Cerebral ischaemia, traumatic injury to the brain, inflammatory neurological disorders and HIV infections are amongst the most prevalent causes of neurodegeneration. Neuroprotective strategies are usually to limit the progressive secondary injury that generally occurs, thus limiting overall tissue damage. Neuroprotective strategies are usually to limit the progressive secondary injury that generally occurs, thus limiting overall tissue damage. Sodium channel blockers have been often used for this matter as they prevent the cascade of events culminating in free radical generation and eventually neuronal apoptosis. Newer compounds, such as antiperoxidants and free radical scavengers, show encouraging experimental results, but their clinical use is still very limited. Phenytoin being a popular drug in the treatment of epilepsy has also been used as a neuroprotectant during certain neurological emergencies and in pharmacological prophylaxis of post-traumatic epilepsy. Furthermore this agent functions by prolonging inactivation of voltage gated sodium channels. In these sets of experiment the neuroprotective properties of phenytoin were examined. The histological study revealed that phenytoin confers protection to the CA1 and CA3 regions of the hippocampus under the insult of QUIN. Cells maintain their characteristic shape and minimal tissue necrosis occurs in the presence of this agent. The in vitro effect of this antiepileptic drug on free radicals generation shows that phenytoin does not reduce or prevent the formation of these reactive species. Lipid peroxidation was induced using QUIN and iron (II), two known neurotoxins. The study reveals that only lipid peroxidation induced using iron (II) is reduced by phenytoin. These experiments were carried out in whole rat brain homogenate. These studies show that phenytoin possesses poor free radical scavenging properties. However, the dose-related reduction of iron-induced lipid peroxidation allows for speculation that phenytoin interacts with iron in order to reduce neuronal damage. Metal binding studies were performed using UV, IR and electrochemical analysis to examine the interaction of phenytoin with iron (II) and iron (III). Phenytoin, when added to iron (II) in solution, first oxidises the latter to iron (III) and maintains it in that form. A shift in the peak was observed in the UV spectrum when iron was added to phenytoin. Moreover, electrochemical studies indicate that the interaction between the metal and the ligand is very weak. The IR analysis it shows that phenytoin may be coordinating with iron through the Nitrogen atom on the phenytoin molecule. These studies show that phenytoin maintains iron in its oxidised form, which is a good property to possess as a neuroprotectants. Pineal organ culture showed that phenytoin does not increase melatonin production but slightly and non-significantly reduces the levels of this pineal hormone. However there is a significant rise in precursor NAS levels. As melatonin is known to possess antioxidant and free radical scavenging properties, this could mean that this drug can cause the CNS to become more susceptible to attacks by reactive oxygen species.

Osteoarthritis (OA) is a complex, multifactorial, and slowly progressive disease where there is currently no effective medical treatment. Research in understanding the mechanisms of OA has been advanced by preclinical studies in rodent models of OA. Recent evidence highlights the role of different classes of lipids in OA pathogenesis. Therefore, the main aim of this thesis was to apply both targeted and untargeted (global) lipidomics mass spectrometry based an alytical methods, in conjunction with univariate and multivariate statistical analysis, in various tissues from three established rodent models of OA; meniscal transection (MNX), monosodium iodoacetate (MIA), and destabilization of the medial meniscus (DMM). The overall goal was to identify statistically differentiated lipids between controls versus OA rodents that may reflect changes in the pathophysiology of OA and associated pain. In addition, a global lipidomics workflow was developed by me, following the latest trends used within the wider metabolomics community, ensuring robustness and reproducibility in the identification of putative metabolite/lipid biomarkers for diseases. Experiments in this thesis using a targeted oxylipin liquid chromatography tandem mass spectrometry (LC-MS/MS) method