomesfrom D ,whichhasunitsm 2 /s. D = k B T 6 R H .6 Thehydrodynamicradiusreferstotheradiusofahardspherethatdiusesatthe samerateasD.Thediusionoftheliposomesareinrealityaectedbytheirhydration andglobularshapeeects.Since1XPBSwasusedassolvent,theviscosity, ,isclosely approximatedbytheviscosityofwater,whichhasatemperaturedependenceaccording totherelation, = : 414 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(5 247 : 8 = T )]TJ/F22 7.9701 Tf 6.586 0 Td [(140 .7 whereTistheaveragetemperatureofthesolutionduringmeasurementinKelvins. 32

PAGE 47

4.1FormationofSmallUnilamellarVesicles 4.1.2Path1:DilutionAboveCriticalTemperature CoherentDLSdatadoesnotexistforliposomescreatedusingPath1sincenoneofthe samplescreatedabovethecriticaltemperatureresultedinamonodispersesolutionof SULVs,evenafterextrusion.AtlowanglestheDLSdatawashighlyerratic,jumping betweenanundenedradiusto5000nm.Thesenonsensereadingsindicatedthatthe detectorwasnotmakingmeasurementsappropriatetothescaleof l andthereforethe anglewasadjustedtoanarrowerrange.Betweenapproximately50 and110 the readingsconvergedtoarangebetween950nmand2900nmforthe2.5%solution. Theremainingsolutionsdidnotstabilizeeventothisdegreeatmedianangles.For lowangles,theradiuspredictionwasonceagaininniteandhighlyerratic,moving momentarilytopredictionsapproximating100nm.Thevolatilesizereadingsimply thattherewasmorenoisethansignalevenatnerscaleresolutions. Toensurethesampleswerenotfaultyorgiventoomuchtimetoaggregate,the procedurewasrepeatedagainforasecondbatchofliposomesthatwereDLStested withinhoursofextrusion.DLSmeasurementsonceagainwerenotrecordedbecause themoredilutesolutionsdidnotproducestablereadingsatanyangle,andthe1.25% and2.5%solutionsgavesomewhatstablereadingsonlywithinaverysmallrangeof angles.The1.25%solutioninthissecondtrialvariedbetween3000and9000nmfrom 45 to110 ,andthe2.5%solutionconsistentlygavereadingsbetween600and1500 nmfromapproximately40 to80 DLSmeasurementsontherstbatchoccurreddaysaftertheparticleswereextruded whereasthesecondbatchonlysatundisturbedforapproximately2hoursbeforetesting. Perhapsbecauselittletimewasaordedforsolutionsfromthesecondtrialtoaggregate, the1.25%solutionfromthesecondtrialstabilizedwithina6000nmrange.The2.5% solutionalsostabilizedtosmallersizeswithinasmallerrangeofanglesinthesecond trial.Thereforeaggregationdidhaveasmallcontributiontothesizesofthe2.5% solutionfromthersttrial,indicatingthatPath1liposomesareunstableandcan coalesceoverthespanofafewdays.ThePath1solutionswerehypothesizedtoyield smallerliposomesasthedilutionofthephospholipidsincreased,butthelargeraverage radiusofthe1.25%solutionthanofthe2.5%solutionindicatestheopposite. ThepossibilitywasstillconsideredthatSULVsmaybepresentinPath1solutions,butundetectablebytheDLSeitherduetothenatureofthesolutionordueto systematicerrorintheDLSsetup.OnepossiblereasontheSULVsinthesesolutions couldbeoverlookedisduetorogueLULVsinsolutionformedduringthehighpressure conditionsofextrusion.Tearingofthenanoporemembraneisaknownriskofhigh pressureextrusion,buttheextentoftearingisoftenunknown.Becausethevolumeof 33

PAGE 48

4.DATAANDANALYSIS asphereincreasescubicallywiththeradius,onlyafewliposomesoneorderofmagnitudegreaterinradiuscouldhaveasignicanteectonthewaylightscattersthrough thesolution.EvenifLULVsmakeup5%oftheparticlesinsolution,theytogethercan occupyalargervolumethantheremainingSULVS.Sinceparticlesofgreatervolume willscattermorelight,thescatteringeectsofjustafewLULVscanmakeSULVs weaklydetectableonlythroughmultiplescatteringeects,ifatall.Thiswouldresult inaveryweaksignalforsmallparticles,andisoneexplanationforwhySULVsmight notbedetectedatsmallerangleseveniftheyarepresentinsolution.TheLULVsalso contributetopolydispersity,increasingthenoiseinthesignalandcausinginstability inthecontinuousrstorderreadingoftheradiusdisplayedbythesoftware,makingit diculttotakeascriptedmeasurement.TheLULVsalsodiusemoreslowlythanthe smallerparticlesandthesporadicjumpsinradiusintheDLSreadingsarelikelydue toanLULVdiusingataslowerratethroughthespaceilluminatedbythelaserlight. Extrusioninthiscasewasineectiveinlteringoutlargeparticlessincethehigh pressureenvironmentduringextrusioncanactuallycreateLULVs.AsPath2andPath 3datawillshow,extrusionwashighlyeectiveatlteringoutlargeparticlesinthe othersolutionswhichdidnotrequireasmuchpressure.Thereasonsforthisaredue tophospholipidconcentration;becausePath1solutionscalledforhigherconcentrationsthesolutionsbeingextrudedwerenotasdiluteandhencemorelikelytotearthe nanoporemembrane.ThehighpressureextrusionalsoindicatesthatthelteringprocessisheavilyreliedupontosizetheparticlesforPath1,ratherthantheaggregates spontaneouslyequilibratingintoSULVsaswillbedemonstratedfortheotherpathways.SinceDLSfunctionwascheckedbeforeeverysampleusing100nmpolystyrene beads,andbecausemultipletrialsofPath1solutionsgavesimilardata,thepresenceof LULVsinsolutionisacertainty.Thegoalofthisportionofthestudywastotestthe formationofSULVs,andsincethepresenceofafewLULVsconfoundstheDLSdata, analternatemethodisneededtotestwhetherSULVsarepresentinPath1samples. Thisalternatemethodreliesonthescalabilityofthemeasurementby q ,which reliesontheangleofdetection.ThelightscatteringotheLULVscanberendered largelyundetectableif ishighenoughsuchthatthevalueof l ismuchlessthanthe radiusofthelargerparticles,andcomparabletothatofSULVs.Thisessentiallymeans thatathighanglesmostofthedetectedlightwillbeduetosmallparticlescattering,so observinghowtheintensityoflightvarieswith canprovideinformationoftherelative concentrationsofdierentsizedparticles.EventhoughDLSdatadidnotstabilizelong enoughtorecordascriptedmeasurementforthe2.5%solutioninthesecondtrial,the softwarewasusedtoproduceaplotofhowtheintensityvarieswiththeangle.Thisis showninFigure4.3below. 34

PAGE 49

4.1FormationofSmallUnilamellarVesicles Figure4.3: RelationshipbetweenIntensityandtheAngleofDetection Thenon-linearityoftherelationshipindicatestheabsenceofsmallparticlesas l scalesdownatgreaterangles. Theplotshowsthatbelowa of80 theintensitywasverylowandconstant.At smallangleswhere l islonger,theintensityishigh,onceagainconrmingthepresence ofLULVs.Ifthereexistedanormaldistributionofliposomeswithinthesizerange of l takenfromthelargesttosmallestangles,theintensitywoulddroplinearlywith theangleofthescatteringvector.Theplotaboveinsteadshowsanexponentialdecay. Thismeansthatas l decreasesandthescaleofthemeasurementshrinkstodetectthe smallerparticles,intensityrapidlydecaysandverylittlemeaningfulsignalisobserved. Athighangles,thereadingsoftenremainedundenedindicatingthatonlyparticles signicantlylargerthanthescaleof l werebeingobserved.Theplotabovethusconrms thatthelackofDLSdetectionofSULVsforPath1solutionsisduetotheirabsence insolution,andnotbecausemultiplescatteringeectsorothersystematicerrorsare interferingwithaproperreading. UltimatelyPath1solutionsdidnotyieldamonodispersesolutionofSULVs,and wasinsteadfoundtobeapolydispersesolutionofLULVs.Thiswasananticipated stageofPath1solutionsasSANSstudiesbyNiehetal.demonstratethatatconcentrationsaslowas0.1%polydisperseULVsareformedaboveT c .[1]However,thisstudy aimedtoreducethatpolydispersitythroughtheaddedstepofextrusiontoeitherlter outLULVsorsizethembyforcingthemthroughsmallerpores.Tostillretainadecent SULVyieldafterltrationhigherconcentrationswereusedwiththispathway.The solutionswithconcentrationslessthan0.5%likelygavenonsensedataduetothehigh 35

