Gallas, Andrzej

165 Pages·2017·14.2 MB·English

by  Christine Middleton

DEVELOPMENT OF IN VITRO SCREENING
APPROACHES TO OPTIMISE FORMULATION
PERFORMANCE.
ANDRZEJ GALLAS, mgr farm.
ThesissubmittedtotheUniversityofNottingham
forthedegreeofDoctorofPhilosophy.
September2013
Abstract.
The pharmaceuticalindustry has been criticised foralack of innovation associated with the
drug discovery and development process, for example when compared with the computer
ormusicindustries.Infact,bringinganewmedicinetothemarketrequires,onaverage,the
screeningofupto 10 000molecules, anexpense inthe range of$500million-$2 billionand
roughly 10-15 years ofresearch.Suchasituationnot only hasadirect impact onthe health
and life expectancy of every single human being on the planet, but also indicates that
alternativestrategiesfordrugdevelopmentshouldbeinvestigated.
In this thesis, studies of direct formulation-membrane interactions, both in a high
throughput (HT) manner and at a nanometre scale, were initially identified asan important
approach that could offer advantages for in vitro-in vivo correlations of in-man drug
behaviours. Subsequently, supported lipid bilayers (SLBs) of physiologically-relevant lipid
compositions were indicated as experimental models of preference for pre-clinical drug
development. For that reason, the characterisation and assessment of physicochemical and
behavioural properties of the model SLBs at a nanometre scale, as well as development of
anSLBmicroarrayforHTapplicationswerethefocusofthisresearch.Here,theoptimisation
and characterisation of model lipid films was performed using atomic force microscopy
(AFM), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron
spectroscopy (XPS). Additionally, the AFM-investigated assessment of the interactions
between model SLBs and formulation components (e.g. Pluronics®, siRNA, DNA polyplexes)
enabled both the correlation of in vitro observations with literature-reported in vivo
performances of the components of interest and the development of hypotheses with
regards a number of phenomena in biology. Furthermore, the development of a SLB
microarrayprototypesuitableforHTapplicationsisreported.
Directly, this research improves: the understanding of SLB behaviours and experimental
investigation at a nanometre scale of the mechanisms of interactions between membranes
and: Pluronics®, nucleic acids and their complexes, as well as the technology of SLB
microarray development. Indirectly, this research contributes towards the progress in a
number of research areas within pharmaceutical sciences, potentially resulting in new
scientificdisciplines,suchasimmunolipidomicsornanopharmacology.
2
Acknowledgements.
IwouldliketothankEPSRC,AstraZenecaandUniversityofNottinghamforfunding.
I would like to thank my supervisors: Dr Stephanie Allen, Prof. Cameron Alexander, Prof.
Morgan Alexander, Prof. Martyn Davies and Dr Sanyogitta Puri for help and guidance
throughouttheproject.
IwouldliketothankProf.XinyongChen,DrDavidScurr,MrsEmily Smith,DrAndrew Hook,
Dr Adam Celiz, Dr Francisco Fernandez-Trillo and other members of the Laboratory of
Biophysics and Surface Analysis and the Drug Delivery groups for advice and hands-on help
inlaboratory.
Mamo, Tato, Haniu, Leszku, babciu Zosiu i Mirciu, dziadku Stasiu i Jurku – serdecznie
dziękuję za wsparcie, pomoc i wszystko co umożliwiło mi zrobienie tego doktoratu.
IwouldalsoliketothankMrsChristineGrainger-Boultby,MrPaulCooling,MrsJuliaCrouch,
DrChristinaGrindonandDrClaudiaMatzfortechnicalsupportduringmyPhD.
IwouldparticularlyliketothankSarah,Gökçen,Vanessa,Marek,Alim,SelinaandBeppefor
friendshipandsupportthroughoutmytimeinNottingham.
Finally, I would like thank my assessors: Prof. Clive Roberts and Dr Patrick Gunning for
feedbackandtimetheyspentonimprovingthisthesis.
AndrzejGallas.
3
ListofContents.
