1,721,041 research outputs found
Microcavity supported lipid bilayers; biomimetic models of the cell membrane
Full text linkBiomimetic models of the cell membrane are sought after as they have the potential to provide a realistic representation of an organism’s lipid bilayer. They can be used to understand lipid dynamics, signalling, drug permeability and membrane protein diffusion in an environment that is away from the complexity of the real living cell. This thesis examines the application of a new type of lipid membrane model, the micro-cavity supported lipid bilayer (MSLB), to study drug-membrane interactions and glycolipid containing bilayers using electrochemical impedance spectroscopy (EIS). Chapter 1 outlines the structure and function of the cell membrane and describes current models used to replicate the functions of the cellular bilayer. The limits of these models are also discussed particularly in the context of stability, lipid fluidity and addressability of both sides of the bilayer. The biomimetic MSLB system is then explored as a viable alternative in this thesis and is described in Chapter 2. 2.80 ± 0.04 μm diameter gold arrays were used and their surfaces were chemically modified to render them hydrophilic which aided the assembly of lipid bilayers using Langmuir Blodgett to form the initial monolayer and vesicle disruption to create the final bilayer structure.
This model is applied in Chapter 3 as a means of assessing drug plasma membrane interactions of two representative non-steroidal anti-inflammatory drugs; ibuprofen and diclofenac. These drugs were chosen as their log P values are well established and their interactions with membranes have been characterized by other methods. Their impact on the cavity array supported lipid membrane was investigated using EIS. Chapter 4 uses the MSLB model to study the interactions between the ganglioside, GM1, and disease relevant lectins by fabricating asymmetric GM1 containing lipid bilayer membranes. The influence of lipid/sterol composition on GM1-lectin recognition and aggregation was also considered.
Overall, this work demonstrates that, using EIS as the interrogation method, it is possible to sensitively explore interactions between external molecules and the lipid bilayer using these MSLBs. The MSLBs are a significant advance on current lipid membrane models as they permit accurate representations of cell membrane in elements of composition, fluidity, asymmetry and deep aqueous well on either side of the membrane
Integrin αIIbβ3: from platelet membrane to biomimetic models
Full text linkThe cell membrane is complex mixture of phospholipids, sphingolipids, sterols and proteins that combine to provide a semi-permeable barrier between the extracellular and intracellular environments, while also providing a functional role in cell-signalling, cell adhesion, and membrane transport. This complexity means that it is often better to study both the lipid and protein constituents of the cell membrane using artificial membrane models. Here, the transmembrane protein integrin αIIbβ3 was used as a model protein to be reconstituted into physiologically-relevant artificial lipid systems. αIIbβ3 is an integral membrane protein found in platelets and is a key mediator of thrombosis. Upon activation αIIbβ3 undergoes significant
conformational rearrangement, clustering, and ligand-binding to enable complex bidirectional signalling. It is this structural rearrangement, aggregation and ligand
binding that was the key focus of this project.
In chapter 2, and before reconstitution into artificial lipid models, αIIbβ3 was first studied in its native environment, the platelet. Here, DTT and Mn2+ were used to induce the activated form of αIIbβ3. It was found that both activators lead to structural changes in the integrin protein and varying degrees of platelet
aggregation, without the full range of response normally associated with physiological agonists. Chapter 3 focused on the production of αIIbβ3-reconstituted liposomes. It was found that integrin-ligand binding lead to a reduction in αIIbβ3 mobility, as well as integrin clustering. αIIbβ3 was also found to preferentially
excluded from cholesterol rich regions of lipid vesicles. Chapter 4 focused on the insertion of αIIbβ3 into a novel, cavity-spanning, lipid bilayer. Here, it was possible to determine αIIbβ3 diffusion co-efficients and induce protein aggregation. Finally, in chapter 5, cytoskeletal mimics were incorporated alongside a supported lipid bilayer in order to better imitate the conditions encountered by the cell membrane
The application of Ru(II) polypyridyl complexes to cellular imaging and sensing
Full text linkFluorescent microscopy is the key bio-imaging tool that is used to study live cells. Luminescent transition metal complex have been explored extensively for many years
across a range of applications from solar energy to molecular therapeutics, but it is only over the last decade that they have been seriously considered as cellular imaging probes. Their unique photophysical properties including large Stokes shift, red emission
wavelengths, good photostability, and sensitivity to molecular oxygen mean they are more than just contrast agents, making them invaluable in diagnostics and theranostics.