PAGE 50

4.DATAANDANALYSIS pressureextrusionsinwhichtheULVswereeithercompletelyltered,orcompromised theintegrityofthenanoporemembrane,andonlyresultedinmoreaggregatesdueto shearstressthroughtearsinthemembrane.Inthislightitisparticularlyinteresting thatthesolutionsofhighestconcentrationgavemorestablereadingsthantheother solutionsbecausetheconcentrationof2.5%isapproximatelythethresholdatwhich thelamellartoULVtransitionoccursatanytemperature.[1]ExtrudingintheneighborhoodofthisconcentrationactuallyfacilitatedtheformationofULVsfromlamellar sheets.Thiswasstillahigh-pressureextrusion,butthe2.5%solutionstabilizedthe mostbecausenewlydissociatedsheetswereconveredtoULVsintheprocessofbeing pushedthroughandtearingthepores. Asanalnote,thispathwayisverysimilartotheprocessofmakinglargerliposomesusedintheGBMstudies.Themaindierencesbetweenthispathwayandthe IBandDXRloadedliposomesaretheinclusionofcholesterolratherthanDHPCas astabilizingagent,andtheabsenceofsonicationorcentrifugationtosizedownthe liposomes.SimplyusingDHPCdoesnotautomaticallylowermembranecurvature, orSULVswouldhavespontaneouslyformedinthispathway.Toachievethesizing downofliposomesusingDHPC,itmustberstincorporatedintothestructure,as willbeexploredintheotherpathways.Path1alsoindicatesthatliposomesformed abovechain-meltingtemperaturesrequireasizingdownmechanismwithoutwhichthe solutionwillremainhighlypolydisperse.Italsoshowsthatextrusionisnotaneectivemeansofsizingparticles,butmayoeranovelmeansofaectingthestructures aroundthresholdparameters.Anotheravenueforfurtherexplorationwouldbecreating DMPC/DHPCSULVsthroughtypicalsonicationmethodstotestwhetherincorporationofDHPCintoSULVstructurecanoccuraboveT c 4.1.3Path2:Disk-likePrecursorsBelowCriticalTemperature Path2solutionswereformedbydilutingtolowconcentrationsat4 C,equilibrating aboveT c ,andthenextruding.Figures4.4-4.7showthedecayrateofcorrelatedintensityplottedagainstthescatteringvectorforeachSULVsolutionmadeviathebicellar phase.Theslopesofthelinesdenotethediusioncoecient, D ,whichisthenusedin Equation4.6todeterminetheaverageradiusofthevesiclesineachsolution. 4.1.3.1 =0.75 Figure4.4displaystheDLSdataforliposomeswithdisk-likeprecursorsmadeata concentrationof =0.75%.Thedatapointsfallneatlyalongthetrendlineshowing thatthedecayrateincreasedproportionallywiththedetectionofsmallerparticles. Sincesmallerparticlesdiusefastertheirdecayrateshouldbehigherthanthedecay 36

PAGE 51

4.1FormationofSmallUnilamellarVesicles Figure4.4: LiposomesFormedviaBicellarPhaseat0.75%w/v rateoftheintensitycorrelationoflargerparticles.Ifthereexistapproximatelyasmany smallparticlesaslargerones,thedecayratewillhaveacloselylinearrelationshipwith q 2 asdepictedinFigure4.4,indicatinganunskewednormaldistributionofparticlesize. Thediusioncoecient,theaveragetemperature,andviscosityareneededinorder toaccuratelydeterminetheaveragehydrodynamic R H .Theaveragetemperatureand itsstandarddeviationwasdeterminedfromtherecordedtemperatureateachangle. Equation4.7wasusedtocalculate withthevariancein givenby, var = @ @T 2 dT 2 .8 var = : 414 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(5 247 : 8 = T )]TJ/F22 7.9701 Tf 6.587 0 Td [(140 ln 247 : 8 T )]TJ/F15 10.9091 Tf 10.909 0 Td [(140 )]TJ/F15 10.9091 Tf 8.485 0 Td [(247 : 8 T )]TJ/F15 10.9091 Tf 10.909 0 Td [(140 2 2 dT 2 .9 TheaveragevalueforTandthestandarddeviationintemperaturedTforthe =0.75solutionaregiveninTable4.1.BysubstitutingthesevaluesintoEquations4.7 and4.9thevaluesanduncertaintiesfor werecalculatedandarealsolistedinTable 4.1. 37

PAGE 52

4.DATAANDANALYSIS Quantity Units ValueStd.Dev.Variance T K 310.6450.3140.099 Pa s 6.837 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(4 6.82 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(7 4.64 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 D m 2 /s 9.714 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(12 6.87 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(14 4.72 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(27 Table4.1: Temperature,ViscosityandDiusionCoecientfor =0.75 TheradiusoftheseliposomesisdeterminedbyrearrangingEquation4.6, R H = k B T 6 D = : 380648810 )]TJ/F22 7.9701 Tf 6.587 0 Td [(23 : 6453 : 837 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(4 : 714 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(12 .10 andtheuncertaintyinliposomesizeisgivenbytheformula, var R H = @R H @T 2 dT 2 + @R H @ 2 d 2 + @R H @D 2 dD 2 .11 var R H = k B 6 D 2 dT 2 + )]TJ/F24 10.9091 Tf 8.485 0 Td [(k B T 6 2 D 2 d 2 + )]TJ/F24 10.9091 Tf 8.485 0 Td [(k B T 6 D 2 2 dD 2 .12 Asimpleunitanalysisfor R H showsthat, R H = k B T 6 D = J K s K Pa s m 2 = kg m 2 m s 2 s 2 kg m 2 = m .13 Thehydrodynamicradius,R H ,anditsvarianceandstandarddeviationwerecalculated inmetersfromtheaboveformulasandarelistedinTable4.4. 4.1.3.2 =1.0 Figure4.5displaysDLSdataforSULVsformedviabicelleprecursorsataconcentration of =1.0.Notably,thissolutiondisplaysaclearoutlierforthetwodatapointstaken at =135degrees.Becausetherestofthedatafollowsawell-denedlineartrend, theoutlierslikelyoccurredduetointerferencewiththelaserlightbyanobject,which couldbeadustparticleorsmudgeatthewallofthedecylenecontaineratthisangle.A noisysignalforsuchreasonswouldresultinamuchhigherdecayrateastheintensity wouldnotcorrelatewelltoitself.Ifthetwomisplacedpointswereduetoajumpin theamountofsmallervesicles,thesurroundingpointswouldalsoshowsomedeviation fromthetrend.Sincetheseoutliersmostlikelydonotholdanymeaningwithregards tothesizeofvesiclesdetectedatthatangle,theywereomittedandinterpolatedusing thetrendlineinthecorrectedplotdisplayedinFigure4.6. 38

PAGE 53

4.1FormationofSmallUnilamellarVesicles Figure4.5: LiposomesFormedviaBicellarPhaseat1%w/v-Uncorrected Thetemperature,viscosity,anddiusioncoecientforthissolutionarelistedin Table4.2.Thevarianceformulasandtheunitanalysiswereprovidedassamplecalculationsfortherstsolutionandwillnotbereproducedhere.Justasforthe = 0.75solution,thevaluesinTable4.2weresubstitutedintotheformulastocalculate thevalueanduncertaintyof R H ,listedinTable4.4. Quantity Units ValueStd.Dev.Variance T K 311.9750.2790.078 Pa s 6.663 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(4 5.69 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(7 3.24 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 D m 2 /s 9.674 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(12 1.29 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 1.66 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(26 Table4.2: Temperature,ViscosityandDiusionCoecientfor =1.0 4.1.3.3 =1.5 Finally,SULVsinthelastbicellardisksolutionwitha of1.5weremeasured,giving theplotshowninFigure4.7andvalueslistedinTable4.3.Itisevidentfromthe 39