TitlePage………………………………………………………………………………………………………………………………………………………………1
Abstract…………………………………….…………………………………………………………………………………………………………………….……2
Acknowledgements…………………….………………………………………..………………………………………………………………………………3
ListofContents…………………………………………………………..…………………………………………………………………………………………4
ListofFigures…………………………………………………………………………………………..……………………………………………………………7
ListofTables……..………………………………………………………………………………………………………………………………………………..10
Preface………………………………………………………………………………………………………………………………………………………………..11
1 Chapter1:IntroductionandAims.
Supportedlipidbilayersasmodelsforpre-clinicaldevelopmentofnewdrugs…………………..…12
1.1. Abstract………………………………………………………………………………………………………………………..…………………………12
1.2. Introduction……………………………………………………………………………………………………….……………………………………13
1.3. Theimportanceofmembraneinteractionsinpharmaceuticalsciences…………………………………….……………14
1.4. Modelsformembraneinteractions:developmentandtypes.…………………………………………………………………17
1.5. SLBsasamodelformembraneinteractions……………………………………………………………………………………………21
1.6. CurrentlimitationsassociatedwithSLBs…………………………………………………………………………………………………26
1.7. Conclusionsandfutureprospects.……………………………………………………..……………………………………………………27
1.8. References………………………………………………………………………………………………………………………………………………30
2 Chapter2:MaterialsandMethods.
2.1. Materials………………………………………………………………………………………………………………………………………………33
2.2. Methods………………………………………………………………………………………………………………………………………………36
2.2.1. Preparationofsupportedlipidbilayers……………………………………………………………………………………………..…36
2.2.1.1. Calculationoflipidamountsforliposomepreparation……………………………………………………………………………….…36
2.2.1.2. Bilayerself-spreadingtechnique(BSST).…………………………………………………………………………………………………..…36
2.2.1.3. Vesicledepositiontechnique(VDT).……………………………………………………………………………………..……………………36
2.2.2. AtomicForceMicroscopy(AFM)……………………………………………………………………………………………………….…37
2.2.2.1. Background……………………………………………………………………………………………………………………………………………37
2.2.2.2. Experimental……………………………………………………………………………………………………………………………………….…38
2.2.2.2.1.BasicSLBimaging(chapter3and4).………………………………………………………………………………………………………..…38
2.2.2.2.2.SLBinteractionstudies(chapter5)………………………………………………………………………………………………………………38
2.2.2.2.3.SLBairstabilitystudies(chapter1and6)……………………………………………………………………………………………………39
2.2.2.2.4.SLBmicroarrayimaging(chapter6)…………………………………………………………………………………………………..………39
2.2.2.2.5.ImagingofPolyphosphonium-DNApolyplexes(chapter5)……………………………………………………………………………39
2.2.2.2.6.PreparationandimagingofFluidArray®-likesurfaces(chapter6)………………………………………………………………..…40
2.2.2.2.7.AFMdataanalysis……………………………………………………………………………………………………………………………………40
2.2.2.2.7.1. EvaluationofSLBthickness…………………………………………………………………………………………………….…40
2.2.2.2.7.2. QuantificationofSLBcoverage………………………………………………………………………………………………..…41
2.2.2.2.7.3. Evaluationofparticlesize…………………………………………………………………………………………………….……41
2.2.3. Time-of-FlightSecondaryIonMassSpectrometry(ToF-SIMS)………………………………………………………………41
2.2.3.1. Background.……………………………………………………………………………………………………………………………………………41
2.2.3.2. Experimental…………………………………………………………………………………………………………………………………….……42
2.2.3.3. ToF-SIMS:dataanalysis……………………………………………………………………………………………………………………………42
2.2.4. X-rayPhotoelectronSpectroscopy(XPS)………………………………………………………………………………………………43
2.2.4.1. Background……………………………………………………………………………………………………………………………………………43
2.2.4.2. Experimental…………………………………………………………………………………………………………………………………….……44
2.2.4.2.1. XPS:samplepreparation……………………………………………………………………………………………………………………..……44
2.2.4.2.2. XPS:dataanalysis……………………………………………………………………………………………………………………………………45
2.2.4.2.2.1. Calculationofthetheoreticallipidcontentandratiobetween:-N+(CH),=N+(H)and–NH-nitrogen
3 2
groupsforlipidfilmsamplesanalysedwithXPSintheN1sregion……………………………………………………46
2.2.4.2.2.2. CalculationoftheoreticalintensityoftheP2ppeakforlipidfilmsamplesanalysedwithXPS
intheP2pregion..…………………………………………………………………………………………………………………..47
2.2.5. DynamicLightScattering(DLS)……….……………………………………………………………………………………………………47
2.2.5.1. Background……………………………………………………………………………………………………………………………………………47
4
ListofContents.