showed that statistical significant changes in the levels of certain oxylipins were observed. More specifically, 11,12-DHET (mean concentration: 0.26 pmol/g in control, 0.54 pmol/g in MNX; p < 0.01), 14,15-DHET (0.46 pmol/g in control, 0.75 pmol/g in MNX; p < 0.05) and 8-HETE (5.46 pmol/g in control, 7.40 pmol/g in MNX; p < 0.05) were statistically increased in the MNX compared to control (sham) rats in ventral spinal cord in the MNX rat model of OA. These findings are supported by literature since these three lipids exhibit pro-inflammatory properties and thus are expected to increase in the OA group where inflammation is the main feature of OA. Regarding the MIA rat model levels of other oxylipins in synovial fluid were differentially expressed in the MIA compared to saline (control) rats. Arachidonic acid (AA), (272.3 pmol/g in control, 435.3 pmol/g in MIA; p < 0.05) was increased in the MIA-treated compared to saline-treated rats, while 9-HODE (4.42 pmol/g in control; 1.21 pmol/g in MIA; p < 0.05) was statistically decreased in the MIA compared to saline rats. Since AA has been reported to be released from membrane phospholipids in OA, the observation that AA is statistically increased in synovial fluid in MIA- compared to saline-treated rats bears strong significance. In addition, maps of oxylipins metabolism were generated to visualize the pathways underlying the changes of lipid concentrations in plasma between control and OA rats for both MNX and MIA rat models. Therefore, applying a targeted oxylipin LC-MS/MS method in different tissues of MNX and MIA rat models of OA is a successful approach and informative about changes in pathophysiology of OA, underlying significant alterations in oxylipins concentrations. Although the global lipidomics approach was able to measure different classes of lipids that might account for differences in plasma between MNX/MIA and sham/saline-treated rats, this approach exhibited weak MVA (multivariate analysis) models. In contrast to MNX and MIA rat models, the global LC-MS lipidomics profile in plasma from a DMM mouse model of OA exhibited excellent MVA models with good prediction scores. Twenty-six statistically significant lipids were identified, using the lipidomics workflow that I have developed, and when four of these lipids were used to build Receiver Operative Curves (ROC) the model produced high prediction (84%) power in separating sham from DMM mice. The identity of these four lipids was classified as being fatty acids (FAs), sterols, sphingolipids, and diacylglycerols (DAG). In addition, MS/MS experiments were performed to confirm the identity of significant lipids. Thus, it was shown herein that applying a global lipidomics LC-MS approach in plasma from the mouse DMM model, using only a small number of mice (15 in total), can be informative about significant changes in the “lipidome” in OA and can be used as a robust means of predicting OA in mice based on their global lipidomics profile. Lastly, correlation statistical analysis was applied between levels of lipids in the various tissues, pain behaviour, and histopathology parameters in the three rodent models of OA. Although many oxylipins/lipids levels were found to be statistically correlated with the aforementioned parameters, the most striking finding is that 9-HODE and AA were both found to be positively correlated with Weight Bearing (WB), a parameter of pain behaviour, in plasma and synovial fluid in the MIA rat model of OA. Since plasma reflects systemic inflammation and synovial fluid reflects local (inflammation) 9-HODE (p < 0.01 in plasma; p < 0.05 in synovial fluid) and AA (p < 0.01 in plasma and synovial fluid) are oxylipins that potentially depict systemic and local changes in WB differences, and subsequently in OA related pain. This finding is supported by literature since both AA and 9-HODE are both agonists of a pain receptor (i.e. transient receptor potential vanilloid 1, TRPV-1). Thus, it was proved in this thesis that correlation analysis can be used as an additional and complementary statistical tool in an effort to determine the role of lipids in OA pathogenesis in rodent models of OA. In conclusion, applying both targeted oxylipin LC-MS/MS and global lipidomics LC-MS analytical methods capable of measuring either oxylipins or the whole “lipidome” in vivo, have provided novel findings to support the involvement of these lipids in OA and associated pain.

