Peptide Mediated Nanoparticle Assembly investigated using Small Angle X-ray Scattering

155 Pages·2012·20.79 MB·English

Peptide Mediated Nanoparticle
Assembly investigated using
Small Angle X-ray Scattering
Piotr Gryko
Thesis submitted for degree of
Doctor of Philosophy
Supervisors:
Dr Mary Ryan
Professor Molly Stevens
Department of Materials
Imperial College London
12 2012
th March
PiotrGryko: PeptideMediatedNanoparticleAssemblyinvestigatedusing
SmallAngleX-rayScattering,DoctorofPhilosophy, 12thMarch2012
Agoodtravelerhasnofixedplans
andisnotintentonarriving
—LaoTzu
Toloveistorecognizeyourselfinanother
—EckhartTolle

ABSTRACT
May you live
Nanoparticle systems arranged through bio-chemical self-assembly
in interesting
mechanismsdisplaynovelelectronicandopticalcharacteristicsapplic-
times
abletobiosensing,tissueengineering,therapeuticsanddrugdelivery.
-Ancient
Metallic Nanoparticles such as those composed of gold or CdSe are
Chinese pro-
expected to replace standard chemical fluorophore due to their high
verb
optical scattering cross-section. The colloidal nature of nanoparticles
necessitatestheengineeringofNPssurfacechemistrytocontrolparticle
stabilityandassemblyorder.Thehighintensityofmodernsynchrotron
sources,combinedwiththehighelectrondensitycontrastofmetallic
NPs,allowsforfast,sensitive,in-situsynchrotronmeasurements.Here
Small Angle X-ray scattering is used to observe nanoscale structural
changesandrelatethemtochangesinpolypeptidemediatedinteraction
potential,particlesurfacechargeandmobilitywithinaggregates.These
results enable strategies for optimising the structure and the optical
characteristicsofNPassemblies,whicharevitalfortheirperformance
ascolourimetricsensors.HerethreeNPsystemsareinvestigatedand
characterised:
(1) Gold nanoparticles assembled using de novo designed poly-
peptides developed by D. Aili (Chapter 3). The assembly is achieved
by the folding dependent bridging of the particles induced by the
heteroassociationofimmobilisedhelix-loop-helixpolypeptidesanda
complementarynon-linearpolypeptidefoundinsolution.Changesin
theopticalpropertiesoftheassemblyasafunctionofparticlesizeand
linker concentration are observed, with SAXS used to obtain in-situ
structural information on the aggregates. The particles are found to
assemble into highly ordered close packed structures with an inter-
particlespacingof4.8±0.4nm,correspondingtothelengthofafolded
polypeptide.Changesinparticleordering,aggregationdynamicsand
massfractaldimensionareobservedasafunctionoflinkerconcentra-
tionandparticlesize.Thesetransitionsarerelatedtochangesinlinker
mediated interaction potential, particle surface charge and mobility
withintheaggregates.
(2) Interaction of BSA with AuNP aggregates (Chapter 4). Bovine
serumalbumin(BSA)isavitalcomponentofstandardimmunological
assays and it is often extended for use in de-novo assays based on
peptidemodifiedmetallicnanoparticles.Whilstawiderangeofplasma
proteins such as BSA and HSA have been shown to associate with
nanoparticles, there is little work done on the effect of BSA on NP
assemblies,whicharehighlydependentonparticlestability.Herethe
effect of BSA on AuNP aggregates is characterised, using stability
assays, DLS, UV-VIS and SAXS. Far from being an inert component,
the introduction of BSA directly alters the structure and interaction
of AuNP aggregates. Temperature is observed to be a factor in these
interactionswithaspecificaffinityforhydrophobicsystems.
(3) A Quantum Dot - Gold Nanoparticle conjugate assay for the
detection of urokinase plasminogen activator (uPA) (Chapter 5). Pro-
teolytic enzymesare usedas diseasebiomarkers forseveralformsof
cancer. The surface assembly of gold nanoparticles onto the surface
of quantum dots is demonstrated, with the growth in conjugate size
v
characterisedusingin-situSAXS.Completeconjugatedisassemblyis
observedupontheadditionoftheenzymeuPA,howeverQDslumines-
cencedoesnotfullyrecover.Thisindicatesthatothereffectsmayresult
inthepermanentquenchingofquantumdots.
Theworkpresentedhereisdesignedtoguideinthesynthesisofgen-
eralprinciplesforassemblyofNPsbybiomolecularmediatedbridging,
whilstcontributingtothefundamentalunderstandingoftheassembly
process.
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PUBLICATIONS
TheworkdescribedinChapter3hasbeenpublishedinNanoletters:
PolypeptideFolding-MediatedTuningoftheOpticalandStructural
PropertiesofGoldNanoparticleAssemblies-DAili,PGryko,BSepul-
veda,JDick,NKirby,RichardHeenan,LarsBaltzer,BoLiedberg,Mary
P.RyanandMollyM.Stevens-NanoLetters,2011-ACSPublications
Theworkpresentedinlaterchaptersiscurrently(asofsubmission)
intheprocessofbeingpreparedforpublishinginresearchjournals.
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Acceptthefactthatnothingiscertainandnolawisfixed,
beasfluidandformlessaswater,
neverbetonstabilityorlastingorder.
Everythingchanges.
—RobertGreene[71]
ACKNOWLEDGMENTS
Iamextremelygratefulfortheextensivesupportandcheerfulenthusi-
asmofDrMaryRyanduringthepastfouryears.Withoutyoursupport
IwouldhavedroppedoutofthePhD;ithasbeenanhonourtowork
withyou.IwouldliketothankImperialCollegeandProfessorMolly
Stevens for the support and opportunity to study for a PhD and the
EPSRCforfunding.Themultidisciplinarybiochemistryaspectsofmy
workwerebasedontheexperimentalsystemsdevelopedbyDrDaniel
Aili and Mr John Dick. Much of this thesis came from collaborative
workwiththemandIamgratefuloftheirsupport.
The opportunity to travel during my PhD provided experiences
for personal growth and was immensely enriching. I would like to
thanktheJapaneseSocietyforthepromotionofscience(JSPS)forthe
opportunitytotraveltoJapanandDrTamotsuZakoandProfMaedafor
theirhospitalityandexcellentscientificsupportattheRikenInstitute
(Tokyo).
All of the synchrotron measurements in this thesis were possible
duetothehighperformanceSAXS/WAXSbeamlineattheAustralian
synchrotron. The beamline scientists provided outstanding support,
oftenworkingintotheearlyhoursofthemorning.Withouttheirhelp
thisthesiscouldnothavebeenwritten.
I would like to thank Prof Ian Robinson for his extensive supervi-
sionduringmymastersprojectandsummerresearchinternship.Your
supportencouragedmetopursueaPhD.
Myclosefamilyandfriendshaveprovidedkindsupportoverthese
years;thankyouKristinaforyourpatienceandlove.
FinallyIwouldliketothankmyparentsfortheirstrength,loveand
support over the decades. My achievements have grown out of your
strengths.
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