2.2.5.2. Experimental……………………………………………………………………………………………………………………………………….…48
2.2.5.2.1. DLS:samplepreparation………………………………………………………………………………………………………………………..…48
2.2.5.2.2. DLS:dataanalysis……………………………………………………………………………………………………………………………………48
2.2.6. Piezoelectricinkjetprintheadtechnology……………………………………………………………………………………………48
2.2.6.1. Background……………………………………………………………………………………………………………………………………………48
2.2.6.2. Experimental……………………………………………………………………………………………………………………………………….…49
2.2.7. PreparationofPluronic®solutions(chapter4and6)……………………………………………………………………………50
2.2.8. PreparationofDNAcomplexes(chapter5)….………………………………………………………………………………………50
2.2.9. Preparationoftextandfigures………………………………………………………………………………………………………….…51
2.3. References……………………………………………………………………………………………………………………………………………51
3 Chapter3:FabricationandCharacterisationofModelSLBs.
Advancedsurfaceanalysistechniquesforthedevelopmentofasupportedlipidbilayer
model………….……………………………………………………………………………………………………………………………………………………53
3.1 Abstract……………………………………………………………………………………………………………………………………………….………53
3.2 Introduction…………………………………………………………………………………………………………………………………………………54
3.3 MaterialsandMethods……………………………………………………………………………………………………………………………..…54
3.4 ResultsandDiscussion…………………………………………………………………………………………………………………………………55
3.4.1 StudyingSLBmorphologyandcomposition…………………………………………………………………………………………………55
3.4.1.1 AFM:optimisationofmodelSLBfabricationprotocols……………………………………………………………………………….…55
3.4.1.2 ToF-SIMS:qualitativeandsemi-quantitativeevaluationofmodellipidfilms………………………………………………….…57
3.4.1.3 XPS:quantitativeevaluationofmodellipidfilms……………………………………………………………………………………….…62
3.4.2 AFM:studyingSLBbehaviour.……………………………………………………………………………………………………………….…71
3.5 Conclusion……………………………………………………………………………………………………………………………………………………79
3.6 References………………………………………………………………………………………………………………………………………………..…79
4 Chapter4:ModelSLBs-ExcipientInteractionStudies.
Supportedlipidbilayerstoaidunderstandingofpharmaceutical
excipientperformancesinvitro…………………………………………………………………………………………………………….…81
4.1 Abstract……………………………………………………………………………………………………………………………………………………….81
4.2 Introduction…………………………………………………………………………………………………………………………………………………82
4.3 MaterialsandMethods……………………………………………………………………………………………………………………………..…82
4.4 ResultsandDiscussion…………………………………………………………………………………………………………………………………83
4.5 Conclusion……………………………………………………………………………………………………………………………………………………93
4.6 References…………………………………………………………………………………………………………………………………………………..93
5 Chapter5:ModelSLBs-NucleicAcidFormulationInteractionStudies.
Supportedlipidbilayers:stop-changeinnucleicacidpharmacology………………………………………….94
5.1 Abstract……………………………………………………………………………………………………………………………………………………….94
5.2 Introduction…………………………………………………………………………………………………………………………………………………95
5.3 MaterialsandMethods………………………………………………………………………………………………………………………………..96
5.4 ResultsandDiscussion…………………………………………………………………………………………………………………………………96
5.4.1 Nucleicacid-SLBinteractions…………………………………………………………………………………………………………………….96
5.4.2 DNApolyplex-SLBinteractions…………………………………………………………………………………………………………………106
5.5 Conclusion…………………………………………………………………………………………………………………………………………………111
5.6 References…………………………………………………………………………………………………………………………………………………112
6 Chapter6:DevelopmentofSLBMicroarrayforNano-HTSApplications.
Developmentofsupportedlipidbilayermicroarrayfor
highthroughputscreeningapplicationsatnanoscale……………………………………………………………………114
6.1 Abstract……………………………………………………………………………………………………………………………………………………..114
6.2 Introduction……………………………………………………………………………………………………………………………………………….115
5
ListofContents.
