Polymer chemistry enables the design and development of synthetic cationic gene delivery systems with varying polymer architectures. Branched polymers have been shown to have advantages for drug delivery purposes including nucleic acid delivery. The objective of this work is to utilise advanced polymer synthesis methods to synthesise a range of cationic polymers with well controlled branched architectures and investigate their cytotoxicity, nucleic acid complexation, resulting polyplex morphology, and gene transfection efficiency. Firstly, the synthesis of hyperbranched polymers using thiol-yne chemistry is explored with a semi-batch process to form hyperbranched polymers with well-defined molecular weights and dispersities. Following this, cationic moieties are introduced onto thiol-yne hyperbranched polymers using the ring opening polymerisation of 2-ethyloxazoline and an additional hydrolysis step to form PEI-POx copolymers with hyperbranched architectures. An investigation of plasmid DNA complexation, and in vitro toxicity and GFP plasmid gene transfection is then conducted. RAFT polymerisation is then utilised to form highly branched polymer architectures by copolymerisation of a divinyl branching comonomer. This strategy has the advantage of being able to introduce tuneable degradation and nucleic acid release. Finally, the possibility of using RAFT to synthesise branched polymers with phosphonium cationic moieties is also investigated, and their DNA complexation, toxicity, and gene transfection efficiency compared to the equivalent cationic polyammoniums. Overall, this thesis describes a number of advanced polymer synthesis methods to create hyperbranched and highly branched cationic polymers suitable for nucleic acid complexation, and also investigates their structure-function characteristics relating to aspects of nucleic acid delivery.

This thesis explores the nucleophilic thiol-yne reaction as a crosslinking method for the synthesis of hydrogel materials under biologically relevant conditions. The reaction, using simple functional groups, can be carried out without the use of an external catalyst. This thesis aims to portray the immense potential this reaction has in creating hydrated polymer networks for a wide range of biomedical applications. In the review of the literature (Chapter 1), the popularity and future of hydrogels in tissue engineering has been discussed and the advantages of using alkyne functional groups to crosslink polymers has been highlighted. The main aim of this thesis is to further develop the nucleophilic thiol-yne reaction and to prepare poly(ethylene glycol) (PEG) hydrogel materials with superior performance for application as tissue engineered scaffolds (e.g. extracellular matrix (ECM) mimics or injectable scaffolds). This aim has been approached through a variety of experimental pathways in this thesis demonstrating the suitability of this reaction in the biomaterials field. In Chapter 2, the nucleophilic thiol-yne reaction has been presented as a highly efficient chemistry for producing robust, high water content hydrogels which could be repeatably compressed without hysteresis. Through a straightforward blending process of PEG thiol precursors, the material properties were easily tuned to a range of relevant biological environments. In a similar manner, using the PEG precursors to tune the resultant properties, Chapter 3 addresses the swelling profiles of the thiol-yne hydrogels. By increasing the number of hydrophobic crosslinking points within the networks, nonswelling, cytocompatible hydrogel material were created when immersed in aqueous environments. The monoaddition product of the nucleophilic thiol-yne reaction results in a vinyl thioether bond which can favour different isomers, depending on the reaction conditions. To exploit this in hydrogel synthesis, Chapter 4 describes the formation of sterecontrolled hydrogels. Significantly, an impressive range of mechanical properties was achieved, without affecting the structure or swelling behaviour of the materials. To achieve a structure with more advantageous properties (e.g. self-healing and stretchability) thiol-yne interpenetrating networks (IPNs) were synthesised through the inclusion of natural polymer hydrogels (Chapter 5). These IPNs achieved the advantageous properties required in a simple and effective manner, while retaining the characteristics already exhibited by these materials. To improve on this aim, the thiol-yne PEG hydrogels successfully encapsulated breast cancer cells with enhanced viability compared to the widely used radical thiol-ene reaction (Chapter 6). Controlled matrix degradation allowed for cell proliferation and the formation of cell clusters. Chapter 7 investigates the kinetics of the nucleophilic thiol-yne reaction with different activating groups (e.g. adjacent group to the alkyne), to reduce the toxicity of the PEG alkyne precursors and degradation of the resultant thiol-yne hydrogels. This chapter highlights key requirements of the functionalisation reaction to form alkyne and thiol precursors for successful hydrogel synthesis. Chapter 8 provides a summary of the key findings from Chapters 2-7 and Chapter 9 states the experimental procedures of this thesis.