A key aim of this thesis was to drive forward the demonstrated applications of luminescent Ru(II) and Os(II) probes conjugated to cell penetrating peptides in cellular imaging and sensing using confocal microscopy, and the relatively new phenomenon of super resolution microscopy, specifically, STimulated Emission Depletion (STED) microscopy.
Chapter 1 overviews the general photophysical and optical of ruthenium and osmium polypyridyl complexes, examining the current status of their application in live cellular
imaging and sensing. Microscopy techniques such as confocal laser scanning microscopy, stimulated emission depletion (STED) microscopy and fluorescence lifetime imaging microscopy (FLIM) are discussed and how they are used alongside luminescent probes for cellular sensing.
Despite its NIR emission and high photostability Os(II) complexes have not been reported as general imaging probes before this report. Chapter 2 describes a novel Os(II) complex conjugated to the octa-arginine (R8) cell-penetrating peptide sequence as a cellular imaging probe, and compares it to its Ru(II) analogue. The photophysical
properties of [Os(bpy)2(pic-arg8)]10+ and [Ru(bpy)2(pic-arg8)]10+ are examined and compared, as well as its ability to be taken up by live CHO and Sp2 cells, examined by confocal microscopy.
Chapter 3 outlines a novel Ru(II) oxygen sensing probe, conjugated to the mitochondrial localising peptide sequence FrFkFrFk, in order to direct the complex to the mitochondria of live HeLa cells using confocal microscopy to confirm location. Fluorescent lifetime imaging microscopy (FLIM) allowed [(Ru(bpy)2phen-Ar)2-FrFKFrFK]7+ to measure oxygen concentrations and reactive oxygen species (ROS) from within the mitochondria of live HeLa cells.
Chapter 4 focuses on ratiometric oxygen sensing in live cells. Here, two probes are examined – a ratiometric molecule and a ratiometric core-shell nanoparticle. In both cases, the oxygen-sensitive probe consists of a Ru(II) complex, while the oxygeninsensitive reference probe is a BODIPY complex.
Finally, Chapter 5 describes the application of Ru(II) probes to STimulated Emission Depletion (STED) microscopy. Here, signal peptides conjugated to two novel Ru(II) probes directs them to the endoplasmic reticulum (ER) and nucleus of HeLa cells. The probes performance under confocal microscopy and STED is compared in the improved image resolution achieved as well as their photostability under the intense STED xii depletion laser. High resolution images of the ER and nuclear DNA during the stages of mitosis are captured using STED microscopy
Fluorescent probes for lipid droplet and lipid membrane imaging in cells and models
Full text linkCell membranes are believed to be laterally ordered into micro and nano-domains comprising of more fluid liquid-disordered (Ld) and more viscous liquid-ordered (Lo) phases. The latter subphases contain high concentrations of cholesterol and glycosphingolipids. These so call lipid rafts are experimentally distinguishable on the basis of their resistance to detergent solubilisation and are believed to play important roles in membrane function including in protein trafficking and signalling as they can drive protein-protein interactions through sequestering of proteins to these domains. Membrane domains in living cells are difficult to interrogate as they are dynamic and at sub-microscopic length scales they are outside the range of most conventional microscopies. However, they can potentially be imaged using recently developed super-resolution methods and as they are dynamic structures their diffusion can be measured using correlation methods. Therefore, new fluorescent probes are needed that can (a) partition selectively to membranous regions, (b) target the Lo and Ld phases selectively (c) that have appropriate photophysical properties compatible with the above techniques. These include large Stokes shift, high selectivity, excellent photostability, high molecular brightness, low cytotoxicity and high quantum yields. A key aim of this thesis was to design and synthesize new fluorescent probes that sequester specifically to lipid rich regions of cells or models and can distinguish Lo/Ld regions or lipid droplets, using confocal microscopy, fluorescence correlation spectroscopy (FCS), fluorescent lifetime imaging (FLIM) and the relatively new technique of super resolution microscopy, specifically, STimulated Emission Depletion (STED) microscopy