6.3 MaterialsandMethods………………………………………………………………………………………………………………………………115
6.4 ResultsandDiscussion……………………………………………………………………………………………………………………….………116
6.4.1 OptimisationofSLBfabricationmethodonmicroscopeslide-sizedmicasheets………………………………………………116
6.4.2 AFMstudiesofdry,modelSLBsinhighhumidityenvironments……………………………………………………………………119
6.4.3 OptimisationofthepiezoelectricinkjetprintingmethodologyforSLBmicroarraydevelopment…………………….…122
6.4.4 OptimisationoftheToF-SIMSchemicalimagingoftheSLBmicroarrays…………………………………………………………126
6.5 Conclusion…………………………………………………………………………………………………………………………………………………131
6.6 References…………………………………………………………………………………………………………………………………………………132
Chapter7:ConcludingRemarksandFutureProspects……………….………………………………………………...133
References…………………………………………………………………………………………………………………………………………………….136
AppendixI:AnalysisofAFMImages………………………………………………………………………………………………….....137
AppendixII:SupportingInformationonAFMStudies.………………………………………………………..……….....145
AppendixIII:MolecularIonAssignmentsforToF-SIMSSpectraofSingleLipids……..……………....154
AppendixIV:ChemistryandFormulationsforsiRNAtherapeutics…………………………………………......165
6
ListofFigures.
1. Chapter1:IntroductionandAims.
1.1. Thegraphicalsummaryofthefactorsinvolvedinthedevelopmentofthesupportedlipidbilayer
(SLB)-basedmodelforstudyingformulation-membraneinteractions..……………………………………………………12
1.2. Illustrationdepictingtheimportanceofinteractionsbetween
drugs(orformulations)andmembranes(orlipids)inpharmaceuticalsciences………………………………………15
1.3. Graphicalsummaryofthefactorsinvolvedinthedevelopment
oftheParallelArtificialMembranePermeabilityAssay(PAMPA)……..……………………………………………………..17
1.4. StrategiesprovidingairstabilityforSLBs…………………………………………………………….…………………………………..27
2. Chapter2:MaterialsandMethods.
2.1. Thechemicalstructuresoflipids,PEO-PPO-PEO(Pluronic)tri-blockco-polymers
and19bpDNAstrand………………………………………………………………………………………………………………………………35
2.2. TheschematicofAFMoperationprinciple…………………………………….………………………………………………………..38
2.3. TheschematicofToF-SIMSoperationprinciple…………………………….………………………………………………………..42
2.4. TheschematicofXPSoperationprinciple………………………………………………………………………………………………..44
2.5. TheschematicofDLSoperationprinciple………………………………………………………………………………………………..48
2.6. Theschematicofpiezoelectricinkjetprintheadtechnologyoperationprinciple…….………………………………49
3. Chapter3:FabricationandCharacterisationofModelSLBs.
3.1. Graphicindicatingthenatureofstudiesdiscussedinchapter3………………….…………………………………………..53
3.2. AFMimagesofBSST-fabricatedmodelSLBsandVDT-fabricatedmodellipidfilms……………….…………………56
3.3. ToF-SIMSspectraformicaandbothmodellipidfilmsamples
inthe0–300umassregions……………………………………………………………………………………………..…………………..57
3.4. TheToF-SIMSchemicalimagespresentingthespatialdistributions
ofsinglelipid-representativepeakswithinthemodellipidfilmsamples…………….…………………………………..60
3.5. Graphspresentingthesemi-quantitativeevaluationof
singlelipid-representativepeakintensitieswithintheToF-SIMSspectra
ofthemodellipidfilmsinbothpositiveandnegativemodes…………………………..……………………………………..61
3.6. Wide-scanXPSspectraindicatingtheelementalcompositions
ofthesolidsupport(MICA)andtwomodellipidfilm(BSST,VDT)samples……………………………………………..62
3.7. Thegraphandequationdepictingthecalculationsofthelipidfilmcoverage