Drug delivery systems in the size range of ~ 10-250 nm are enabling tools for site-specific targeting and controlled release applications. To take advantage of these capabilities, various nanocarriers e.g., micelles, dendrimers, liposomes, nanoparticles, nanocapsules, nanotubes, and nanogels, have been designed for drug delivery. Specifically, micelle-based drug carrier systems have emerged as promising tools for site-specific delivery and controlled release applications. Despite several advantages over conventional drugs, some limitations of micelle-based drug delivery have also been reported. These drawbacks include low stability in vivo, poor penetration, modest accumulation in tumour tissues, and inadequate control over drug release. To overcome these limitations, stimuli-responsive or smart polymeric nanocarriers have been developed for drug delivery and tumour therapy, previously. The most well-known internal stimuli in cancerous regions include higher acidity associated with dysregulated metabolism in tumour tissues, elevated levels of glutathione in the cytosol and nucleus of cancer cells, and altered degradative enzymes in the lysosomes, and reactive oxygen species in the mitochondria. These intrinsic microenvironments can be exploited as internal stimuli to attain active drug release in the tumour tissues or cancer cells. In particular, the reducing potential inside the cancer cells is considerably higher than found in the extracellular environment and bloodstream. Such varying redox potential can be exploited for tumour-specific drug delivery and controlled release applications. Various types of redox-responsive micelles have been developed, previously. Generally, redox-responsive micelles have disulfide linkages that undergo rapid cleavage in the presence of reducing agents in the intracellular components, however, are stable at oxidising extracellular environment. The redox-responsive disulfide bridges can be incorporated into nanocarriers by placing multiple disulfide bonds in the hydrophobic backbone or by conjugating therapeutic agents to the side chain of the polymer via a disulfide linker. Another strategy to construct redox-responsive linkages is to crosslink the polymeric nanocarriers with a disulfide crosslinker. Studies have shown that polymeric micelles can dissociate, especially upon administration when they are diluted below their critical micelle concentration. The stability of polymeric micelles can be enhanced by chemical crosslinking. Various types of crosslinked micelles can be prepared subjected to the localisation of the crosslinking, e.g. shell crosslinked micelles, and core crosslinked micelles. Introducing redox-responsive bridges through disulfide crosslinkers may not only provide stability to nano-carriers against dilutions during circulation, but also render them responsive to reducing conditions. Specifically, redox-responsive core-crosslinked micelles have demonstrated good stability and better ‘stealth’ properties, nevertheless, the hydrophobic core of most of the existing core-crosslinked micelles have been based on non-degradable polymers such as polyacrylamide or polyacrylate. The non-degradable constituent of the block copolymer may cause complications in clinical applications. Therefore, reduction-responsive core-crosslinked micelles comprising entirely of biologically inert or biocompatible and biodegradable polymers would be better candidates for drug delivery and controlled release application. To overcome these limitations, micelles based on polyesters (a class of aliphatic biodegradable polymers) can used for drug delivery application. In the last few decades, various FDA approved aliphatic polyesters e.g. poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone), and poly(lactic acid), have been intensively studied to exploit their potential in drug, gene and protein delivery and controlled release applications. Nevertheless, most of these polyesters lack functional groups, making it difficult to incorporate redox-responsive linkages to core-crosslink their micelles. To address these issues, we have synthesised functional biodegradable and biocompatible block copolymers based on methoxypoly(ethyleneglycol)-b-poly(-caprolactone-co--azido--caprolactone) (mPEG-b-poly(CL-co-N3CL)). The pendent chloro groups of the block copolymer were converted into azides using nucleophilic substitution reaction to obtain mPEG-b-poly(CL-co-N3CL) block copolymer as a precursor of reactive polymeric micelles. The synthesised polymers were characterised by NMR, FT-IR and size exclusion chromatography (SEC). Micelles were prepared using the dialysis method and methotrexate (an anticancer drug) was loaded into the hydrophobic core of the reactive micelles. Micelles were subsequently crosslinked by a redox-responsive bis-alkyne ethyl disulfide crosslinker. The size distributions and morphology of core-crosslinked micelles were assessed using dynamic light scattering (DLS) and transmission electron microscopy. The drug release studies were performed under simulated non-reducing and reducing conditions. Cellular uptake studies in human breast cancer cells (MCF7 cells) were performed using Oregon-green loaded core-crosslinked micelles. The MTX-loaded core-crosslinked micelles were assessed for their cytotoxicity in human breast cancer cells by MTT assays. The apoptosis inducing potential of MTX-loaded core-crosslinked micelles was analysed using Hoechst/PI assays and was further probed by annexin-V/PI assays. The data from these studies indicate that drug release from these crosslinked micelles can be controlled and that redox-responsive micelles are more effective carriers for MTX than non-cross-linked analogues in the cell lines tested. In another strategy, a multifunctional amphiphilic block copolymer based on -amine-PEG-b-poly(CL-co-N3CL) was synthesised and subsequently was used to conjugate methotrexate on the hydrophilic block for receptor mediated targeting of breast cancer cells. Cellular uptake studies revealed 2.3-fold higher uptake of MTX-conjugated micelles as compared with un-conjugated micelles. The blank micelles showed low cytotoxicities in breast cancer cells, however, MTX-conjugated micelles exhibited greater antitumor activity in contrast to the free-MTX. We hypothesise that these functional micelles could be potentially powerful nanocarriers for stimuli-responsive controlled release, active tumour targeting, and cancer therapy.