Microcavity PDMS and gold substrates for supported lipid bilayers
No full textCell membranes surround all living cells and are comprised of a complex matrix of phospholipids and proteins. The proteins embedded in or bound to the exterior of the membrane are responsible for a wide range of processes, for example cell signalling, and transport of material in and out of the cell. Understanding how transmembrane proteins behave within the lipid membrane system will allow for a better understanding of molecular mechanisms of diseases as well as the development more targeted therapeutics. However, due to the complex nature of the cell membrane environment, it is difficult to selectively study single protein species within the whole cell system. This has driven the development of model membrane systems, which allow for the sub-division of these complex systems into simpler forms and allow for the study of individual membrane proteins. Solid supported lipid bilayers have been widely used as model systems, however they have multiple limitations, the most important being the influence of the underlying substrate on the bilayer. This can impede lipid fluidity and is particularly detrimental to mobility of reconstituted proteins as substrate-protein interactions can impede motion and even cause protein to denature.
This thesis attempts to address this by developing substrates for studying membrane proteins in a biomimetic environment where such interactions are minimized. The initial substrates, designed for optical measurements, comprise of a microcavity array substrate formed in Polydimethylsiloxane (PDMS). A method for spanning bilayers over these PDMS microcavity arrays was developed and lipid diffusion dynamics over the cavities was assessed using Fluorescence Lifetime Correlation Spectroscopy (FLCS). Importantly, diffusion coefficients for lipids over these cavities are 2 to 3 times faster than on flat PDMS, and are more akin to diffusion rates normally observed in liposomes, indicating that the bilayer is minimally influenced by the underlying substrate.
In the second part of this thesis an analogous substrate and bilayer deposition method was developed using gold substrates with the objective of using electrochemical methods to address the bilayer or trigger events within the cavity.
Firstly lipid bilayers are spanned in a similar manner as developed for PDMS and the bilayer modified gold was characterised by electrochemical impedance spectroscopy (EIS). Incorporation of ion transporting molecules into the supported bilayers is also investigated by EIS. Finally a novel means of inducing electrically controlled release of reagent from inside the gold cavities to a lipid bilayer suspended across the cavity was developed using a ferrocene/cyclodextrin complex. To demonstrate this Streptavidin was released to a biotinylated lipid bilayer and its interaction with the bilayer was monitored using electrochemical impedance spectroscopy
Peptide-directed metal complex luminophores: candidates for photodynamic therapeutics
Full text linkDespite their potential to overcome critical limitations of conventional organic dyes, metal complex
luminophores have yet to be truly accepted as probes for cellular imaging and phototherapy. Longlived
and reactive luminophore excited states grant a sensitivity not currently achievable by organic
probes and offer the ability to efficiently photosensitise cellular toxicity. A barrier to their exploitation
to date has been their relatively poor uptake and unpredictable localisation, especially to important
theranostic targets like DNA. However, signal peptides are a powerful strategy towards achieving
precision-targeting of key organelles and were previously successfully implemented to deliver metal
complexes to the nucleus and mitochondria - two locales where cellular DNA resides. The
overarching aim of this thesis was therefore: to explore the candidacy of peptide targeted Ru(II)
luminophores for imaging and photo-destruction of DNA in live cells.