forbothmodellipidfilmsamplespreparedusingvesicledeposition(VDT)
andbilayerself-spreading(BSST)techniques…………………………………………………………………………………………..64
3.8. XPSspectraintheN1sregionindicatingthepeaksassociatedwiththepresence
ofnitrogenonthesolidsupport(MICA),egg-phosphatidylcholine(EPC),sphingomyelin(SPM),
cholesterol(CHOL),dioleoylphosphatidylethanolamine(DOPE),
dioleoylphosphatidylserine(DOPS)andtwomodellipidfilm(BSST,VDT)samples………..……………………….66
3.9. ThegraphindicatingtheXPSspectraintheN1sregionformica,
singlelipidandmodellipidfilmsamples…………………………………………………………….…………………………………..67
3.10. XPSspectraintheP2pregionindicatingthepeaksassociated
withthepresenceofphosphorousonthesolidsupport(MICA),egg-phosphatidylcholine(EPC),
sphingomyelin(SPM),cholesterol(CHOL),dioleoylphosphatidylethanolamine(DOPE),
dioleoylphosphatidylserine(DOPS)andtwomodellipidfilm(BSST,VDT)samples…………………………………69
3.11. AFMimagespresentingtheinstancesofphaseseparationoccurrencewithinthemodelSLBs…….…………72
3.12. Thechemicalstructureofcholesterol(CHOL)……………………………………………….………………………….……………..73
3.13. Thechemicalstructuresofionicandnon-ionicmesomersofsphingomyelin(SPM)……..…………………………74
3.14. Illustrationofthe‘sphingomyelin(SPM)trap’hypothesis……………………………………………..………………………..75
3.15. Thechemicalandthree-dimensionalstructuresofDNA-deoxyribonucleicacid
andRNA-ribonucleicacidmolecules……………………………………………………………………………………………………..77
4. Chapter4:ModelSLB-ExcipientInteractionStudies.
4.1. Graphicalabstractindicatingtheexperimentalfocusofchapter4………………..………………………………………..81
4.2. AFMimagespresentingstepsoftheinteractionsbetweenmodelSLBs
ofdifferentbilayerqualitiesandPluronic®L-62monitoredovertime………….…………………………………………84
7
ListofFigures.
4.3. AFMimagespresentingstepsofinteractionsbetweenmodelSLBs
ofdifferentbilayerqualitiesandPluronic®L-64monitoredovertime….…………………………………………………85
4.4. AFMimagespresentingstepsofinteractionsbetweenmodelSLBs
ofdifferentbilayerqualitiesandPluronic®F-68monitoredovertime…….………………………………………………86
4.5. Schematicillustrationoftwobasicporetypesinalipidbilayer:hydrophilic andhydrophobic……………….87
4.6. Thegraphpresentingthechangesofthecoverageforpatch-like,modelSLBsovertime,
afterexposureto0.001%(w/v)Pluronics:L-62,L-64andF-68inPBS……..……………………………………………88
4.7. Thegraphpresentingchangesofthecoverageofporous,modelSLBsovertime,
afterexposureto0.0001%(w/v)Pluronics®:L-62,L-64andF-68inPBS…….……………………………………………90
4.8. Schematicillustrationofpossibleinteractionmechanismbetween
phaseseparatedSLBsandPluronics®ofdifferentpolyethyleneoxideblockcontent………………………………92
5. Chapter5:ModelSLB-NucleicAcidFormulationInteractionStudies.
5.1. Graphicalabstractdepictingthespontaneousformationof
DNA/sphingomyelin/cholesterol(DNA/SPM/CHOL)lipoplexes………………………………………………………………..94
5.2. TheAFMimagesandgraphspresentingthequalitative
andquantitativechangestotheuniform,modelSLBs:
before and after exposure to a 2.5 μg/ml DNA solution………………..………………………………………………………..97
5.3. TheAFMimagesandtheanalysisofL domainthicknessthroughout
O
the interaction of the model SLB and 5 μg/ml DNA, indicating three phases…………………………………………..98
5.4. TheAFMimagesandgraphspresentingthequalitative
andquantitativechangestothenon-phaseseparated,modelSLBs
over time after exposure to: 5, 10, 62 [μg/ml] DNA solutions……………………………………………………..…………100