PAGE 54

4.DATAANDANALYSIS Figure4.6: LiposomesFormedviaBicellarPhaseat1%w/v plotthatthissolutionwasnotasuniforminparticlesizeastheotheronesmadewith thesamepathway.Outliersareseenthroughoutarangeoflengthsofthescattering vectorindicatingthatratherthanthenormaldistributionofparticlesizesthereis polydispersity.Ifthesolutionwaspolydisperseinsizetherewouldbeanincreasein randomerroraroundthetrendlineduetogreateramountsofnoiseateachscattering anglefromparticlesoutsideofthescale, l .Howeverthepointsontheplotdonotshow increasedrandomnessofallpointsaroundtheline,ratherafewpointsaresignicantly shifted,andallmeasuredalowerdecayrate.Thelowereddecayrateofafewof thepointsindicatesthatthemajorityoftheparticlesinsolutionareSULVbutthat somethinginsolutionisdiusingmuchslower,resultinginhighcorrelationofscattered lightwithtime. Thiscanbeexplainedbyreferringtotheexpectedmorphologyofthesolution accordingtoFigure2.6.Atconcentrationsbetween1.25%and2.5%Niehet.al.observe theunbindingtransitionofthebicelledisksintolamellarsheets.Aconcentrationon thethresholdofthebicellephasewaschosentoobservetheeectonSULVsize.The 40

PAGE 55

4.1FormationofSmallUnilamellarVesicles Figure4.7: LiposomesFormedviaBicellarPhaseat1%w/v plotisnotaccuratelydescribedasanincreaseinSULVsize,butrathertheemergence oflamellaeinsolution.ThesesheetsareDHPC-pore-linedaggregatesofaDMPC bilayeraboveT c andthereforediusemuchslowerthroughsolution,resultinginthe lowerdecayrate.Theyalsointroduceslightanisotropytothesolution,seenbythe curvingofthedatapointsatthehighestandlowestscatteringvectors.Thiswillbe furtherexplainedinanalyzingtheDLSdataforSULVswithellisoidalprecursorssince theeectwillbecomemorepronounced.Interestingly,theverypresenceoflamellaein the =1.5solutioncausedasignicantincreaseintheaveragehydrodynamicradius oftheSULVs.Fromthisitcanbeinferredthattheproliferationoflamellaeisnotjust limitedtothesheetsbutisseeninthebicellarphaseaswell,withtheplanesofthe disksexcludingDHPCfromthestructure,resultinginlargerSULVs. 4.1.3.4ComprehensiveDiscussionofSULVsFormedviatheBicellarPhase Table4.4showstheaveragehydrodynamicradiusandtheuncertaintyinthevaluesof allSULVsolutionstakenthroughthebicellarphase.Themolarratio, q ,ofDMPCand 41

PAGE 56

4.DATAANDANALYSIS Quantity Units ValueStd.Dev.Variance T K 312.0100.3110.096 Pa s 6.658 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(4 5.68 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(7 3.23 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 D m 2 /s 5.952 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(12 2.19 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 4.80 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(26 Table4.3: Temperature,ViscosityandDiusionCoecientfor =1.5 R H mStd.Dev.Variance 0.75 3.426 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(8 2.423 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(10 5.871 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(20 1.0 3.545 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(8 4.469 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(10 1.997 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(19 1.5 5.767 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(8 2.406 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(9 5.790 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(18 Table4.4: AverageHydrodynamicRadiiforallLiposomeswithDisk-likePrecursors DHPCinthesesolutionswas3.2,whichaccordingtorenedbicelletheory,detailedin Chapter2,shouldresultinbicellediskswithaplanarradiusof, R = krq 2 [ + p 2 + k=q ].14 Table2.1showsthattheratiooftheheadgroupsofDHPCtoDMPC, k ,is1.71and thevalueof r is2.39nmleadingtoaplanarradiusof, R = : 71 : 39 : 2 2 [ + p 2 + : 71 = : 2]=45 : 14nm.15 Withaplanarradiusof45.14nmandacross-sectionalrimradiusof2.39nmthe lengthofaDHPCmolecule,alsogiveninTable2.1,thecombinedradius r 0 is r 0 = R + r =45 : 14+2 : 39=47 : 53 nm .16 allowingcalculationofthetheoreticalhydrodynamicradiusforSULVswithdisk-like precursorsusingEquation2.14,reproducedhere, R H = 3 2 r 0 0 @ s 1+ t 2 r 0 2 + 2 r 0 t ln 2 4 t 2 r 0 + s 1+ t 2 r 0 2 3 5 )]TJ/F24 10.9091 Tf 16.872 7.38 Td [(t 2 r 0 1 A )]TJ/F22 7.9701 Tf 6.587 0 Td [(1 .17 Thethicknessofthebilayerdisk, t ,isdoublethelengthofaDMPCtail.Referring toTable2.1 t isfoundtobe5.06nm.SubstitutingintoEquation4.17gives R H =36 : 60nm.18 UsingthisvalueasthetheoreticalradiusofSULVsformedthroughthebicellar phase,thepercenterrorintheaverageradiusforSULVsinthe =0.75solutionis6.4% 42

PAGE 57

4.1FormationofSmallUnilamellarVesicles andtheerrorinSULVsizeinthe =1.0solutionis2.9%.Thelowpercenterrorsof thesesolutionsindicatethatbicelleswereformedunderlargelyidealconditions,with q verycloseto q eff .ThusbyfollowingPath2,andmodulatingthemolarratiowithinthe rangeshowninFigure2.5,SULVscanbecreatedwithinaverynarrow,predetermined sizerange.Thiscouldbeparticularlyusefulintargeteddrugdeliveryapplications wherethedrughastopenetratetissuesorsemipermeablemembranesthatonlyallow particleswithinanarrowsizerangetodiusethrough.Theprimarymotivationfor creatingtheseSULVswastocreatestabledrug-carriersthatcouldpenetratefenestrae intheinterendothelialjunctionsoftheneovasculatureinGBMtumors.Atabout35 nm,theSULVscreatedthroughthebicellarphaseareabletofreelydiusethrough poresinneovasculature.[11] 4.1.4Path2:EllipsoidalPrecursorsBelowCriticalTemperature Path3solutionswerehighlydilutedat4 CandequilibratedaboveT c .Becausethe precursorsareellipsoidsandincorporatefreeDHPCmoleculesratherthanmicellar DHPCastherim,idealbicelletheoryisnotapplicable.ThesignicantlylowerconcentrationsinducedagelphaseinDMPC,andlossofmicellarDHPC.Thedatafrom thispathwaywillthusfocusontheeectofconcentrationonSULVsize. 4.1.4.1 =0.3 Figure4.8showsDLSdataofthe =0.3solutionandTable4.5listsitstemperature, viscosity,andmeasureddiusioncoecient. Quantity Units ValueStd.Dev.Variance T K 311.0590.2920.085 Pa s 6.782 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(4 6.22 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(7 3.86 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 D m 2 /s 5.056 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(12 1.18 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 1.39 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(26 Table4.5: Temperature,ViscosityandDiusionCoecientfor =0.3 TheaveragehydrodynamicradiusofthissolutionisgiveninTable4.8. 4.1.4.2 =0.6 Figure4.9showsDLSdataforthe =0.6solution. Notethatthedatapointsexhibitaconcavityinthemiddleandshiftupwards towardstheends.Theupswingindecayratesatthehighestandlowestanglesindicates polydispersityastheintensitylosesitscorrelationataratethatdoesn'ttthediusion oftheSULVs.Sincethescaleofmeasurementvariesinverselywith q ,thepolydispersity 43