The aim of this project was to prepare, optimize, characterize and compare a subcutaneous/oral delivery system for insulin. The effect of various low molecular weight chitosans (LMWCs) on the stability of insulin, using USP HPLC methods, was investigated. Insulin was found to be stable in a polyelectrolyte complex (PEC) consisting of insulin and LMWC in the presence of Tris-buffer at pH 6.5. In the presence of LMWC, the stability of insulin increased with decreasing molecular weight of LMWC; 13 kDa LMWC was the most efficient molecular weight for enhancing the physical and chemical stability of insulin. The bioactivity of insulin in the PEC was assessed using enzyme-linked immunosorbent assay (ELISA) testing; results showed that insulin is still functionally active in the presence of chitosan. Solubilization of the PEC in a reverse micelle formulation (RMF) and administration to diabetic rats resulted in an oral delivery system for insulin with acceptable bioactivity. The effect of reduced (GSH) and oxidized (GSSG) glutathione on the bioactivity of insulin was studied. A PEC of insulin with low molecular weight chitosan (13 kDa) was prepared and characterized. The PEC was then solubilized, in the presence and absence of GSH and GSSG, in a reverse micelle consisting of oleic acid and two surfactants (labrasol and plurol). The in vitro and in vivo performances of the reverse micelle formulations (RMFs) were evaluated in rats. At pH 6.5 the association efficiency of the PEC was 76.2%. In vitro insulin release from the RMs was negligible at pH 1.2 and was markedly increased at pH 6.8. The hypoglycemic activity of insulin in the PEC was reduced when administered via the subcutaneous route, regardless of the GSH content. On the other hand, the presence of GSSG significantly enhanced hypoglycaemia. When the RMF was administered via the oral route, the presence of GSH had no effect on the hypoglycemic activity of insulin compared with the GSH free system. However, the presence of GSSG in the oral preparation increased the hypoglycemic activity of insulin; probably by inhibiting insulin degradation, thereby prolonging its effect. Thus, incorporation of GSSG in the RMF reduces blood glucose levels in rats and protects insulin from degradation. The effect of glucosamine HCl (GlcN⋅HCl) on the bioactivity of insulin, administered via subcutaneous (SC) and oral routes, in rats was also investigated. The oral insulin delivery system (IC-RM) was prepared by solubilizing insulin-chitosan (13 kDa) polyelectrolyte complex (IC-PEC) in a RM system consisting of oleic acid, PEG-8 caprylic/capric glycerides and polyglycerol-6-dioleate. The blood glucose levels were measured using a blood glucose meter. The results revealed that the extent of hypoglycemic activity of SC insulin was GlcN⋅HCl dose dependent when they were administered simultaneously. A significant reduction in blood glucose level (p < 0.05) was found for the insulin:GlcN⋅HCl at mass ratios of 1:10 and 1:20, whereas lower ratios (e.g. 1:1 and 1:4) showed no significant reduction. Furthermore, enhancement of the action of SC insulin was achieved by oral administration of GlcN⋅HCl for five consecutive days prior to insulin injection (p < 0.05). For oral insulin administration via the IC-RM system, the presence of GlcN-HCl increased the hypoglycemic activity of insulin (p < 0.05). The relative pharmacological availabilities (PA) were 6.7% and 5.4% in the presence and absence of GlcN⋅HCl (i.e. the increase in the relative PA was about 23% due to the incorporation of GlcN⋅HCl in the IC-RM system), respectively. The aforementioned findings offer an opportunity to incorporate GlcN⋅HCl in oral insulin delivery systems in order to enhance a reduction in blood glucose levels.