Two prominent Ru(II) complexes were established as candidate complexes to derivatise under the
scope of this work. The first was [Ru(bpy)2(dppz)]2+ - a molecular light switch for DNA that is nonluminescent
in water but switches on upon intercalating DNA. The second was [Ru(tap)2(bpy)]2+
- a
complex which possesses an excited state reduction potential sufficiently positive to photo-oxidise
and damage DNA. Chapter 3 explored efficient synthesis routes to conjugatable derivatives of Ru(II)
luminophores with a highlight being the development of a novel protocol to prepare tris-heteroleptic
Ru(II) complexes in unprecedented yield. Chapter 4 investigated the interaction of Ru-dppz
conjugates with DNA in vitro and in live cells, where remarkably, both nuclear and mitochondrial
DNA were successfully targeted permitting high resolution imaging of structure and cellular phase.
Phototoxicity was induced at higher irradiation intensities leading to cellular apoptosis. Chapter 5
investigated the photo-reactivity of a nuclear-targeted Ru-tap conjugate in live cells where singlet
oxygen independent photo-oxidation of DNA led to photosensitised destruction of HeLa cells with
spatiotemporal control. Finally, Chapter 6 explored additional imaging and biophysical applications
of Ru(II) luminophores
The synthesis and characterisation of inorganic and organic luminophores suitable for biomolecule conjugation
Full text linkInorganic transition metal complexes have been under extensive investigation for many years in supramolecular assemblies due to their favourable photophysical and redox properties including; absorbance and emission in the visible region of the spectrum, large stokes shifts, long lifetimes, intense luminescence, good photostability and useful photosensitising properties for photodynamic therapy. Their properties make them potentially very valuable biological probes but to date relatively little application of transition metals in this area have been made. This thesis focuses on a range of novel ruthenium and iridium luminophores, their bioconjugates and nanoparticle conjugates which were prepared for applications in cell imaging. A key aim of this thesis was the synthesis, characterisation and identification of novel bioconjugates suitable for applications in cellular imaging. Some preliminary studies of their application in cell imaging are also presented.
Chapter 1 outlines how metal complexes have been used previously in cellular imaging and how conjugation of these transition metal complexes to biomolecules has lead to more targeted and improved applications in medical diagnostics, photodynamic therapy, cellular imaging and pharmaceutical drug delivery. Chapters 3 & 4 detail the synthesis and photophysical characterisation of a series of Raman and oxygen sensitive, water soluble and water insoluble ruthenium (II) and novel iridium (III) polypyridyl complexes suitable for biomolecule coupling. Following conjugation of these luminophores to gold nanoparticles in Chapter 3 and cell penetrating peptides in Chapter 5, the dye-conjugates were shown to transport efficiently across the cellular membrane of mammalian SP2 and CHO cells and locate throughout the cell’s organelles. Whereas, using confocal fluorescence microscopy, the parent complexes were shown not to internalise within the cellular structures. The inherent properties of the dyes, such as Raman and lifetime sensitivity, may then be used to determine pH and oxygen levels inside the cell. This could provide critical information for the early detection of certain diseases, as abnormal pH and oxygen levels are indicative of cancerous tumours. Furthermore, the generation of singlet oxygen following light absorption by the luminophores is known to cause additional cell apoptosis.