The objective of this work is to investigate and expand the use of methacrylic macromonomers as chain transfer agents. Although chain transfer activity had been demonstrated previously, the limits of the technique have not been fully explored. As such, a new approach for the efficient synthesis of methacrylic polymers in emulsion is presented, aiming at fully exploiting the vinyl end-group of the CCTP-derived macromonomers and consequently their chain transfer activity. Moreover, the preparation of higher MWt copolymers as well as more complex structures (e.g. triblocks etc) by this method will be investigated as research so far has only been focusing on certain degrees of polymerisation, mainly resulting in diblock copolymers of relatively low MWt. In addition, macromonomers based on diverse methacrylic monomers will be employed, as most studies to date have focused on a narrow monomer pool. In parallel, another aspect of radical polymerisation in the presence of macromonomers is the livingness of the system. Even though living-like characteristics have been observed, previous studies did not reach definitive conclusions, according to the generally set criteria of livingness. At the same time, the use of macromonomers as precursors for comb-like polymers will be described. Despite the technique being known and well-reported, the aim is to successfully employ solvents that satisfy the needs of automotive applications, such as mineral oil. In detail, both the macromonomer synthesis and the subsequent comb formation will be attempted in this solvent. A similar approach has not been reported so far. It needs to be noted, that this part is an ongoing work with the Lubrizol Corporation and as such it only demonstrates a few initial steps towards developing materials with interesting properties and applications.

Reversible addition fragmentation chain transfer (RAFT) polymerization is a very versatile way to generate synthetic polymeric materials. Multiblock copolymers have received enormous scientific interest recently due to the ability to mimic the sequence-regulated microstructure of biopolymers. The objective of this thesis was to investigate RAFT polymerization and explore its potential in the synthesis of sequence-controlled multiblock polymeric chains, and their use to tune the micro-structure of the polymers, engineer single chain polymeric nanoparticles, and fabricate functional polymeric nanomaterials. This work firstly addresses the investigation of the enormous ability of sequence-controlled multiblock copolymer to tune the physical properties by altering their microstructure. A series of sequence controlled multiblock copolymers were synthesized by RAFT polymerization using ethylene glycol methyl ether acrylate and tert-butyl acrylate as monomers. These block copolymers were synthesized with an alternating order of the two monomers with a similar total degree of polymerization. The number of blocks was varied by decreasing the length of each block while keeping the ratio of monomers constant. Their microphase separation was studied by investigating the glass transition temperature utilizing differential scanning calorimetry analysis. Small angel X-ray scattering analysis was also applied to investigate the transition of the microphase separation with the variation of the segmentations of these multiblock copolymers. The study found the microstructure was significantly affected by the number of segments of the polymer chain whilst keeping the total length constant. Having demonstrated the enormous potential of sequence controlled multiblock copolymers to access defined microstructures, further studies were focused on mimicking the controlled folding process of the peptide chain to a secondary and tertiary structure using sequence controlled multiblock copolymers. RAFT polymerization was used to produce multiblock copolymers, which are decorated with pendant cross-linkable groups in foldable sections, separated by non-functional spacer blocks in between. An external cross linker was then used to cause the folding of the specific domains. A chain extension-folding sequence was applied to create polymer chains having individual folded subdomains. In order to achieve a further step on the way to copy nature’s ability to synthesize highly defined bio-macromolecules with a distinct three dimensional structure, linear diblock copolymer precursors were synthesized by RAFT polymerization. One block of the precursor with pendant functional groups was folded using an external cross-linker to form tadpole-like single chain nanoparticles (SCNPs). These tadpole-like SCNPs could then self-assemble into a more complex stimuli responsive 3D structure by adaptation to environmental pH changes. The stimuli responsive assemblies were found to be able to dissociate responding to low pH or exposure to glucose.