PAGE 58

4.DATAANDANALYSIS Figure4.8: LiposomesFormedviaEllipsoidalPrecursorsPhaseat0.3%w/v iscomingbothfromareasofhighandlowcurvature.Thiscanbeexplainedbythe presenceofellipsoidsremaininginsolutionastheirrimsscatterlightaswellastheir planes,meaningthatellipsoidswouldbebetterdetectedatextremeangles.Dueto theiranisotropicdiusionandbecauseoftumbling,whichisnegligibleforspheres,the decayrateofellipsoidswouldbehigherthanSULVs. CallingthestructuresresponsibleforthestructuralpolydispersityseeninFigure4.9 ellipsoidsisnottechnicallycorrect.Forone,thetemperatureduringdatacollectionwas higherthanthechain-meltingtemperatureofDMPC,causingaphasechangetoliquidcrystallinewithintheellipsoid.TheellipsoidsthatdidconverttoSULVsunderwent thisphasechangeastheycoalescedwithotherellipsoids,allowingthedisk-linetension causedbyDHPCtotakeover.Duetothehighlydiluteconcentrations,notallellipsoids mayhaveinteractedwithotherellipsoidsasthesolutionequilibratedto45 C.These loneellipsoidsmostlikelydesegregatedDMPCandDHPCwithinthestructureand adoptedmorphologiesrangingfrommicellestouidbicellesbasedonstudiesbySternin et.al.[22]ThesestructuresaremostlikelyanisotropicasindicatedbytheDLS 44

PAGE 59

4.1FormationofSmallUnilamellarVesicles Figure4.9: LiposomesFormedviaEllipsoidalPrecursorsPhaseat0.6%w/v data,andnotsignicantlyhighinconcentrationrelativetoSULVs,otherwiseagreater variationwouldbeseenaboutthetrendlineortheconcavityoftheplotwouldbemore pronounced.Heretheconcavityissmallenoughthattheerrorbarsremainclosetothe trendline. 4.1.4.3 =0.9 Finally,Figure4.10showstheplotofthe =0.9solution. Thissolutionalsoexhibitstheslightconcavityindicatingpolydispersityinstructure forreasonsexplainedabove.Theaveragehydrodynamicradiusofthissolutionisgiven inTable4.8. 4.1.4.4ComprehensiveDiscussionofLiposomesFormedviaEllipsoidal Precursors OveralltheSULVsmadethroughtheellipsoidalpathwayaresignicantlylargerinsize thanthosethatwereformedthroughthebicellarphase.Thisisbecausetheellipsoids 45

PAGE 60

4.DATAANDANALYSIS Quantity Units ValueStd.Dev.Variance T K 311.0350.3080.095 Pa s 6.785 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(4 6.57 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(7 4.31 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(13 D m 2 /s 5.771 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(12 9.54 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(14 9.10 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(27 Table4.6: Temperature,ViscosityandDiusionCoecientfor =0.6 Quantity Units ValueStd.Dev.Variance T K 311.0110.3000.090 Pa s 6.788 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(4 1.31 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(6 1.73 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(12 D m 2 /s 5.237 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(12 8.74 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(14 4.64 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(27 Table4.7: Temperature,ViscosityandDiusionCoecientfor =0.9 useDHPCmoleculesratherthanaDHPCmicelletostabilizetherim,incorporating lessDHPCintotheSULV q>q eff ,increasingthebendingmodulusofthemembrane, resultinginaspherewithlowercurvature. AlloftheSULVsolutionsmadeat < 0.9demonstratedaslightconcavity,linked topolydispersityinstructure.Specically,structuresofbothhighandlowcurvature areindicatedinsolution.Thesizeofthesestructuresdoesnotseemtobeoutsidethe scalabilityof l sinceotherwisethedatawould'vebeensignicantlynoisier.Sincethe upperandlowerboundanglesareknowntobe140 and20 ,respectively,thebounds of l canbecalculated.UsingEquations4.3and4.4, l wascalculatedtobebetween 1.37 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(6 mat =20 and2.53 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(7 mat =140 4.1.5DiscussionofPreferredPathwayforSubsequentExperiments BecausethebicellepathwayreliablyproducedSULVswithaverageradiilessthan50 nm,liposomeswithdisk-likeprecursorswillbeusedtotestdiusivityandleakagerates againstLULVs.TheseSULVshavebeenexperimentallyshowntobemorerobustthan SULVscreatedthroughsonicationbutjustifyingthisfactisnotatrivialmatter.[23] Thedependenceon q eff inthebicellarphaseandthewayconcentrationdictatesthe divergenceof q eff from q intheellipsoidalpathwayareindicationsthatthesizeof theresultantULVisdictatedlargelybytheamountofDHPCthatwasincorporated intothestructure.IftheamountincorporatedisclosetotheamountofDHPCused whilemakingthesolution,renedbicelletheorycanaccuratelypredictparticlesize. Sonicationusesultrasoundtotransferenergyintothesystem,breakinguplargerparticles,andcausingDHPCandDMPCtomix,makingSULVs.Theend-statesofboth processesrepresentnonequilibriumsystems.Insonication,theenergytransferredto themoleculestoassumetheSULVmorphologyallowedthemtotemporarilyovercome 46

PAGE 61

4.1FormationofSmallUnilamellarVesicles Figure4.10: LiposomesFormedviaEllipsoidalPrecursorsPhaseat0.9%w/v theenergybarrierofthemembrane'scurvatureenergy.Furthermore,insonication notallvesicleshavethesamemolarratiosofDHPCandDMPC,leadingtoawide variationinSULVsize.Interestingly,thisvariationspeedsupthecoalescenceofthe vesiclesasthosewithlittleDHPCseektostabilizetheirmembranecurvatureandfuse withothervesicles.Thiseectalsoexistsintheend-stateSULVscreatedthroughthe bicellephase,buttheseSULVswereprecludedbydiskswhichplacedtightconstraints onthemolarratio,onlyself-foldingintoSULVswhenthethresholddisklinetension wascrossed.Thecrossingofthisboundaryisadiscretestepmarkedbythefusionofthe growingdiskbyjustonemorebicelle.Thismeansthatthevariationin q eff isminimal acrossSULVsformedfromdiskprecursors.WhileSULVsmadethroughbicelledisks arestillnonequilibriummorphologiesthatseektoaggregatetorelievethecurvature energiesintheirmembranes,itisnotimmediatelyfavorableforthemtocoalescewith mostotherSULVsinsolution.Theendresultisasolutionofmetastablestructures thatdegenerateataratefarslowerthansonicatedSULVs. 47

PAGE 62

4.DATAANDANALYSIS R H mStd.Dev.Variance 0.03 6.640 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(8 8.813 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(11 7.767 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(21 0.06 5.818 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(8 9.651 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(10 9.314 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(19 0.09 6.408 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(8 8.450 10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(10 7.140 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(19 Table4.8: AverageHydrodynamicRadiiforallLiposomeswithEllipsoidalPrecursors 4.2DependenceofVesicleLeakageonSize Thisportionofthestudymeasuredtheconcentrationoffreerhodamine-Bovertimein solutionsof200nm,100nm,and35nmSULVsusingmembranedialysistoseparate liposomesfromtheuorescentdye. 4.2.1QuantifyingRhodamineConcentrationsthroughFluorescence Intensity Figure4.11belowplotsthemeasureduorescenceintensitytotheconcentrationofdye in1XPBS. Figure4.11: FluorescenceIntensitywithVaryingRhodamine-BConcentrations 48

PAGE 63

4.2DependenceofVesicleLeakageonSize Theexpectationwasaproportionalrelationshipbetweenthepercentsaturationof rhodamineandtheintensity,butasFigure4.11shows,afterasaturationofapproximately0.5%intensitybeginstodecrease.Thelowintensityreadingsofsaturation percentagesabove20 = photonsemitted photonsabsorbed .19 AstudyofthequantumyieldofrhodamineBbyBindhuet.al.demonstratedthe dependenceofthequantumyieldonconcentration,showninFigure4.12.[30] Figure4.12: DependenceoftheQuantumYieldofRhodamine-BonConcentration [30] At1 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(3 Mthequantumyieldisalreadyatabout80%,decreasingtolessthan 10%atconcentrationsabove1 10 )]TJ/F22 7.9701 Tf 6.586 0 Td [(3 M.Theseeectsareduetonon-radiativeprocessesduetotheformationofdimersandotherhigherorderaggregates.Notonlycan anexcitedrhodamineBmoleculeconvertfromthesinglettotripletstate,therebylosinguorescence,butdimerizationalsoresultsinForster-typeenergytransfersbetween dimers,signicantlyloweringthequantumyield.[30]Theregionfrom0.5%to12.5% isexpandedinaplotofintensityagainstmolarconcentrationshowninFigure4.13. Thedataistwithanexponentialdecayfunctionshowingtheplateauinintensity towardshigherconcentrations. 49