Finally, Chapter 6 describes attempts to functionalise the nucleobase guanine with a fluorescent fluorescein molecule through a short and rigid linker. A range of synthetic
techniques such as Suzuki coupling, Sonogashira coupling, click chemistry and Buchwald-Hartwig coupling were used in an effort to achieve this. Using DNA as a
scaffold for the first time, the modified nucleoside may be incorporated into the sequence of DNA which may be surface immobilised. Thus, providing an efficient light harvesting supramolecular assembly for the conversion of solar energy into electrical potential
Interfacial electroactive assemblies: from molecular electronics to biological applications
Full text linkSpontaneously adsorbed monolayers of di-6A, 6B-deoxy-6-(4-pyrid-ylmethyl)amino-ƴ-cyclodextrin (ƴ-CD-(py)2) were formed on platinum electrodes. AC voltammetry showed significantly lower capacitance values for electrodes exposed to ƴ-CD-(py)2 solutions overnight compared to bare electrode values. Co-adsorption of 1-nonanethiol in the
presence of a 10-fold excess of cavity guest 1-adamantylamine created layers which exhibited greater blocking ability to the solution phase probe [Fe(CN)6]4−. Complete blocking was achieved by insertion of a high-affinity guest 1-adamantylamine into the cavity. Raman spectra of the ƴ-CD-(py)2/1-nonanethiol layer exhibited features associated with both pyridine-functionalised CD and alkane moieties. Significantly, co-adsorption of 1-nonanethiol dramatically effected the ability of the
ƴ-CD-(py)2 layer to complex the electroactive, high affinity guest, [Co(biptpy)2]2+. A redox response for the Co2/3+ couple was not observed at the pure ƴ-CD-(py)2 layer, but the molecular recognition properties were turned on by co-adsorbing the alkanethiol molecules with the
CD layer. The binding of [Co(biptpy)2]2+ to co-adsorbed monolayers depends on the bulk concentration of guest and was modelled by the Langmuir isotherm, yielding a free energy of adsorption, △Gads, of -29 kJ.mol−1 for the Co2+ state and a limiting surface coverage 1.49 ± 0.25
x 10−11 mol.cm−2. The rate of electron transfer from the cobalt metal center to the electrode surface was found to be of the order of 1 x 105 s−1 by high speed chronoamperometry.
Molecular junctions incorporating monolayers of
ƴ-CD-(py)2, co-adsorbed with 1-nonanethiol have been formed by bringing macroscopic platinum and mercury electrodes together. The mercury electrode was either modified with an alkanethiol layer for bi-layer junction formation,
or remained unmodified for monolayer junction formation. The junctions were characterised by determining the effect of junction thickness on the magnitude of the tunnelling current through alkanethiol layers. A tunnelling co-efficient,β, of 0.88 ± 0.01 per carbon atom was determined for these alkanethiol bilayer junctions. Significantly, for bilayer junc tions incorporating CD layers, the tunnelling current depends markedly on the nature of the CD guest. Junctions where nonconjugated guests, such as 1-adamantylamine, were included in the CD showed an order of magnitude lower current than junctions incorporating the conjugated guest C60. Moreover, monolayer junctions of CD backfilled with 1-nonanethiol exhibited potential-dependent currents in the presence of
CD guest molecule [Co(biptpy)2]2+ but not for [Co(tpy)2]2+, which is structurally analogous but cannot associate with CD. The effect of electrode displacement on these potential dependent currents indicated a redox cycling or electron hopping mechanism of electron transport.
Fibrinogen has been adsorbed at planar and 820 nm nano-cavity gold surfaces. AC voltammetry showed that the capacitance values of electrodes exposed to fibrinogen solutions overnight were lower, by 30 ±5 μF.cm−2, than those seen at bare gold electrodes. AFM of the protein at the planar surfaces showed a fibrous network of adsorbed protein. Oregon Green labelled fibrinogen layers were imaged using fluorescence microscopy at both the planar and nano-cavity surfaces. The effect of electrode potential on the fibrinogen layer was investigated. It was found that the protein desorbed at a potential of -1.2 V. The rate of
this desorption process was investigated by capacitance studies, which showed a two stage desorption process from the planar surface, where k1 = 0.400 ± 0.065 s−1 and k2 = 0.011 ± 0.001 s−1. The desorbed protein was collected in solution and UV-visible spectroscopy studies showed
that 6.3 x 10−13 mol.cm−2 of protein is desorbed. SDS-PAGE gel electrophoresis studies showed that the desorbed protein was fragmented by the adsorption-desorption process. Selective modification of the nano-cavity arrays resulted in localisation of the protein predominately inside
the nano-cavities. The desorption of the dye labelled protein was investigated using fluorescence microscopy at both the planar and nano-cavity surfaces. The diffusion of the protein out of the confocal laser volume was seen to be slower at the nano-cavity surfaces, compared to the planar surface, indicating that the exit of the protein from the cavities is a rate limiting step. An increase in the fluorescence signal was observed at the nano-cavity surface and an enhancement factor of 500 is estimated
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