This thesis is concerned with the development and application of novel theoretical and experimental methodologies to study crystal dissolution processes across multiple lengthscales. In particular, it presents a versatile in situ multimicroscopy approach, comprising atomic force microscopy (AFM), scanning ion-conductance microscopy (SICM), and optical microscopy (OM) that is readily combined with finite element method (FEM) simulations. The methodology permits the quantitative 3D visualization of microcrystal morphology during dissolution with well-defined, high mass transport rates, enabling both the measurement of face-dependent dissolution rates and the elucidation of the dissolution mechanism. The approach also allows the determination of interfacial concentrations and concentration gradients, as well as the separation of kinetic and mass transport limiting regimes. The high resolving power and versatility of this new methodology is demonstrated on four different crystalline compounds with very different characteristics. First, the dissolution kinetics of individual faces of single furosemide microcrystals are investigated by OM-SICM and FEM modeling. It is found that the (001) face is strongly influenced by surface kinetics, while the (010) and (101) faces are dominated by mass transport. Dissolution rates are shown to vary greatly between crystals, with a strong dependence on crystal morphology and surface properties. A similar approach is then used to investigate changes in both crystal morphology and surface processes during the dissolution of bicalutamide single crystals, achieving high resolution with in situ AFM. It is shown that dissolution involves roughening and pit formation on all dissolving surfaces, and that this has a strong influence on the overall dissolution rate. FEM simulations determine that mass transport contributions increase as dissolution proceeds due to a continuous increase of the intrinsic dissolution rate constant, promoted by the exposure of high index microfacets. The methodology is further developed to show that kinetic data obtained from OMSICM and AFM, which provide differing measures of kinetic parameters, are in good agreement when the different mass transport regimes of the two experimental configurations are accounted for. The robustness of the methodology is verified via studies of L-cystine crystals, while also providing insights into the dissolution mechanism by visualizing hexagonal spirals descending along screw dislocations. Finally, the ability of the methodology to characterize processes with fast surface kinetics is demonstrated by the study of the proton-promoted dissolution of calcite single crystals. The approach allows the accurate determination of the near-interface concentration of all species during dissolution, as well as the intrinsic dissolution rate constant of the {104} faces, showing that surface kinetics play an important role in the dissolution process. Overall, this methodology provides a significant advance in the analysis and understanding of dissolution processes at a single crystal level, revealing the intrinsic properties of crystal faces and providing a powerful platform from which future studies can be developed.

Studies of the kinetics and mechanisms of the dissolution and growth of crystals and other solids are beneficial in many areas of science. In pharmaceutical science, dissolution testing is a key quality control procedure used to determine the rate at which an active pharmaceutical ingredient (API) is released and is thus available for absorption in the gastro-intestinal tract. However, the general processes governing the dissolution and growth of crystals are poorly understood despite many years of study. This thesis focuses on the implementation of various microscopy and electrochemical techniques as a novel approach to further understand the dissolution and growth of API crystals and amorphous solids. The motive of the first part of the thesis, was the use of atomic force microscopy (AFM) to obtain new insight into API dissolution and growth from both the crystalline form and amorphous solid state. Studies of the crystalline API, bicalutamide have focused on measuring the 3D morphological changes of individual microcrystals in aqueous solution, in real time, from which the intrinsic dissolution rates of each crystal surface exposed to solution have been extracted. In addition, with finite element method (FEM) modelling, interfacial concentrations around the dissolving crystal have been obtained, allowing the elucidation of the kinetic regime of the overall dissolution reaction. A major conclusion of this work is that the dissolution kinetics accelerate significantly during the process, due to changes in nanoscale features on the surface. AFM was then used to examine targeted regions of dissolving amorphous solid dispersions (ASDs), comprising of felodipine API and the water-soluble polymer copovidone, in aqueous solution, together with a localized electrochemical-droplet (flux measuring) technique and Raman spectroscopy. This multi-microscopy approach allowed real-time information about initial API release rates, and changes in solid-state composition and morphology during dissolution. This thesis then transitions to the study of nanocrystallization of APIs using nanopipettes under electrochemical control in a nanoscale anti-solvent configuration using bicalutamide, as an example system. A key feature of the technique is that a bias between an electrode in the nanopipette, and one in bulk solution, can be used to control the supersaturation level at the end of the nanopipette and the current-time response detects nucleation and growth events. Using Raman microscopy the formation of the least stable crystal polymorph of Form II was demonstrated. To highlight the generality of nanopipette-based electrochemical techniques, a final results chapter reports the use of scanning electrochemical cell microscopy (SECCM) to study the electro-oxidation of nicotinamide adenine dinucleotide (NADH), on various carbon electrodes, showing how active surface sites are readily identified and quantified.