PAGE 64

4.DATAANDANALYSIS Figure4.13: FluorescenceIntensitywithVaryingRhodamine-BConcentrations Finally,theregionofthegraphshowingincreasingintensitywithincreasingrhodamineconcentrationisalsoexpandedintermsofmolarconcentrationandshownin Figure4.14below.Theexponentialrelationshipgivenbyttingthetrendlineoughtto havebeenusedtogaugetheconcentrationofrhodamineusedinliposomalsolutionsfor encapsulation.However,duetologisticalconstraintstheserhodaminesolutionswere imagedatthesametimeasthesamplestestingrhodamineleakagefromliposomes,so theinitialconcentrationusedinencapsulationdoesnotreecttheoptimalvaluesas willbecomeevidentinthefollowingsection. 4.2.2DependenceofLeakageRateonVesicleSize Figure4.15showstheuorescenceintensityofthe200 Lsamplestakenfromsolutions inwhichtheliposomes,connedtoamembranedialysiscassette,wereimmersed. TheplotdoesnotshowacleartrendintheleakageratesbetweenSULVsand LULVs.Thiscouldbeduetoseveraldierentreasons,includingtheuseofinappropriate 50

PAGE 65

4.2DependenceofVesicleLeakageonSize Figure4.14: FluorescenceIntensitywithVaryingRhodamine-BConcentrations concentrationsofrhodamine,theeectrhodaminemayhavehadontheformationof theliposomes,orsimplybecausesizedoesnotaectrateofleakage. Theinitialamountofrhodamineaddedwas12.5%,andFigure4.11showsthat thisamounthasaverylowquantumyield.Thisconcentrationofrhodaminewas chosentoensurethatSULVformationwouldencapsulateahighenoughconcentration ofrhodaminesuchthatevensmallpercentagesleakingintothe20mLbuerwouldbe detectablebyuorescence.Theliposomeswerewashedwithbuerinordertowashall thefreerhodamineoutofsolution,buttheconcentrationofrhodaminewerelikelyhigh enoughthatlongerbuerwasheswereneededtoridthesolutionoffreerhodamine. Sinceleakageofthedyeoutoftheliposomebeginsimmediately,withoutestimates forthetimescalesforleakagetherewasnowaytogaugeatwhichpointtheexcess rhodamineinthebuerincludedrhodamineleakingfromtheparticles.Thereforedata wascollectedimmediatelyafterthersthour-longbuerwashwiththeassumption thattheinitialrhodamineconcentrationscouldbenormalizedfor,andthechangein rhodamineconcentrationovertimeforeachsolutionwouldstillyieldeectiveleakage 51

PAGE 66

4.DATAANDANALYSIS Figure4.15: FluorescenceIntensitywithVaryingRhodamine-BConcentrations rates. AsisevidentinFigure4.15therewasverylittlechangeinintensityduetorhodamineconcentrationaftertherst5hours.Thisindicatestheassumptionmade abovedidnothold,mostlikelybecausetheamountofrhodaminediusingoutofthe liposomeswasnegligiblecomparedtothefreerhodamineinsolution.Theliposomes composed0.75%w/voftheapproximately1.5mLsinjectedintothemembranedialysis cassettesandtheconcentrationofrhodaminewassignicantevenfora20mLbuer solution.Theleakageofrhodaminefromtheliposomesdidnotcontributesignicantly totheoverallconcentrationoffreerhodamine.Finally,becausethesampleswerecollectedoveraperiodof36hours,quenchingeectsoftherhodamineduetolightand oxygenexposurenegativelyaectedtheuorescenceintensityovertimebyanunknown amount.Thislossofuorescenceintensitymayhavealsocounteredthesmallincrease inrhodamineinsolutionduetoleakage.ThesequenchingeectsareindicatedbyFigure4.15sincetheintensityindicatestheconcentrationofrhodamineinallsolutions decreasedovertime,whenitoughttohaveincreased. 52

PAGE 67

4.3DiusionthroughaUniformMedium Thehighrhodamineconcentrationalsomayhaveaectedtheformationofbicellesin solution,whicharehighlydependentontherespectivemolarratiosofphospholipidsas discussedintheprevioussection.BecauseDLSdatawasnottakenforthesesolutions, theexactsizesoftheseliposomesareunknown,andtheporesizeoftheextrusionlters areusedasestimates.Togainexperimentallysoundinformationaboutthedependence ofleakageratesonliposomesize,DLSshouldbeusedtoaccuratelysizetheparticles, andtheinitialconcentrationofrhodamineshouldbeadjustedtobeataconcenration ofapproximately10 )]TJ/F22 7.9701 Tf 6.587 0 Td [(4 M.Theuorescenceimagingshouldalsobeperformedasthe samplesarecollected,usingahighgainifnecessarytodetectevensmallamountsof rhodamineleakage. 4.3DiusionthroughaUniformMedium SULVswithanaverageradiusof34.95nmandLULVswithaverageradiusof120nm and211nmwereinjectedintoagelmediuminordertoobservetheirdiusionacross a0.4 mspace.Thegelmedium,Sephadex50,formsarelativelyuniformnetworkof pores,aspicturedinFigure4.16.Figures4.17-4.19showthediusionoftheliposomes. Figure4.16: Sephadex50GelMedium[31] Itisimmediatelyevidentfromobservingthetimescales,measuredinseconds,that theSULVsdiusedmuchfasterthantheLULVs,coveringthe0.4 mspacein330s. The120nmliposomestook1260sandthe211nmliposomestook6600stocoverthe samedistance.Becausetheimagesshowthatthediusionprogressedforeachsolution atarelativelyconstantrate,thedistancecoveredovertimeforeachsolutioncanbe takentobeanaveragerateofdiusion.TheSULVsthusdiusedatarateof1.21nm persecond,the120nmdiusedat0.32nmpersecondandnallythe211nmliposomes diusedatarateof0.06nmpersecond. ThisdemonstratesthatSULVscanpenetrateporesduetopassivediusionmuch fasterthanLULVscan.Theliposomesusedindrugdeliveryrelyonpassivediusion andrecirculationoftheliposomestodeliverthedrugtotumorregions.Thecirculatory 53

PAGE 68

4.DATAANDANALYSIS Figure4.17: Diusionof50nmLiposomesAcrossa0.4 mSpace 54

PAGE 69

4.3DiusionthroughaUniformMedium Figure4.18: Diusionof100nmLiposomesAcrossa0.4 mSpace 55

PAGE 70

4.DATAANDANALYSIS Figure4.19: Diusionof200nmLiposomesAcrossa0.4 mSpace 56

PAGE 71

4.3DiusionthroughaUniformMedium owisinthiscasemimickedbytheminimalhydrationofthegel,causingtheaqueous mediumtoowoutwardsrehydratingtheSephadexbeads.Thelevelofhydration wasestimatedtobeasdryaspossiblewithoutintroducingcracksinthegel,andthe variationsinhydrationlevelsbetweentheplatesisaprimarysourceofuncertainty inthisexperimentasitcausesdierentowpressuresintheowoftheliposomal solutions.Thisportionofthestudyismeanttobelargelyqualitative,andthelarge dierencesindiusionrateswithliposomesizearemorethantheslightvariationin gelhydrationcanaccountfor. TheSULVsdiusedataratenearly50timesfasterthanthe211nmliposomes. AppliedtoabiologicalcontextthismeansthatLULVswouldneedtohave50timesthe circulatoryhalf-lifeofSULVstopenetratetissuestothesameextentasSULVs.LULVs infacthavealowercirculatoryhalf-lifeaslargerparticlesaremoreeasilytargetedby clearancemechanismsandlteredbythekidneys.Thisisassumingthatthetissues targetedfordrugdeliveryhaveporesthatallowLULVstoeasilydiusethrough.As discussedintheIntroduction,GBMneovasculaturehasporesapproximately260nm indiameter.[9]Thesearetoosmallforthe211nmliposomestopenetrate,andeven the120nmliposomesarenotabletofreelydiusethrough.Therefore,basedon thisdiusionstudy,SULVswouldbemosteectiveindeliveringdrugsusingpassive diusionthroughGBMvasculature. 57