The last two decades have seen an increased drive to administer parenteral nutrition (PN) to patients in their home environments, thereby reducing associated hospital costs and improving patient quality of life. The occurrence of deranged nutritional biochemistry results has baffled PN experts for years because PN additives are marketed for the general needs of patients and PN is tailored to each patient’s requirements (both formulation and regimen). This thesis documents the investigations into HPN population characteristics, the extent of nutritional abnormalities (deficiencies and excesses) in a cohort of LT PN patients in Wales. Both cross-sectional and longitudinal retrospective study designs were employed alongside small-scale laboratory efforts to investigate stability of vitamin D in PN additives using High Performance Liquid Chromatography (HPLC). Characteristics of the HPN population in Wales were shown to be variable in terms of PN requirements for a predominantly female sample population (2:1); in whom 78.6% of patients received PN for indications relating to short bowel syndrome (SBS). A database analysis of micronutrient test results revealed a high prevalence of deficiencies of vitamin D and selenium, as well as excesses of manganese and water-soluble vitamins; which can lead to clinically relevant effects in patients. The sample population was shown to have impaired bone health since first receiving PN; respective sites of the femoral neck and total hip presented 58% and 60.8% of patients had osteopenia, while 28% and 19.6% had osteoporosis. Evidence in the literature links these clinical outcomes of metabolic bone disease (MBD) to patients’ inadequate vitamin D status. A final study exploring the adequacy of the trace element (TE) preparation Additrace®, found it lacking in selenium and excessive in manganese for the general requirements of the PN population. Clinician-directed supplementation of PN outside of Additrace® was associated with better micronutrient status in patients and more test results within range.

For the past two decades, the development of protein therapeutics has significantly expanded with numerous biopharmaceutical and biosimilar products entering the medicine market every year, and even more queuing in the pipeline globally. Biologics are very complex molecules and therefore extremely sensitive to minor changes in the manufacturing process, which can result in heterogeneity and affect the stability, potency and immunogenicity of the final product. Public health organisations, such as EMA (European Medicines Agency), require that biological products should be extensively tested for their similarity to the original drug (in the case of a biosimilar) as well as to products from different batches (batch-to-batch comparison). The issued guidelines focus, among other tests, on physicochemical characterisation of these molecules. The suggested analytical techniques, however, are only vaguely named in the specifications, leaving the final decision to the manufacturers. The present work focuses on the use of different combinations of analytical techniques with an aim to demonstrate similarity or dissimilarity between two or more samples. The selected instrumental techniques are characterised by their simplicity and are able to detect structural differences and microheterogeneity of the active ingredient in different samples, aggregation, degradation and post-translational modifications (PTMs). Seven studies were completed in total, each one to a different extent, and these included protein therapeutics such as insulin and monoclonal antibodies. The applied techniques served for primary (MS),* secondary (far-UV CD, FT-IR) and tertiary structure (near-UV CD, fluorescence) comparison of the examined samples. Particle size comparability and detection of aggregation was achieved with DLS, and higher-order structure comparison with 1D 1 H-NMR. Coupling of the techniques with temperature-dependent measurements enabled further comparison on the thermal stability of the samples and provided confidence in the observed (at room temperature) results. The acquired empirical experience pointed out the advantages and disadvantages of each technique compared to the rest of the techniques, possible solutions to the encountered challenges, and the cases that one technique can be used instead of another or as complementary to it. Finally, a potential SOP is suggested, advising on which biophysical techniques should be used depending on the structure of the protein that is examined and its formulation.