Identifying and treating infectious diseases remains a challenge for modern healthcare professionals. Proper identification of infectious diseases and understanding of the means of infection will allow for optimal use of antibiotics and the development of alternative therapies such as anti-adhesion therapy. It is therefore important to develop tools that can probe the processes involved in infection, or that can be used as point of care diagnostics. In vivo glycosylated surfaces are inherently heterogeneous, increasing the complexity of the interactions that take place, and with a corresponding increase in analytical difficulty. Glycopolymers and glycosylated nanoparticles are ideal methods for incorporating synthetic functionalisation into a biological setting to probe interactions between glycosylated surfaces and carbohydrate recognising proteins (lectins). This work utilises heterogeneously glycosylated polymers to probe the inhibitory and kinetic activity of the polymers towards various lectin targets. We see further evidence of the “heterocluster effect” whereby nominally non-binding sugar epitopes give rise to faster association rates and increase overall residence time of bound lectins to the polymers. Highly coloured heterogeneously glycosylated gold nanoparticles are used to develop a high throughput screening library for the identification of binding patterns with lectins that could lead to use as an identification system of unknown lectins. Finally, unnatural azide containing sugars are used to metabolically label the surface of A549 carcinoma cells and tagged using fluorescent polymers. This system provides a robust way of introducing polymeric functionality onto the surface of cells, opening the ability to probe in-depth the cell surface.

This thesis presents investigations into the nature, properties and distribution of defects in diamond grown by the methods of chemical vapour deposition (CVD) and high pressure, high temperature (HPHT) synthesis. The experimental techniques used include electron paramagnetic resonance, optical absorption, cathodoluminescence, photoluminescence and secondary ion mass spectroscopy (SIMS). The optical spin polarisation of the neutral silicon vacancy defect (SiV0) was shown to be strongly enhanced by resonant excitation at the zero-phonon energy, although there was significant sample to sample variation in the magnitude. The spin polarisation mechanism is different to that observed for the negatively charged nitrogen vacancy defect in diamond and more than one mechanism may be generating spin polarisation. The spin-lattice relaxation time (T1) of the SiV0 ground state was found to change by six orders of magnitude between room temperature and 11 K, where T1 exceeded 25 seconds. At room temperature the achievable optical ground state spin polarisation is limited by the rapid spin-lattice relaxation. Irradiation and annealing studies of silicon doped CVD diamond samples showed that the silicon vacancy concentration can be increased by irradiation and annealing. However, the same processing conditions can also reduce the concentration of grown-in silicon vacancy defects. This work suggests that the relative incorporation efficiency of silicon in different forms in homoepitaxial CVD diamond may depend on the orientation of the substrate, and that the details of post growth silicon vacancy defect production, especially in boron doped diamond, are not yet well understood. HPHT samples in which the13 C isotopic abundance had been increased up to approximately 10% were studied. The variation of the abundance of13 C with distance from the seed was studied using Raman spectroscopy and SIMS, and the nitrogen incorporation by infrared microscopy. Possible explanations of the variations in both are discussed. The incorporation of point and extended defects into diamond grown by heteroepitaxial CVD was studied in a nitrogen doped sample and another grown with efforts to exclude nitrogen. The samples were highly birefringent when observed through cross-polarisers and exhibited strong dislocation related photoluminescence, suggesting significant concentrations of dislocations and dislocation bundles. The nitrogen doped heteroepitaxial CVD sample contained point defects in relative concentrations typically observed in nitrogen doped homoepitaxial CVD diamond; the total nitrogen impurity concentration exceeded 2,000 ppb, whereas in the intrinsic heteroepitaxial CVD sample it was less than a few ppb. Both samples contained a significant concentration of silicon vacancy defects and the photoluminescence spectra indicated that the point defects were subject to significant strain arising from both extended and point defects.

Responsive polymer-based gold nanoparticles are capable of altering their unique optical properties, chemical and/or physical properties upon exposure to external stimuli, which allows these materials to use in a diverse range of biomedical applications. Herein, the use of temperature responsive polymers and glycopolymers functionalised gold nanoparticles is given as sensors and biosensors, for controlled and triggered target detection. Chapter 2 investigates the transition characterisation and thermal aggregation co-operative behaviour of thermo-responsive polymer conjugated gold nanoparticles for a detailed understanding of fundamental features to apply in a biosensing system. Chapter 3 develops an optical, gold nanoparticle-based biosensor for the detection of specific biological target. Both temperature responsive and molecular recognizable polymers co-coated gold nanoparticles that change colour by protein-carbohydrate interaction mediated interparticular aggregation are controllable at desired condition. Optimisation of the particle coating is very essential to enhance the sensitivity and specificity of the biosensing system. Finally, this strategy is then extended and improved in Chapter4, with a larger glycans for probing control over the expression of particle surface glycans triggering carbohydrate-carbohydrate interactions with high specificity and selectivity, effectively. In summary, functionalised polymers and gold nanoparticles have been synthesized and developed as a biosensor. These gold nanoparticle conjugates may promise a powerful solution for rapid and reliable identification of disease and point-of-care treatment in modern healthcare issue.

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