PAGE 72

4.DATAANDANALYSIS 58

PAGE 73

5 Conclusions Thisstudyvalidatedthespontaneousformationofsmallunilamellarvesiclesthrough thestructuralprecursorsofbicelledisksandellipsoidsusinga3.2:1DMPC/DHPC formulation.SULVswerenotdetectedforsolutionscreatedoverarangeofconcentrationsaboveT c ,evenutilizingone-stepdilutionsandtwo-stepextrusions.These solutionsindicatedthatwhilelamellarphasesarenotconvertibleintoSULVsthrough highpressure,thresholdconcentrationswherelamellarsheetsarebeginningunbinding transitionsmaybeusedtocreateULVsorotherstructures.Thebicellediskpathway wasfoundtoreliablyproduceSULVswithanaverageradiusofapproximately35nm 0.3nm.Bystudyingthedisk'sgrowthbycoalescenceanditsconversiontoaSULV, itwasdeterminedthatthemolarratioisabletoaccuratelypredictthehydrodynamic SULVradiuswithintheboundsoutlinedbyidealbicelletheory.Theproximityofthe averageradiustotheradiuspredictedbyrenedbicelletheoryalsoindicatesverylow polydispersityinsolution.Thiswasevenfurthercorroboratedbythegrowthmodel ofthediskswhichproceedindiscretesteps,immediatelyfoldingintoaSULVwhen thethresholddisklinetensionovercomesthemembrane'scurvatureenergy.Anideal bicellephasewillthereforegiverisetometastableSULVsthatapproachequilibriumat aratemuchslowerthanSULVsmadethroughothermethods.Theellipsoidalpathway yieldedliposomesbetweenapproximately56and68nminradius,alongwithindicationsofotherstructuresinsolution,possiblyanisotropicmicellesormixedbilayers. Thehighlydiluteconditionsunderwhichtheellipsoidswereformedweredeterminedto excludefreeDHPCmoleculesfromthestructure,alteringthemolarratio,andresulting inlargerliposomesthanthosemadethroughthebicellepathway. ThemetastableSULVscreatedthroughthebicellarphasewerecomparedindiffusivityandleakagetoLULVssimilartothoseusedintheGBMstudy.Theleakage studieswereinconclusiveduetooversaturationofthesolutionswithrhodamine.The diusionstudiesdemonstratedthat35nmvesiclesdiusedatarate50timesthatof 59

PAGE 74

5.CONCLUSIONS 211nmvesicles.NotonlywillSULVsfreelydiusethroughGBMneovascualturethat theLULVsmaynotbeabletopenetrate,buttheresultsofthisstudyindicatethat SULVswillpenetratethetumorstoamuchgreaterextent. TheGBMtargeteddrugdeliveryschemeusingstealthSULVsallowstargetingof thecanceratitsearlystageswhenneovasculatureinthebrainhasn'treacheditspeak size.ByloadingwithIBandDXR,SULVsmaybeabletodeeplypenetratetumorous regions,especiallylocalizingwherethecancerismostmetastatic.Futurestudiesshould testtheleakageofSULVswiththesespecicanti-cancerdrugs,eventuallytestingthe eectsofSULVsinvivo. 60

PAGE 75

6 Appendix 6.1CurvatureEnergy Deformationstothemembranesuchasstretches,bends,andtwistscostthemembrane someamountoffreeenergy.Suchappliedforcescanchangethemembrane'ssurface area,thickness,anddensity,allofwhichcontributetothefreeenergydierence.When abilayerisbent,theinnerlayeriscompressedandtheouterlayer'ssurfaceareais expanded.Betweenthelayersthereexistsaneutralsurfacethatexperiencesnochange inintermembranousforces,andhasafreeenergychangeonlycorrespondingtothe curvatureofthestructure.Thisneutralsurfacewillbeusedtocalculatethecurvature ofthesurfaceateachpoint,fromwhichthecurvatureenergydensitycanbecalculated forvariousstructures. Consideranarbitrarycurvedsurface F x;y withanormalvector n ij x i ;y j and atangentplane F 0 ij x i ;y j .Atanygivenpoint,thetangentplanecontainsallpossible tangentvectorsatthatpoint.Thusthereexists,ateverypointonthesurface,aset ofpossibleplaneseachofwhichcontains n ij x i ;y j andonepossibletangentvector in F 0 ij x i ;y j .Eachplaneintersects F x;y togiveacurve f ij x i ;y j withaspecic curvature at x i ;y j .Dependingonthesymmetryofthesurface, f ij x i ;y j ,and therefore ,canvaryacrossallpossibleplanesat x i ;y j Specically, isthecurvatureofthelargestcircletotthecurveat x i ;y j ,and canbeexpressedintermsoftheradius,whichisinverselyproportionaltotheextentof curvature.Thegoalistodeterminetheprincipalcurvatures max and min fromall possible of f ij x i ;y j at x i ;y j .Theaverageofthesevaluesisthemean-curvatureof thesurfaceat x i ;y j ,andmultiplyingthevaluestogetheryieldstheGaussiancurvature atthatpoint.ThemeanandGaussiancurvaturescanthenbeusedtoconstructthe HelfrichHamiltonian,whichhaseigenvaluescorrespondingtotheenergyofonlythe curvatureoftheneutralsurfaceofthestructure.Formationpathwaysestablished 61

PAGE 76

6.APPENDIX inpreviousstudiesshowthatcertaintemperaturesandconcentrationsgiveriseto structureswithcharacteristiccurvatures.TheprimarygoalindevelopingtheHelfrich Hamiltonianistotestwhetherminimizationofthefreeenergyofcurvatureunder variousconditionscanpredictthestructuresoutlinedintheformationpathways. 6.1.1CurvatureUsingMongeParametrizationinOneDimension Thecurvature, ,willbeformulatedforthecurve f i x i inonedimension,andlater extendedtohigherdimensions.Thiscurverepresentsapossiblecross-sectionofan arbitrarysurfaceat x 0 ;f x 0 ,wheretosimplifynotation, f x 0 = f 0 .Thenormal, n 1 ,at x 0 ;f 0 willintersectthecenterofthecirclewhosecurvaturematchesthelocal curvatureof f 0 .Thecenterofthecircle, x c ;y c ,isdenedasthepointofintersection of n 1 ,withthenormal, n 2 ,at x 0 + dx .Thedistancebetween x 0 ;f 0 and x c ;y c isthe radius. Figure3.1 Thetangentat x 0 ;f 0 hastheslope f 0 0 .Hence,theslopeof n 1 is )]TJ/F22 7.9701 Tf 11.673 4.296 Td [(1 f 0 0 .Bysubstitutingtheslopeandthepoint x 0 ;f 0 intothelineequation y = mx + b for n 1 f 0 = )]TJ/F24 10.9091 Tf 9.68 7.38 Td [(x 0 f 0 0 + b andcalculatingforthey-intercept, b = x 0 f 0 0 + f 0 theequationfor n 1 isdeterminedtobe, n 1 x = x 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x f 0 0 + f 0 Theequationfor n 2 isthen 62