5.5. Graphsillustratingthequantitativerelationship:DNAconcentrationvs.
theincreasesofSLBcoverageovertime………………………………………………………………………….……………………100
5.6. TheillustrationofthekeystepsoftheinteractionbetweenDNAandamodelSLB………………….……………102
5.7. TheillustrationofthesiRNA-cellinteractionmechanismbased
ontheAFM-observed19bpDNA-modelSLBinteraction………………………………..……………………………………..104
5.8. Anillustrationindicatingthedirectionsforfutureresearch
asanimplicationofthespontaneousDNA-sphingomyelin-cholesterol
(DNA/SPM/CHOL)lipoplexformationphenomenon……………………………………………………………………………..106
5.9. Theillustrationofapolyphosphoniumpolyplexwith19-bpDNA(right)
andthechemicalstructureofPPP-phosphoniumpolymer………..………………………………………………………..107
5.10. AFMimagespresentingthechangesinthebehaviourof
thenon-phaseseparated,modelSLBsafterexposureto:
5 (A), 10 (B), 50 (C) [μgDNA/ml] polyphosphonium polyplexes
with 19-bp DNA and 90 μg/ml phosphonium polymer solutions (D)….………………………………………………….108
5.11. Thegraphsillustratingthequantitativerelationship:DNAconcentration
vs.theincreasesofSLBcoverageovertimefor
themodelSLB-polyphosphoniumDNApolyplexinteractions………………………………………………………………..109
5.12. Theillustrationoftheanticipatedcelltransfectionmechanism
forpolyphosphonium-DNApolyplexes…………………………………………………………..……………………………………..111
6. Chapter6:DevelopmentofSLBMicroarrayforNano-HTSApplications.
6.1. Graphicalabstractindicatingthesupportedlipidbilayer(SLB)microarrayapproach
thathasbeenstudiedinchapter6………………………………………………………………………………………………………..114
6.2. ThegraphicsandAFMimagespresentingtheengineeringapproaches
fortheoptimisationofmicasheetpreparationbeforetheexposure tolipidmaterial
andlipidfilmmorphologiesrecordedforbothapproaches,respectively……………………………………………..118
6.3. AFMimagesindicatingthebehaviourofmodelSLBsoncetheliquidexcess
hasbeenremovedfromthesurfaceatthreedifferentrelativehumidity(RH)values:
~25%,~50%and~70%.…………………………………………………………………………………………………………..…………..122
6.4. TheopticalimageofanAFMprobeapproachingtheliquid-excess-free,
SLB-coatedmicasurfaceincubatedina~70%relativehumidityenvironmentatRT……………………………..123
8
ListofFigures.
6.5. Opticalimagespresentingink-jetprintedspotsonmicasheetssurfaces
incubatedat70%relativehumidityand28°Ctemperatureofthesamplestage..……………………..…………..125
6.6. TheIllustrationpresentingToF-SIMSchemicalimagesthathavebeencollected
fromglassslide-attachedmicasheetsoftiltedsurfacetopography……………………………….……………………..127
6.7. ToF-SIMSimagesofanSLB-Pluronic®interactioninamicroarrayformat……………………………………………..128
6.8. AFMimagesandillustrationdepictingtheFluidArray®surface-basedpreliminarystudies…….……………..130
9
ListofTables.
Chapter1:IntroductionandAims.
1.1 Examplesofdrug-membraneinteractionsofclinicalimportance……………………………………………16
1.2 Examplesofinsilico,invitroandinvivomodelsinpre-clinicaldrugdevelopment…………….……19
1.3 Experimentalandtheoreticalmodelsforstudyingdrug-membraneinteractions……………………20
1.4 Comparisonofcellmembranesandsupportedlipidfilms…………………………………...………..……….22
1.5 KeytechniquesemployedforSLBcharacterisationstudies…………………………………………………….23
1.6 ExamplesdemonstratingalterabilityofSLBproperties…………………………………………….…………….23
1.7 KeytechniquesusedtofabricateSLBs…………………………………………………………………………………….25
Chapter3:FabricationandCharacterisationofModelSLBs.
3.1 Thesinglelipid-representativepeakswithintheToF-SIMSC spectraofmodellipidfilms…………59
60
3.2 TheXPS-measuredlipidcontentratioswithinthemodellipidsfilms..……………………………….……….68
Chapter4:ModelSLBs-ExcipientInteractionStudies.
4.1 ThephysicochemicalandcellviabilityparametersfortherepresentativePluronics®.………..………83
10