Malaysia’s rainforests have a great biodiversity, a lot of which has yet to be studied. This presents researchers with the opportunity for the discovery of new chemical structures which may be new leads in drug discovery. With problems such as antibiotic resistance and the need for improved drugs for the treatment of diseases such as cancer and neurodegenerative diseases, there is need for more research into drug discovery. The focus of the research covered in this document is on Pericampylus glaucus, a common climbing plant for which there is limited literature available. The aim of this study is to determine its pharmacological activities, based on what is known of its traditional use, in the hope that it can be used in the development of pharmaceutical drugs in the future. The literature review of the traditional uses of P. glaucus suggests that the plant may be used in ethnomedicine for its anti-inflammatory and/or antibacterial activity as it is used to treat many conditions in which inflammation and infection is indicated. Components of P. glaucus extracts were investigated through a variety of phytochemical screening methods and were found to include polyphenols such as tannins and flavonoids, terpenes, sterols, alkaloids and saponins and similarities between the different extracts were observed. P. glaucus was found to possess antioxidant activity, with the stem chloroform extract possessing the greatest radical scavenging activity. The antibacterial assays revealed only mild antibacterial activity by P. glaucus extracts, making the use of the plant for treating infections seem unlikely, although this may be due to activity against specific strains of bacteria. The anti-inflammatory assays suggested a number of different mechanisms by which P. glaucus could exert anti-inflammatory activity, with many of the extracts and known compounds inhibiting COX-1 and COX-2 enzymes, and only the fruit hexane extract providing less than 50% inhibition of COX-1 and COX-2 at 500 μg/mL. The hexane extracts generally prevented protein denaturation to a greater extent compared to other extracts, as well as palmitic and stearic acid. The extracts showed a varying ability to prevent lysis of red blood cells, which were used as a model for lysosomes, depending on whether lysis was induced by hypotonicity or by heat. Some anticancer activity, cytotoxicity and growth promoting activity was recorded when cells were cultured with P. glaucus extracts. The ethanol extracts were found to exert the greatest cytotoxic activity, with the exception of the fruit ethanol extract. The root ethanol extract was the most toxic towards HK1 and MRC-5, while the stem hexane extract increased HK1 proliferation by over 200% and the root hexane extract increased MRC-5 proliferation by over 600%. Because of the growth promoting activity that was observed and the traditional use of P. glaucus for the treatment of wounds, the wound healing activity of the extracts was tested and found to be much less effective than the control of 10% FBS, although the greatest wound healing activity of around 20% wound coverage after 24 h was seen by the fruit extracts. Since P. glaucus is traditionally used for the treatment of venomous snake bites, its antivenom activity was assessed and found to have some antivenom activity, with the fruit hexane extract increasing cell proliferation by over 50% at 200 μg/mL and the alkaloid, periglaucine A, increasing proliferation by over 70% demonstrating significant antivenom activity. The antioxidant activity is most likely attributable to the polyphenols as the total polyphenol content correlated with the results of the DPPH radical scavenging activity of the extracts. The mild antibacterial activity suggested that P. glaucus may be used more for its anti-inflammatory activity in traditional medicine rather than antibacterial activity. The findings of toxicity towards cancer cells supports the traditional use of P. glaucus for the treatment of cancer. The results of the wound healing activity would indicate that P. glaucus would have a mild effect in the treatment of wounds due to its ability to promote cell proliferation and migration, however, its anti-inflammatory activity and mild antibacterial activity may be beneficial in treating wounds. It did possess antivenom activity, which supports its use for the treatment of envenomation by snakes, although the fruit extracts had stronger antivenom activity compared to the root, which is the plant part traditionally used. The mild antibacterial activity of P. glaucus may partly contribute to its medicinal effects, however, this study reported stronger evidence of anti-inflammatory, cytotoxic and antivenom activity which may be the properties that practitioners of traditional medicine have found valuable when selecting this plant for treatment. The pharmacological activities reported support a number of the traditional uses for the plant, P. glaucus, suggesting that the traditional use may have a scientific ground and could be investigated further, possibly leading to the discovery or development of new anti-inflammatory, chemotherapeutic or antivenom drugs.