PAGE 77

6.1CurvatureEnergy n 2 x = x 0 + dx )]TJ/F24 10.9091 Tf 10.909 0 Td [(x f 0 x 0 + dx + f x 0 + dx f x 0 + dx and f 0 x 0 + dx canbeexpandedusingtheTaylorseries, f x 0 + dx f x 0 + f 0 x 0 dx + 1 2! f 00 x 0 dx 2 + ::: n 2 x x 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x + dx f 0 0 + f 00 0 dx + f 0 + f 0 0 dx Since n 1 and n 2 intersectat x c x 0 )]TJ/F24 10.9091 Tf 10.91 0 Td [(x c f 0 0 + f 0 = x 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c + dx f 0 0 + f 00 0 dx + f 0 + f 0 0 dx x 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c f 0 0 = x 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c + dx f 0 0 + f 00 0 dx + f 0 0 dx x 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c f 0 0 + f 00 0 dx = f 0 0 x 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c + dx + f 0 2 0 f 0 0 + f 00 0 dx dx x 0 f 0 0 + x 0 f 00 0 dx )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c f 0 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c f 00 0 dx = x 0 f 0 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c f 0 0 + f 0 0 dx + f 0 3 0 dx + f 0 2 0 f 00 0 dx 2 x 0 f 00 0 dx )]TJ/F24 10.9091 Tf 10.909 0 Td [(x c f 00 0 dx = f 0 0 dx + f 0 3 0 dx + f 0 2 0 f 00 0 dx 2 x c f 00 0 dx = )]TJ/F24 10.9091 Tf 8.485 0 Td [(f 0 0 dx )]TJ/F24 10.9091 Tf 10.91 0 Td [(f 0 3 0 dx )]TJ/F24 10.9091 Tf 10.909 0 Td [(f 0 2 0 f 00 0 dx 2 + x 0 f 00 0 dx x c = )]TJ/F24 10.9091 Tf 10.447 7.38 Td [(f 0 0 f 00 0 )]TJ/F24 10.9091 Tf 12.104 7.38 Td [(f 0 3 0 f 00 0 )]TJ/F24 10.9091 Tf 10.91 0 Td [(f 0 2 0 dx + x 0 Asdxgoestozero, x c = )]TJ/F24 10.9091 Tf 8.485 0 Td [(f 0 0 1+ f 0 2 0 f 00 0 + x 0 y c = n 1 x c y c = x 0 )]TJ/F15 10.9091 Tf 10.909 0 Td [( )]TJ/F24 10.9091 Tf 8.485 0 Td [(f 0 0 1+ f 0 2 0 f 00 0 + x 0 f 0 0 + f 0 y c = x 0 + f 0 0 1+ f 0 2 0 f 00 0 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 0 f 0 0 + f 0 63

PAGE 78

6.APPENDIX y c = 1+ f 0 2 0 f 00 0 + f 0 R = p x c )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 0 2 + y c )]TJ/F24 10.9091 Tf 10.909 0 Td [(f 0 2 R = s )]TJ/F24 10.9091 Tf 8.485 0 Td [(f 0 0 1+ f 0 2 0 f 00 0 2 + 1+ f 0 2 0 f 00 0 2 R = p + f 0 2 0 3 f 00 0 Thecurvature, K ,istherefore, K = 1 R = f 00 0 p + f 0 2 0 3 = f 0 0 p 1+ f 0 2 0 0 Thistechnique,Mongeparameterization,wasuseddetailedforonedimensionto demonstratehowexpressionsforcurvaturecanbederivedintermsoffx.When extrapolatedtohigherdimensionstheformulaforcurvaturelookssimilar: K = r r h r p 1+ r h r 2 Liposomeshapewillbeexpressedasaheightfunction, h r ,where r isavectorin twodimensions, ^x and ^y .Thecurvatureisthen: K = r 0 @ h x ^x + h y ^y q 1+ h 2 x + h 2 y 1 A where h x = @h @x and h y = @h @y K = @ h x + h 2 x + h 2 y )]TJ/F22 7.9701 Tf 6.587 0 Td [(1 = 2 @x + @ h y + h 2 x + h 2 y )]TJ/F22 7.9701 Tf 6.586 0 Td [(1 = 2 @y K = h xx + h 2 y )]TJ/F24 10.9091 Tf 10.909 0 Td [(h x h y h xy + h 2 x + h 2 y 3 = 2 + h yy + h 2 x )]TJ/F24 10.9091 Tf 10.909 0 Td [(h x h y h xy + h 2 x + h 2 y 3 = 2 K = h xx + h 2 y )]TJ/F15 10.9091 Tf 10.909 0 Td [(2 h x h y h xy + h yy + h 2 x + h 2 x + h 2 y 3 = 2 Theequationabovereferstothemeancurvatureofasurface,andisequivalentto theaverageoftheprincipalcurvaturesofthesurface h x;y .Themeancurvatureisan 64

PAGE 79

6.1CurvatureEnergy extrinsicpropertyofthesurface,meaningthatitisdependentuponthespaceinwhich itisembedded.Whileextrinsiccurvatureonlyexistswithrespecttoaspaceseparate fromthecurvedsurface,theintrinsic,orGaussian,curvatureisaninherentpropertyof thesurface,andwouldbeinvisibletoanyofitsinhabitants.Gaussiancurvatureisthe productoftheprincipalcurvatures,andcanbedeterminedforasurface h x;y using thefollowingformula: G = h xx h yy )]TJ/F24 10.9091 Tf 10.909 0 Td [(h 2 xy + h 2 x + h 2 y 2 BothmeanandGaussiancurvaturesaecttheenergyofastructure,andneedtobe explicitlycalculatedtodeterminethecurvaturecontributiontotheoverallfreeenergy. Thecurvatureenergydensityofaspherewillnowbedeterminedusingtheformulations above.Theheightfunctionforasphereis h x;y = p R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 h x = )]TJ/F24 10.9091 Tf 8.485 0 Td [(x p R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 h y = )]TJ/F24 10.9091 Tf 8.485 0 Td [(y p R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 h xx = y 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 h yy = x 2 )]TJ/F24 10.9091 Tf 10.91 0 Td [(R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 h xy = h yx = )]TJ/F24 10.9091 Tf 8.485 0 Td [(xy R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 h xx + h 2 y = y 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 1+ y 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 = )]TJ/F24 10.9091 Tf 8.485 0 Td [(R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 y 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 5 = 2 h yy + h 2 x = x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 1+ x 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 = )]TJ/F24 10.9091 Tf 8.485 0 Td [(R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 y 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 5 = 2 )]TJ/F15 10.9091 Tf 8.485 0 Td [(2 h x h y h xy = )]TJ/F15 10.9091 Tf 8.485 0 Td [(2 xy R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 )]TJ/F24 10.9091 Tf 8.485 0 Td [(xy R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 = 2 x 2 y 2 R 2 )]TJ/F24 10.9091 Tf 10.91 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 5 = 2 + h 2 x + h 2 y )]TJ/F22 7.9701 Tf 6.586 0 Td [(3 = 2 = 1+ x 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 + y 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 )]TJ/F22 7.9701 Tf 6.587 0 Td [(3 = 2 = R )]TJ/F22 7.9701 Tf 6.587 0 Td [(3 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 Themeancurvatureisthen, K = )]TJ/F15 10.9091 Tf 8.485 0 Td [(2 R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F15 10.9091 Tf 10.909 0 Td [(2 x 2 y 2 +2 x 2 y 2 R 3 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 5 = 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = 2 = 2 R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 R 3 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.91 0 Td [(y 2 65

PAGE 80

6.APPENDIX K = 2 R h xx h yy )]TJ/F24 10.9091 Tf 10.909 0 Td [(h 2 xy = R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 + x 2 y 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 )]TJ/F24 10.9091 Tf 38.597 7.38 Td [(x 2 y 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 3 = R 2 R 2 )]TJ/F24 10.9091 Tf 10.91 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 2 + h 2 x + h 2 y 2 = 1+ x 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 + y 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 2 = R 4 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 2 G = R 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.91 0 Td [(y 2 2 R 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(x 2 )]TJ/F24 10.9091 Tf 10.909 0 Td [(y 2 2 R 4 = 1 R 2 TheHelfrichHamiltonianisdescribedby E bend = Z membrane dA 0 + 0 K )]TJ/F15 10.9091 Tf 10.909 0 Td [(2 c 0 2 2 + 0 K G Usingthevaluesderivedfor K and K G ,theHelfrichHamiltonianforasphereis E bend = Z membrane dA 0 + 0 2 2 R )]TJ/F15 10.9091 Tf 10.909 0 Td [(2 c 0 2 + 0 1 R 2 E bend = Z membrane dA 0 +2 0 1 R )]TJ/F24 10.9091 Tf 10.909 0 Td [(c 0 2 + 0 1 R 2 E bend = Z membrane dA 0 +2 0 1 R 2 )]TJ/F15 10.9091 Tf 12.105 7.38 Td [(2 c 0 R + c 2 0 + 0 1 R 2 66