Cancer is one of the leading causes of death worldwide with approximately 14 million new cases reported in 2012. Systemic chemotherapy is widely used to treat cancer and can often result in toxicity and adverse side effects. To reduce these adverse side effects and potentially increase efficacy, the focus of this thesis is to create a new intratumoural drug delivery platform for patients who are too ill to receive systemic chemotherapy or with inoperable tumours. Herein, an injectable formulation composed of a hydrogel and drug-loaded polymeric nanocapsules (NCs) was developed, to deliver the chemotherapeutic locally within the tumour and hence reduce the adverse side effects. The hydrogel is used to control the release of the NCs within the tumour, while small NCs (< 100 nm) aim to promote NC penetration throughout the tumour mass. Self-emulsification, a low energy and solvent-free method, was used to produce the polymeric NCs. The lipid core of the NCs was suitable for encapsulation of anticancer drugs: docetaxel (DCX) and 4N-myristoyl-gemcitabine (GEM C14). NCs with diameters of 40 and 80 nm and with zeta potential values of ~ -4 ± 1 to –19 ±1 mV were produced. Both anticancer drugs, DCX and GEM C14 were encapsulated with efficiencies of greater than 90%. GEM C14 was released from the NCs by diffusion with 63 ± 8 and 66 ± 10 % released in 30 days from 40 and 80 nm NCs respectively. Additionally, GEM C14-loaded NCs showed activity against various cancer cell lines in vitro where the following GI50 values were obtained: 15 ± 6, 10 ± 9, 13 ± 3 nM in HCT 116, MIA PaCa-2 and Panc-1 cell lines respectively. Although not as potent in a GEM-resistant pancreatic cancer cell line (Panc-1 GEM resistant), GEM C14 NCs (GI50= 410 ± 463 nM) were significantly more active than GEM C14 alone (GI50=8300 ± 5523 nM). Hydrogels were prepared using a low molecular weight (LMW) gelator - N4-octanoyl-2’-deoxycytidine. This amphiphilic molecule self-assembles in water forming a 3D nanofibre network with the ability to reform after the application of stress, making it suitable for injection through a syringe/needle. N4-octanoyl-2’-deoxycytidine was used to gel the NC suspension via a heat-cooling cycle. The strength of gel-NC composite was determined by oscillatory rheology and its 3D nanofibre structure containing spherical NCs was confirmed by TEM. The gel-NCs formulation reformed instantly into a gel after injection through the needle and formed a depot when injected in an in vitro simulation of intratumoural delivery i.e. chicken tissue. In summary, a novel NCs-gel composite has been designed demonstrating appropriate characteristics for intratumoural drug delivery.

VanS is an integral membrane protein, and a receptor histidine kinase representing on half of the VanSR two component system. In the presence of vancomycin, as well as certain other antibiotics, VanS undergoes autophosphorylation at a conserved histidine residue and then transfers this phosphoryl group to a conserved aspartate on VanR. The phosphorylated form of VanR binds to the promoter region of the vanHAX operon, inducing expression of these genes, which confer glycopeptide antibiotic resistance to the cell. Although the vancomycin resistance mechanism is mostly well-characterised, questions surrounding the structure and mode of activation of VanS remain unanswered. This thesis describes work towards the structural and ligand-binding characterisation of the extracellular \sensor" domain of VanS. Little is known of the structure of this domain, and while there exists evidence in the literature to support numerous theories surrounding the identity of the VanS ligand, no report has yet been made of a direct observation of a binding event involving any VanS protein and its ligand To identify the ligand to VanS, the catalytically active, full-length VanS proteins of Enterococcus faecium (VanSA) and Streptomyces coelicolor (VanSSC) were heterologously expressed, solubilised and purified and binding studies were carried out by solution-state NMR. We present evidence of interactions between vancomycin and both VanS proteins, indicating that direct binding of vancomycin may be the mechanism by which both proteins are activated. The VanSSC extracellular domain was isolated as a synthetic peptide, its structure characterised, and the binding interface between this domain and vancomycin was investigated. Evidence of binding at the N-terminal end of the extracellular domain, in agreement with the findings of Koteva et al. (2010), is presented here. An interaction between vancomycin and DPC detergent micelles was also observed and characterised, and the biological significance of such an interaction is discussed.

No description available.