PAGE 81

References [1] Mu-PingNieh,VelayudhanA.Raghunathan,SteveR. Kline,ThadA.Harroun,Chien-YuehHuang,andJohn Katsaras SpontaneouslyFormedUnilamellarVesicleswithPath-DependentSizeDistribution Langmuir 21 ,2005.1,6,11,12,13,16,17,18,19,35, 36 [2] MaksymV.Yezhelyev,LifengQi,RuthM.O'Regan,ShumingNie,andXiaohuGao Proton-Sponge-Coated QuantumDotsforsiRNADeliveryandIntracellularImaging JAmChemSoc. 130 :9006{9012, 2008.2 [3] PubuduM.Peiris,ErikSchmidt,MichaelCalabrese,and EfstathiosKarathanasis AssemblyofLinearNanoChainsfromIronOxideNanosphereswithAsymmetricSurfaceChemistry PLoSONE 6 ,2011. 2 [4] NationalBrainTumorSociety GlioblastomaMultiformeGBM ,2013.2 [5] KathleenMcNeeleyKristinLoomisandRaviV.Bellamkonda Nanoparticleswithtargeting,triggered release,andimagingfunctionalityforcancerapplications SoftMatter 7 :839{856,2011.3 [6] JenniferM.Munson,LeviFried,SydneyA.Rowson, MichaelY.Bonner,LohitashKarumbaiah,BegoaDiaz, SaraA.Courtneidge,UllaG.Knaus,DanielJ.Brat, JackL.Arbiser,andRaviV.Bellamkonda AntiInvasiveAdjuvantTherapywithImipramineBlue EnhancesChemotherapeuticEcacyAgainst Glioma SciTranslMed 4 ,2012.3,4,5 [7] BruceI.TermanandKonstantinV.Stoletov VEGF andtumorangiogenesis EinsteinQuart.J.Biol.and Med. 18 :59{66,2001.3 [8] W.GregoryRoberts,JoanDelaat,MotooNagane, SuHuang,WebsterK.Cavenee,andGeorgeE.Palade HostMicrovasculatureInuenceonTumorVascularMorphologyandEndothelialGeneExpression AmericanJournalofPathology 153 ,1998.3 [9] W.GregoryRobertsandGeorgeE.Palade NeovasculatureInducedbyVascularEndothelialGrowth FactorIsFenestrated CancerRes 57 :765{772,1997. 3,57 [10] C.C.MICHELandF.E.CURRY MicrovascularPermeability PhysiologicalReviews 79 ,1999.3 [11] RaziaNoreen,RaphaelPineau,Chia-ChiChien,MariangelaCestelli-Guidi,YeukuangHwu,AugustoMarcelli, MichelMoenner,andCyrilPetibois FunctionalhistologyofgliomavasculaturebyFTIRimaging Anal BioanalChem 401 :795{801,2011.4,43 [12] A.B.Madhankumar,BeckySlagle-Webb,AkivaMintz, JonasM.Sheehan,andJamesR.Connor Interleukin13receptor?targetednanovesiclesareapotential therapyforglioblastomamultiforme MolCancer Ther 5 :3162{3169,2006.5 [13] AnneS.Ulrich BiophysicalAspectsofUsingLiposomesasDeliveryVehicles BioscienceReports 22 ,2002.8 [14] J.N.Israelachvili,D.J.Mitchell,andB.W.Ninham Theoryofself-assemblyofhydrocarbonamphiphiles intomicellesandbilayers. J.Chem.Soc.,Faraday Trans. 2 ,1976.9 [15] JohnTexter ReactionsAndSynthesisInSurfactantSystems .CRCPress;1edition,2001.10 [16] AvantiPolarLipids PreparationofLiposomes ,2013. 10 [17] AvantiPolarLipids CriticalMicelleConcentrations CMCs ,2013.10 [18] S.DerenGulera,D.DiponGhoshb,JianjunPana,JohnC. Mathaic,MarkL.Zeidelc,JohnF.Naglea,andStephanie Tristram-Naglea Eectsofethervs.esterlinkage onlipidbilayerstructureandwaterpermeability ChemistryandPhysicsofLipids 160 :33{44,2009.11,12 [19] NelsonS.Haas,P.K.Sripada,andG.GrahamShipley Eectofchain-linkageonthestructureofphosphatidylcholinebilayers;Hydration studiesof1-hexadecyl2-palmitoyl-sn-glycero-3phosphocholine Biophys.J. 57 :117{124,1990.11, 21 [20] M.A.Kiselev,E.V.Zemlyanaya,andV.K.Aswal SANS STUDYOFTHEUNILAMELLARDMPCVESICLES.THEFLUCTUATIONMODELOFLIPID BILAYER. 2009.11 [21] N.E.GabrielandM.F.Roberts. Spontaneousformationofstableunilamellarvesicles. Biochemistry. 23 :4011{4015,1990.12 [22] EdwardSternin,DavidNizza,andKlausGawrisch TemperatureDependenceofDMPC/DHPCMixing inaBicellarSolutionandItsStructuralImplications Langmuir 17 ,2001.12,13,17,18,44 [23] J.Leng,S.U.Egelhaaf,andM.E.Cates StructuralEvaluationofPhospholipidBicellesfor Solution-StateStudiesofMembrane-Associated Biomolecules. BiophysicalJournal 85 :1624{1646, 2003.13,18,19,46 [24] Mu-PingNieh,CharlesJ.Glinka,SusanKrueger,R.Scott Prosser,andJohnKatsaras SANSStudyontheEffectofLanthanideIonsandChargedLipidsonthe MorphologyofPhospholipidMixtures. Biophysical Journal 82 :2487{2498,2002.13,18 [25] KerneyJ.Glover,JenniferA.Whiles,GuohuaWu,Nan junYu,RaymondDeems,JochemO.Struppe,RuthE. Stark,ElizabethA.Komives,andRegitzeR.Vold StructuralEvaluationofPhospholipidBicellesfor Solution-StateStudiesofMembrane-Associated Biomolecules. BiophysicalJournal 81 :2163{2171, 2001.14,15,17,18 [26] R.R.VoldandR.S.Prosser Magneticallyorientedphospholipidbilayeredmicellesforstructuralstudiesofpolypeptides.Doestheidealbicelleexist? J.Magn.Reson.B. 113 :267{271,1996. 14,16 [27] J.ChungandJ.H.Prestegard Characterizationof eld-orientedaqueousliquidcrystalsbyNMRdiffusionmeasurements. J.Phys.Chem. 97 :9837{9843, 1993.18 [28] M.-P.Nieh,V.A.Raghunathan,G.Pabst,T.A.Harroun, K.Nagashima,H.Morales,J.Katsaras,andP.M.MacDonald Temperaturedrivenannealingofperforations inbicellarmodelmembranes Langmuir 27 :4838{ 4847,2011.18 67

PAGE 82

REFERENCES [29] EugeneMachlin AnIntroductiontoAspectsofThermodynamicsandKineticsRelevanttoMaterialsScience:3rd Edition .Elsevier;3rdEdition,2010.19 [30] C.V.Bindhu,S.S.Harilal,GeethaK.Varier,RijuC.Issac,V.P.N.Nampoori,andC.P.G.Vallabhan Measurementoftheabsoluteuorescencequantumyield ofrhodamineBsolutionusingadual-beamthermallenstechnique J.Phys.D:Appl.Phys. 29 :1074{ 1079,1996.49 [31] AmershamBiosciences GelFiltration:Principlesand Methods .AmershamBiosciences.ix,53 68


ERROR LOADING HTML FROM SOURCE (http://ncf.sobek.ufl.edu//design/skins/UFDC/html/footer_item.html)